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Epigenetic Chromatin Modifiers in Barley: II. Characterization and Expression Analysis of the HDA1 Family of Barley Histone Deacetylases During Development and in Response to Jasmonic Acid
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DOI: 10.1007/s11105-009-0121-4
- Cite this article as:
- Demetriou, K., Kapazoglou, A., Bladenopoulos, K. et al. Plant Mol Biol Rep (2010) 28: 9. doi:10.1007/s11105-009-0121-4
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Epigenetic regulation of gene expression plays an important role in various aspects of eukaryotic development and is associated with modifications of chromatin structure. These are accomplished, in part, through the reversible process of histone acetylation/deacetylation, catalyzed by histone acetyltransferases (HATs), and histone deacetylases (HDACs), respectively. Eukaryotic HDACs are grouped in three major families, RPD3/HDA1 (thereafter cited as HDA1), SIR2 and the plant-specific HD2. Histone deacetylase genes have been studied in Arabidopsis and rice, but little is known about these genes from important crop plants. In this work, cDNAs encoding members of the HDA1 family and representing all four classes, Class I, Class II, Class III, and Class IV, were isolated and characterized from barley (Hordeum vulgare), a cereal crop of high agronomic importance. Expression analysis of the barley HDA1 family genes, HvHDAC1-I-1, HvHDAC1-I-3, HvHDAC1-II-1, HvHDAC1-III-1, and HvHDAC1-IV-1 demonstrated that they are expressed in all tissues and seed developmental stages examined. Differences in transcript abundance both in vegetative and reproductive tissues were observed among the different genes suggesting functional diversification of the HDA1 members. Differential expression was also evidenced for some of the HDA1 genes in two barley cultivars differing in various characteristics, such as seed size and resistance to stress, implying a possible association of these genes with different traits. Furthermore, the HDA1 genes were found to respond to the stress-related hormone jasmonic acid (JA), suggesting an association of these genes with barley responsiveness to biotic and abiotic stress. The expression pattern of the HDA1 genes suggests possible roles in the epigenetic regulation of barley development and stress response.
KeywordsHistone deacetylasesRPD3/HDA1 familyChromatin modificationEpigenetic regulationBarley
Epigenetic regulation of gene expression is achieved, in part, through changes in chromatin structure. Highly organized chromatin consisting of DNA wrapped around histone proteins in nucleosomal structures can switch between relaxed and condensed states associated with transcriptional activity and transcriptional repression, respectively (Goldberg et al. 2007). This is accomplished by a variety of enzymatic complexes which catalyze the modification of DNA and histones (Kouzarides 2007; Wu et al. 2009). Post-transcriptional modifications of histones include acetylation, methylation, phosphorylation, and ubiquination.
Acetylation of nucleosomal core histones is a reversible process which plays a crucial role in the regulation of eukaryotic transcriptional activity. Acetylation of histones is often associated with increased gene activation, whereas deacetylation of histones is correlated with transcriptional repression and gene silencing (Chen and Tian 2007). This is achieved through the action of specific enzymes. Histone acetyltransferases (HATs) transfer an acetyl moiety to the ε-amino group of highly conserved lysines in the N-terminal extensions of core histones, thereby neutralizing the positive charge of lysines and resulting in less affinity to the negatively charged DNA molecules. Highly ordered condensed chromatin is then unfolded and accessibility of promoters to transcription factors is increased. Histone deacetylases (HDACs) remove the acetyl moieties from lysines resulting in reestablishment of the positive charge and compact chromatin configuration (Chen and Tian 2007). HATs and HDACs and other factors involved in chromatin structure are highly conserved in yeast, animals, and plants suggesting a fundamental mechanism in transcriptional regulation (Loidl 2004).
The first histone deacetylase, HDAC1, was discovered in mammalian cells and showed striking similarity with the yeast protein RPD3 (reduced potassium deficiency; Taunton et al. 1996). Since then, a number of histone deacetylases have been identified in a variety of higher eukaryotes. Sequence analysis of these proteins demonstrated that most of these enzymatic proteins contain conserved domains: a large domain of homology with the yeast RPD3 that covers most of the N-terminal, and a short C-terminal region with a more variable sequence. Mutagenesis of this homology domain confirmed its involvement in deacetylase enzymatic activity (Hassig et al. 1998; Kadosh and Struhl 1998).
Eukaryotic HDACs can be grouped into three major families based on their primary homology to the yeast HDACs, HDA1, and SIR2 (sirtuins). These are: (1) the HDA1 family, (2) the SIR2 family, and (3) the plant-specific HD2 family (Pandey et al. 2002; Yang and Seto 2003). The presence of a unique HDAC protein family in plants, HD2, possibly reflects the divergence of developmental processes and environmental responses of plant organisms from those of yeast and mammals. SIR2 proteins differ from the other HDACs in that they depend on nicotinamide adenine dinucleotide for enzymatic activity and have no structural similarity to other HDACs. Sequence analysis by Pandey et al. (2002) revealed that the first family of histone acetyltransferases, HDA1, can be subdivided further into three major clades, Class I, Class II, and Class III. Recently, sequence and phylogenetic analysis of the rice genome (Fu et al. 2007) identified the HDA1 family in rice, and showed that rice HDACs belonging in this family can also be divided in Class I, Class II, and Class III. In addition, the sequence analysis in rice revealed a fourth clade, Class IV. Expression analyses of 18 rice HDAC genes from HDA1, SIR2, and HD2 families demonstrated distinct spatial expression patterns and differential responses to environmental stresses and hormones (Fu et al. 2007). Characterization of HDAC mutants in Arabidopsis also revealed functional diversification among the members of different HDAC families and classes. AtHDAC6 gene (HDA1-Class I) for instance was shown to be involved in transgenic and repetitive DNA silencing and in the nuclear organization of rDNA loci (Ausfatz et al. 2002; Lippman et al. 2003; Probst et al. 2004). AtHDAC19, another member of the HDA1-Class I type was shown to be important for proper vegetative development as hdac19 mutants displayed various developmental abnormalities (Tian and Chen 2001; Tian et al. 2005; Zhou et al. 2005; Long et al. 2006). Mutations in the AtHDA18 gene (HDA1-Class II) resulted in alterations in the cellular patterning in the root epidermis in Arabidopsis (Xu et al. 2005). Recently, it was shown that HDA6 and HDA19 are involved in repression of embryonic-specific gene functions during germination (Tanaka et al. 2008). Similar to the HDA1 family, the plant-specific HD2 family has been suggested to be crucial for proper reproductive development. Silencing of the AtHD2A gene resulted in aborted seed development (Wu et al. 2000) and overexpression of AtHD2A resulted in repression of a number of genes involved in seed development and maturation (Zhou et al. 2004). Furthermore, HDAC genes from both families have been associated with the response to biotic and abiotic stress. AtHDAC19 was suggested to mediate jasmonic acid (JA) and ethylene signaling during pathogen defense (Tian and Chen 2001; Tian et al. 2005; Zhou et al. 2005) and HDA6 was shown to be required for jasmonate response, senescence, and flowering, in Arabidopsis (Wu et al. 2008). In addition, AtHD2C, belonging to the HD2 family was proposed to play a role in abscisic acid (ABA) signaling and abiotic stress (Sridha and Wu 2006).
Nevertheless, many aspects of the role that HDACs may play in plant development and the mechanism by which they regulate chromatin structure and gene activity remain obscure. Moreover, very little is known about the function of HDACs in crop plants. The best characterized crop system so far is that of maize where 15 HDAC genes have been identified (ten HDA1, one SIR2, and four HD2-like genes). Members of all three HDAC families have been identified and biochemically characterized from this important cereal crop (Lusser et al. 2001; Rossi et al. 2003) and overexpression of the gene hda101 (HDA1-Class I) showed that it is involved in the process of vegetative to reproductive transitions and it mediates other chromatin modifications such as histone methylations and phosphorylations (Rossi et al. 2007).
Working with another important cereal crop, barley, our group recently reported the isolation, mapping, and expression analysis of the histone deacetylase genes belonging to the HD2 family (Demetriou et al. 2009). Two full-length cDNAs, HvHDAC2-1 and HvHDAC2-2, were cloned and characterized and their expression investigated during seed development and in two cultivars with varying seed size. The two genes displayed differential expression during seed development, suggesting possibly different functional specificity. One member, HvHDAC2-1, exhibited significantly different levels of expression in different seed developmental stages. Interestingly, it also displayed higher levels of expression in all seed developmental stages in a small-seed cultivar, as compared to a large-seed cultivar. In the current work, we have extended these investigations presenting the characterization of the barley HDA1 gene family members as well. Representatives of each of the four classes of the HDA1 family: Class I, Class II, Class III, and Class IV were identified and the putative protein sequences analyzed and classified in a phylogenetic tree of other monocot and dicot HDA1 proteins. The expression of each class was analyzed in different tissues and developmental stages, in two barley cultivars with different characteristics, and in response to treatment with JA.
Materials and Methods
Plant Material
Two commercial barley cultivars, Caresse and Kos were planted in the field and were the source of RNA for expression analysis. Caresse has larger grain and Kos is more resistant to various stresses and diseases. For Caresse, the weight of 1,000 grains is 55 g, and 98% of seeds have diameter longer than 2.5 mm. For Kos, the weight of 1,000 grains is 36–40 g and only 36–49% of seeds have diameter longer than 2.5 mm. (www.cerealinstitute.gr).
Hormonal Treatment
Seven-day-old seedlings (Caresse) grown in a growth chamber (16 hours (h) light, 8 h darkness, at 22°C) were sprayed with 100 µM JA (methyl jasmonate, ALDRICH). Aerial parts of plants were collected at 6 h and 24 h after treatment and immediately thrown in liquid nitrogen. Aerial parts from five plants were pooled together for RNA extraction for each time point. Control plants were sprayed with water plus 0.2% Tween.
RNA Isolation and First-Strand cDNA Synthesis
Total RNA was isolated from roots, shoots, apical meristems, first leaves of seedlings, flowers before fertilization (immature flowers), seeds 1–3, 3–5, 5–10, 10–15, and 15–20 DAF (days after fertilization), and aerial parts of 7-day-old seedlings before and after hormonal treatment, respectively, using the TRIZOL method (TRI REAGENT, Sigma) according to the instructions of the manufacturer.
For the first-strand cDNA synthesis, 1 µg of total RNA was used for each reaction with the M-MLV Reverse Transcriptase kit of Invitrogen, according to the specifications of the manufacturer.
Isolation of cDNAs
A search of the barley EST database in TIGR (www.tigr.org) identified tentative consensus sequences corresponding to different HDACs belonging to different families and classes.
HvHDAC 1-I-1
RT-PCR performed using two specific primers, Barley RPD3CIF2 and Barley RPD3CIR2, based on TC135897 from the barley EST database (TIGR), and total RNA from barley seeds (cultivar Caresse) 1–3 DAF resulted in a HvHDAC 1-I-1 fragment of 762 bp. To obtain the 5′end of the HvHDAC1-I-1 cDNA, the RCA-rolling circle amplification (RACE) technique was utilized according to Polidoros et al. (2006). Circular cDNA synthesized from Caresse seeds 1-3 DAF total RNA was used as template. Specific primers, Barley RPD3CIF5a and HvHDAC1R1, HvHDAC1R2, and HvHDAC1R3 were used as forward and reverse primers, respectively.
HvHDAC 1-I-3
RT-PCR was performed using the primers Barley RPD3C1F1a and Barley RPD3C1R1a (based on the sequence of the TC132159, from the barley EST database, TIGR) and first-strand cDNA synthesized from a mixture of total RNA from Caresse seeds 5–10 and 15–20 DAF, as template. A fragment of 1,700 bp was obtained including the ATG translation initiation codon. To obtain the 3′ end of the HvHDAC 1-I-3 cDNA 3′RACE was performed using first-strand cDNA prepared from a mixture of total RNA from Caresse seeds 5–10, 10–15, and 15–20 DAF. The primers, Barley RPD3CIF1a and Abridge Universal Amplification Primer (AUAP, Invitrogen, Paisley, UK) were used as forward and reverse primers, respectively. A nested PCR with primers Barley RPD3CIF1b and AUAP produced a PCR product of 1,300 bp which contains the 3′UTR.
HvHDAC I-II-1
RT-PCR was performed using the specific primers Barley RPD3CIIF1/Barley RPD3CIIR1 (based on the sequence of the TC 137977) and first-strand cDNA synthesized from total RNA from Caresse seeds 1–3 DAF, as template. This produced a fragment of 742 bp. In order to obtain the 3′end of the HvHDAC I-II-1 cDNA, 3′RACE-PCR was performed using the primer pair BarleyRPD3CIIF1/AUAP for the first PCR reaction with RNA/cDNA from 1–3 DAF seed and HvHDACIIF2/AUAP for the nested PCR reaction. The 5′end of the HvHDAC I-II-1 cDNA was obtained using the 5′RACE System for Rapid Amplification of cDNA Ends, Version 2.0, Kit (Invitrogen, Paisley, UK). The primer pairs used were HvHDAC1II-1GST1/
Abridge amplification primer (AAP) and HvHDACIIR1/AUAP (Abridge Universal Primer) for the first and second PCRs, respectively, were used. A second nested PCR was performed with HvHDACIIR2 as forward primer, and AUAP as reverse primer and the second PCR as template at 0.1% and 0.05% dilution.
HvHDAC I-III-1
RT-PCR was performed using the specific primers Barley RPD3CIIIF1/Barley RPD3CIIIR1 (based on the sequence HDA1005 from ChromDB) and first-strand cDNA synthesized from total RNA from Caresse seeds 5–10 DAF, as template. This amplifies a fragment of 532 bp. 3′RACE-PCR then followed with primers HvHDACIII-F1/AUAP.
All PCR products of interest were cloned into pCR 2.1 TOPO vector using the TOPO TA cloning Kit (Invitrogen) and sequenced.
Protein Sequence Analysis
The protein sequence alignments was generated with the Clustal W Method (Thomson et al. 1994) of MegAlign of the LaserGene software and processed with Bioedit software. The phylogenetic tree was calculated using MEGA 3.1 software (Kumar et al. 2004) by the neighbor-joining method with p distance correction (Saitou and Nei 1987). Bootstrap values were obtained from 1,000 bootstrap replicates.
Expression Analysis
Semiquantitave RT-PCR was carried out with total RNA from roots, shoots, apical meristems, and first leaves of seedlings and from flowers before fertilization (immature flower) and seeds 1–3, 3–5, 5–10, 10–15, and 15–20 DAF from each cultivar, Caresse and Kos, respectively. The PCR conditions for all genes were: initial denaturation at 96°C for 2 min, then 30 cycles of denaturation at 96°C for 30 s, annealing for 30 s, extension at 72°C for 1 min and final extension at 72°C for 10 min. Tannealing was 53°C, 54°C, 63°C, 52°C, and 57°C for HvHDAC1-I-1, HvHDAC1-I-3, HvHDAC1-II-1, HvHDAC1-III-1, and HvHDAC1-IV-1, respectively. Primer pairs used were as follows: BarelyRPD3CIF2/BarelyRPD3CIR2, BarelyRPD3CIF1b/BarleyRPD3CIR1a, BarleyRPD3CIIF1/BarleyRPD3CIIR1, BarleyRPD3CIIIF1/BarleyRPD3CIIIR1, and BarleyRPD3CIVF1/BarleyRPD3CIVR1, respectively. To verify the presence of equal amounts of RNA/cDNA across samples during the PCR reactions, actin amplification was carried out. The pair of primers ActinF and ActinR amplify 1,111 bp of the actin coding EST sequence of barley AY145451. The PCR conditions for the amplification of actin were: initial denaturation at 96°C for 3 min, then 30 cycles of 96°C for 30 s, 70°C for 1 min, 72°C for 2 min and final extension at 72°C for 15 min.
Primers used for cloning and expression analysis
Barley RPD3CIF2
Barley RPD3CIR2
Barley RPD3CIF5a
Barley RPD3C1R5
Barley RPD3CIF1a
Barley RPD3CIR1a
Barley RPD3CIF1b
Barley RPD3CIR1b
Barley RPD3CIIF1
Barley RPD3CIIR1
Barley RPD3CIVF1
Barley RPD3CIVR1
HvADC2 F
HvADC2 R
Barley Actin F
Barley Actin R
HvActin F
HvActin R
The barley gene HvADC2 (arginine decarboxylase 2), which is known to be induced by JA (Walia et al. 2007) was used as a positive control.
Identification, Cloning and Protein Sequence Analysis of HvHDAC Members Belonging to the HDA1 Family of Barley HDACs
Two full-length cDNAs sequences belonging to Class I of the HDA1 family were isolated from Caresse and named HvHDAC1-I-1 and HvHDAC1-I-3, respectively.
One full-length cDNA belonging to Class II of the HDA1 family was isolated from barley, cultivar Caresse, and named HvHDAC1-II-1. A barley EST (TC 137977) corresponding to Class II of the HDA1 family of HDACs was used to design specific primers which amplified a fragment of the EST using total RNA from seeds 1–3 DAF. The cDNA was completed with 5′ and 3′ RACE-PCR and contains 2,391 bp encoding a putative protein of 614 amino acids. An alignment of different HDA1-Class II, proteins from different plant species is shown in Fig. 1. The HvHDAC1-II-1 protein sequence has 95% amino acid identity with TaHDAC4 from wheat. HvHDAC1-II-1 is classified together with HDA1-Class II HDACs, being more closely related to the monocot Class II sequences as shown in the phylogenetic tree (Fig. 2).
For HvHDAC1-III-1, firstly, a partial cDNA was obtained with specific primers based on the sequence of the EST HDA1005 from the ChromDB database (www.chromdb.org) and total RNA from Caresse seeds 5–10 DAF. This cDNA was completed to the 3′ end by 3′RACE-PCR to give a total of 225 amino acids which contain the entire histone deacetylase domain. It has 88% identity with rice OsHDA706 and 73% identity with Arabidopsis AtHDA2 (Fig. 1). HvHDAC1-III-1 groups out into the HDA1-Class III HDAC sequences as shown in the phylogenetic tree (Fig. 2).
A full-length cDNA containing a HDA1-Class IV protein sequence from the cultivar Haruna nijo was identified in the barley DB database (http://www.shigen.nig.ac.jp/barley/) encoding 389 amino acids. The putative protein sequence has highest homology with OsHDA712 (86% identity) and OsHDA716 (70% identity; Fig. 1). The relationship of HvHDAC1-IV-1 with the other HDA1 family members is shown in the phylogenetic tree (Fig. 2). HvHDAC1-IV-1 is classified together with the newly identified Class IV clade in rice (Fu et al. 2007).
Expression of HDA1 Genes During Barley Development
Expression of Barley HDA1 Genes after JA Treatment
In this work, we have presented the cloning and characterization of members of the HDA1 gene family of histone deacetylases from barley. Four cDNAs, encoding HvHDAC1-I-1, HvHDAC1-3-1, HvHDAC1-II-1, and HvHDAC1-III-1 were isolated from barley and one cDNA encoding HvHDAC1-IV-1 was identified in a public database. Previous sequence analysis of HDA1 protein sequences from different organisms (Pandey et al. 2002; Fu et al. 2007) had demonstrated that the HDA1 proteins are divided into four classes, Class I, Class II, Class III and Class IV. Phylogenetic analysis of the barley HDA1 sequences revealed that they are also grouped in Class I, Class II, Class III, and Class IV. Specifically, we have identified and characterized two members of Class I, HvHDAC1-I-1, and HvHDAC1-I-3, and one member of each of the other classes, HvHDAC1-II-1, HvHDAC1-III-1, HvHDAC1-IV-1, respectively. Class IV was recently identified in rice (Fu et al. 2007). Interestingly, our analysis reveals that AtHDAC8, a previously unclassified Arabidopsis HDAC sequence (Hollender and Zhong 2008), and ZmHDA117 a newly identified maize sequence (ChromDB database) also group out in Class IV of HDA1, increasing the size of this previously unknown class.
Protein sequences used for alignments and phylogenetic tree construction
Gene name
Accession number
Bankit 1047187 EU348772
Bankit 1047198 EU348773
Bankit 1047277 EU348774
Bankit 1206526 FJ898365
At HD2a
At HD2b
At HD2c
At HD2d
Os HD2
Zm HD2a
Zm HD2b
Zm HD2c
Zm HDA106
Ta HDAC2
Bankit 1047282 EU348775
Bankit 1047284 EU348776
The expression of barley HDA1 genes was examined in different tissues, seed developmental stages and in two cultivars, Caresse and Kos, with different agronomical characteristics. For instance, Caresse has larger grain size, and Kos is superior with respect to resistance to diseases and general adaptability. Differential expression was observed for each gene during vegetative and reproductive development with notable variation in expression across classes. Likewise, differential expression was evidenced between the cultivars Caresse and Kos for each individual gene. The differences in expression patterns across developmental stages among the different classes of the family may reflect distinct functions of different HDA1 genes during barley development. Similarly, the differences in gene expression for each HDA1 gene between cultivars might be associated with the different characteristics of each cultivar. Further experiments will elucidate the roles of HDA1 genes and the likely functional diversification among family members. Equally, additional experiments with a large number of cultivars will be necessary in order to reach to a conclusive assessment about any potential HDA1-trait association.
The barley HDA1 genes were also examined for their responsiveness to the stress-related hormone, JA. Four genes, HvHDAC1-I-3, HvHDAC1-II-1, HvHDAC1-III-1, and HvHDAC1-IV-1, were found to be induced by exogenous JA application. In our previous work on the barley HD2 family, HvHDAC2-1 and HvHDAC2-2 were also shown to be induced with JA treatment (Demetriou et al. 2009), suggesting that a general JA-mediated HDAC induction may exist in barley. In rice, certain HDA1 genes were found to be induced by external JA whereas others were downregulated. According to Fu et al. (2007), two members of the rice HDAC class I (HDA 702 and HDA 705) and one member of class II (HDA 704) were induced by exogenous JA application, whereas another member of class II (HDA 714) as well as a member of class III (HDA 706) did not show any significant changes in their expression upon JA treatment. Conversely, the expression of a member of class IV (HDA 712) was reduced upon JA application.
In barley, except for HvHDAC1-I-1, the rest of HDA1 genes examined (one of each class, I, II, III, and IV, respectively) were induced upon JA treatment. Induction by JA agrees with that for rice at least for one class I and one class II genes. Differential gene expression between the rice and barley HDA1 members of the same HDAC class may be due to the fact that the representative HDA1 genes examined are not functional homologues. Additionally, it may reflect differences in the expression of these genes between the two plants, implying differences in the HDAC-associated JA response within monocots. Certainly, examination of the expression of all members within a class would be needed in order to make more conclusive comparisons of the HDA1 gene expression in response to external JA, between the two plants. The barley HDA1 genes were also examined for their response to ABA, but no significant changes in gene expression were observed (data not shown).
Histone modifications, including acetylation, are involved in the responses of plants to stress (Chinnusamy et al. 2008; Chinnusamy and Zhu 2009). Biotic and abiotic stress factors trigger the production of certain hormones by plants. The plant hormones JA, ABA, SA (salicylic acid), and ethylene mediate the regulation of gene expression during the adaptive responses of plants to abiotic and biotic stresses. It has been suggested that histone acetylation/deacetylation through the action of HATs and HDACs, respectively, may epigenetically regulate the integration of hormonal signals controlling stress response genes (Sridha and Wu 2006). For example, in Arabidopsis, AtHDA19, a HDA1-Class I gene, was found to mediate histone deacetylation in response to abiotic and biotic stress. AtHDA19 is induced by wounding, JA, ethylene and infection by pathogens (Zhou et al. 2005). Overexpression of AtHDA19 resulted in reduced histone acetylation levels and upregulation of the stress-related genes ERF1 (Ethylene Response Factor-1) and PR (Pathogenesis Related). Conversely, silencing of AtHDA19 led to increased histone acetylation and downregulation of ERF1 and PR. AtHDA6, another HDA1-Class I gene was also found to be induced by exogenous JA and ethylene (Zhou et al. 2005). In addition, in AtHDA6 mutants and in HDA6-RNAi plants the expression of the Arabidopsis JA-responsive genes PDF1.2, VSP2, JIN1, and ERF1 was downregulated. Furthermore, histone modifications have been suggested to play a major role in ABA-mediated response to abiotic stress (Chinnusamy et al. 2008). ABA treatment caused severe reduction in expression of the histone deacetylase, AtHD2C, in Arabidopsis, whereas overexpression of AtHD2C resulted in enhanced abiotic stress tolerance to salt and drought stress, and both repression of several ABA-responsive genes and induction of others (Sridha and Wu 2006). Finally, cold, osmotic and salt stresses, and external application of hormones such as JA, ABA, and SA, in rice, were shown to increase the expression of certain HDA1 genes, and reduce the expression of others (Fu et al. 2007).
The observed induction of certain barley HDA1 genes upon treatment with the hormone JA may suggest a role for these genes in the epigenetic regulation of stress responses in barley.
The study described above on the HDA1 family of barley HDACs in conjunction with our recently published work on the plant-specific HD2 family (Demetriou et al. 2009), provide a basis for future studies aiming to elucidate the role of both HDA1 and HD2 families in epigenetic regulation of barley development and stress response.
This work was supported by a PENED grant (Ο3ΕΔ402/2003). Continuous support for the Institute of Agrobiotechnology/CERTH from the General Secretariat of Research and Technology of Greece is also acknowledged.


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Meet Mr JWoww: Jersey Shore star gets upstaged by boyfriend Roger Matthews and his colourful workout attire
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But all eyes were on the 26-year-old Jersey Shore star's metrosexual boyfriend Roger Matthews as the couple stepped out in Manchester, New Jersey yesterday.
The tattooed bodybuilder stole glances in colourful workout attire as the on/off couple began filming the second season of her MTV spin-off, Snooki & JWoww.
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Peacocking: JWoww was upstaged by her boyfriend Roger Matthews' colourful workout attire as they began filming the second season of Snooki & JWoww
With his waxed eyebrows and thin goatee, Matthews strut out of the gym, putting his bulging biceps on display in a lime green tank top paired with red Nike sneakers and Famous Stars & Straps riptide boardshorts.
Persistent male grooming is practically engrained in the macho culture behind MTV's Jersey Shore.
How else could Pauly D, the
Situation, Ronnie, or Vinnie ever 'smush' with guidettes without their
GTL - gym, tanning, and laundry - lifestyles?
J-Who? With his waxed eyebrows and thin goatee, the tattooed bodybuilder strut out of the gym in a lime green tank top and red Nike sneakers
Low key: JWoww revealed only a hint of cleavage in her white Gaspari tank top and looked much more subdued than usual
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Meanwhile JWoww selected a much more subdued workout ensemble of black
capri-length yoga pants, black Nike sneakers, and revealed only a
hint of cleavage in her white Gaspari tank top.
The turbulent pair, who've been
together for two years, looked much happier than they were on the last
episode of JWoww's MTV-spinoff.
After returning early from their
Cancun vacation, the feisty brunette is seen angrily telling Roger 'go
f*** yourself' and 'you can burn in hell' after accusing him of cheating
on her.
Best friends forever: The buxom brunette recently threw her pregnant costar Nicole 'Snooki' Polizzii a lavish baby shower
'You can burn in hell': The on/off couple are frequently seen having nasty fights on both the Jersey Shore and her MTV spinoff
The outburst was prompted after
Roger, who was partying at Hemingways, called Jenni 'the most insecure
person in the world' and suggested she might be happier with her
allegedly abusive ex-boyfriend Tom Lippolis.
Back in July, Roger confessed that he
was planning to propose to the buxom brunette, who has said she hopes
for a Las Vegas wedding.
'Soon, soon. I see that. I see that,' Roger told JustTheFab.com.
'Soon, I see that': Roger confessed in July he was planning to propose to the buxom brunette, who has said she hopes for a Las Vegas wedding
'I don't have any date set, but soon. Things are going really well with us, so you know...'
JWoww recently threw a lavish baby shower for her pregnant
co-star Nicole 'Snooki' Polizzi, who's expecting a baby boy in early
Snooki & JWoww, which airs on Thursdays on MTV, follows the ladies' highly-successful turn on Jersey Shore.
The controversial show, which follows a group of roommates as they spend the summer in a home share in Seaside Heights, New Jersey, is set to premiere its final season with the original cast on October 4.
Watch video here
Get More: Snooki & JWOWW, Full Episodes
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The Duchess Of Alba Hits The Beach With Her Boytoy
The 86-year-old Spanish royal wasn't afraid to flaunt her bikini bod with new (and much younger) husband Alfonso Diez.
1. If you find this hard to look at I suggest you stop scrolling.
CRISTINA QUICLER / Getty Images
2. You were warned.
5. Hopefully her helper is lathering her with sunscreen. We wouldn't want her to develop anymore wrinkles.
6. The sun really isn't good for her.
Via screened.com
7. Oh wait! She wasn't in "Hannibal." My bad.
Marcelo del Pozo / Reuters
Preview Your Response
In The News Today
- Donald Trump's campaign chief Stephen Bannon said "he doesn't like Jews," according to his ex-wife.
- Facebook shifts to algorithms to decide which stories to highlight in its trending news section after allegations of liberal bias at the company.
- Far-right protesters climbed Berlin's famous Brandenburg Gate to protest against refugees. About 1.1 million asylum-seekers arrived in Germany last year.


text:
|Publication number
|Publication type
|Publication date
|Sep 6, 1898
|Filing date
|Mar 19, 1898
|Publication number
|Edmond B. Slater
|Export Citation
|BiBTeX, EndNote, RefMan
|Referenced by (2), Classifications (1)
|External Links: USPTO, USPTO Assignment, Espacenet
No. 610,309. Patented Sept. 6, 1898.
(Application filed Mar. 19. 1898.,
(N0 Model.)
MT I\' E 55E 5 I .JXIJWM I {7m UNITED STATES P TENT Orrrcs.
EDMOND B. SLATER, OF MERIDEN, CONNECTICUT, ASSIGNOR TO THE FOSTER, MERRIAM dz COMPANY, OF SAME PLACE.
SPECIFICATION forming part of Letters Patent No. 610,309, dated September 6, 1898.
Application filed March 19, 1898. Serial No. 674,472. (No model.)
To all whom it may concern.-
Be it known that I, EDMOND B. SLATER, a citizen of the United States, residing at Meriden, in the county of New Haven and State of'Connecticut, have invented a new and useful Improvement in Furniture-Casters, of which the following is a full, clear, and exact description, reference being had to the accompanying drawings,forming a part of this specification, in explaining its nature.
The invention relates especially to the horn of a furniture-caster; and it consists in providing a peculiar-shaped blank of fiat metal and the horn made therefrom.
Referring to the drawings, Figure 1 is a view in plan of the blank. Fig. 2 is a view in perspective of the horn made from the blank. Fig. 3 is also aview in perspective of the said horn turnedfrom the position represented in Fig. 2. Fig. 4 is a View in side elevation of the complete caster. Fig. 5 is a View, partly in elevation and partlyin vertical section, of the complete caster.
The blank A from which the horn of the caster is formed is punched or otherwise made from sheet metal, and it has integrally-connected sectionsa a, which form the arms of the complete horn-sections a a which are adapted to form the upper and lower pintlebearings, and the connecting-section a, unit ing the arm-forming sections and the two bearing-forming sections. The arm-forming sec tions preferably are curved from the end of the connecting-section a and have at their outer ends the holes a The arm-forming sections also have at their. upper ends the shoulders at. The pintle-bearing sections a a each have a pintle-hole a and each preferably has the curved back a on each side of its juncture with the cross-bar a the shoulder a", and the straight edge a although the form of this edge is immaterial. The blank thus shaped or similarlyshaped is formed at one operation of a punch and die, and it is then submitted to a further forming operation whereby it is converted into the complete horn and whereby the connecting-section a is curved or bent to form a side wall I) of the complete horn B and the arm-forming sections a a brought parallel with each other to form the arms Z) of the horn and to bring the holes a in line with each other, the arms being separated from each other by a space sufficient to receive the caster-roll C and the holes a acting to receive the cross-pin c, which secures the caster to the horn-arms.
The pintle-bearing sections a a are bent to a right angle to the wall I) and form the horizontal supports 1) b for the pintle D, which are separated from each other by the Width of the Wall. They are turned in the same direction, so that the pintle holes a are brought into line with each other, and their curved edges rest upon or bear against the top and bottom edges of the said wall I), while their shoulders 67, a interlock with the shoulders a at the upper ends of the arms and the intermediate section between the arms and form spacing or distance pieces which prevent the further closing of the arms toward each other.
The pintle is attached to the supports 5 b in any of the usual ways. I have represented a conventional one in which the pintle is represented as having a head d at its lower end, beneath the lower support b and awasher d, which is pressed or otherwise united to the pintle and is carried by it immediately above the upper support b The advantages of the invention arise from the simplicity of the construction,its strength, and the cheapness with which it can be made, as but two operations are necessary for forming the complete horn. I do not wish to be understood as confining myself to the especial form of blank shown or to the particular shape of horn obtained from it, as there may be various changes in these forms and shapes which will not modify the principle of the invention.
Having thus fullydescribed myinvention, I claim and desire to secure by Letters Patent of the United States- I 1. In a blank for forming horns, the armforming sections a, a, having the holes a and shoulders afithe pintle-forming supports 60 ,11 having the shoulders a and the holes a and the integral connection a 2. As an improved article of manufacture the caster herein described, the same'comprising a horn B formed from a one-piece flat metal blank having a vertical rear wall Z), forwardextending parallel roll-supporting arms I), fiat pintle-supporting plates b 13 extending forward from the upper and lower edges of said back wall I), the upper of which has both sides extended upon said arms I) and is supported thereby as well as by the back, the pintle D carried by said supporting-plates b b and the caster-roll attached by a pin to said arms.
3. 111 a caster-horn a vertical wall, roll-supporting horns extending from and integral with said walhand having shoulders a,pintlesupports integral with said wall and parallel with each other, arranged to bear upon or against the edges of said wall, and provided with'shoulders a which cooperate with the EDMOND B.
In presence of- JAMEs R. SUTLIFF, J. L. RUTHERFORD.
|Citing Patent
|Filing date
|Publication date
|Sep 19, 1979
|Aug 23, 1983
|Herder, N.V.
|Dual wheel caster
|Feb 25, 1980
|Sep 4, 1980
|R Mccarroll
|Dual wheel caster
|Cooperative Classification


text:
Relevant offers
The man arrested near the Boston Marathon finish line carrying a backpack containing a rice cooker on the anniversary of the bombings was sent to a state psychiatric facility for an evaluation on Wednesday (local time) following his initial court appearance.
Kevin "Kayvon" Edson, 25, was sent to Bridgewater State Hospital and ordered held on US$100,000 bail at an appearance in Boston Municipal Court on charges of threatening battery, possession of a hoax explosive device, threats to commit a crime, disturbing the peace, disturbing a public assembly and disorderly conduct. His next court date was scheduled for May 7.
Edson's arrest occurred hours after ceremonies to mark last year's Boston Marathon bombings, in which two pressure cooker bombs hidden in backpacks exploded, killing three people near the finish line and injuring more than 260 others.
Edson, with addresses in Boston and Wakefield, was stopped late Tuesday (local time) after passers-by told an officer they saw him yelling, walking barefoot down the middle of a street, veiled in black, in pouring rain. His face was painted yellow and blue, the traditional colors of the marathon, police said. The street was open to pedestrians at the time, and police said his presence was not a security breach.
The backpack was destroyed. Police determined that the bag's contents were not explosive.
According to a police report read aloud in court, after Edson was read his rights, he told an officer: "I knew what I was doing, it was conceived in my head. It's symbolism, come on. The performance got the best of me."
Edson's mother, Joie Edson, said her son has bipolar disorder.
Parsons The New School for Design and the Fashion Institute of Technology, both in New York City, confirmed that Edson had taken classes at their schools but never graduated. The Massachusetts College of Art and Design posted a statement on its website saying that an undergraduate student had been arrested by police and the school was cooperating with authorities, but it did not mention Edson by name.
A second suspicious backpack also was found. Officers determined it had been left behind by a media outlet and was not dangerous, but it too was destroyed.
The backpacks rattled nerves days ahead of this year's marathon. Police kept people away from the finish line area for about three hours and trains bypassed the nearby Copley Square station.
"With the marathon coming, our officers are taking it seriously," police Superintendent Randall Halstead said. "The safety of the public is utmost."
Earlier Tuesday (Wednesday, NZ time), survivors, first responders and family members earlier marked the one-year anniversary of the bombing with a ceremony attended by Vice President Joe Biden.
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Spot your bag: Olympic fail for team GB


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"We were very pleased with the quality of the card we received. It looks exactly like it does in the digital proof. The colors are brilliant, and the print quality is very crisp. The standard paper is thick enough to stand up to mailing and display without creasing. The envelopes are smooth and creamy, and take the calligraphy ink very nicely. The printing/shipping was much faster than promised. Strongly recommend."
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سکس با اسب سفید The novel on which the anime series of the same name is also based is also now available in paperback.
سکس با اسب سفید Lip Sucking Kiss - When kissing gently suck on their lower lip.
The grand prize winner will also receive an all expenses paid trip to Japan - home of Cosplay. سکس اریانا سعید Lick Kiss - Just before kissing, gently run your tongue along you partners lip whether it be the top or bottom one depending on the position of your lips. سکس ترکی And then there`s the Lottie`s discovery of Sara`s true identity, a potent bit of melodramatic plot convergence that for the length of a single episode transforms the series into something that may not actually jerk a few tears from the more tender-hearted.
سکس با اسب سفید Can consumer-generated media {CGM} work as a monetizeable business model for video like it has for text? What are the challenges with incorporating video into public Wikis? And, can traditional news companies use the wiki model for incorporating CGM video and build back their own news communities, or are the challenges too great?However, traffic and consumer activity doesn�t equate profit.
Wake Up Kiss - Before your partner awakes lean over and kiss their cheek and move over giving soft kisses until you reach their lips. Simply brush your lips lightly across the crown of their head. سکس ترکی Your 11 liter diesel engine has a long stroke. The car, which is sponsored by DragStar Racing, will be part of Cunningham Ford Pro Stock Racing program. No likeable lead, no interesting relationships, no laughs {that work�plenty that don`t}, and, aside from the scattered low-angle shot and an episode devoted to lusty lesbian hi-jinks, surprisingly little fan-service.
سکس با اسب سفید Gently breathe out of your mouth on the cheek/neck, and listen to them giggle and groan with delight! Asian music encompasses numerous different musical styles originating from a large number of Asian cultures.


text:
Between higher wages and new labor regulations, restaurants are facing a labor crisis — and their existing, outdated processes are doing anything but helping the problem.
Switching into fast casual dining requires more than getting food to the table quicker. It demands new strategies from menu decisions to customer experience elements.
Three fast casual restaurant operators and other experts share their insights about the industry and how they've found success and value in it.
Establishing a 'cult' philosophy in building a robust brand culture isn't a bad thing. In fact, it's one of several valuable and viable approaches.
Entrepreneurs from Burgers n' Fries Forever, Sweet Lorraine's Fabulous Mac n' Cheez and Pincho Factory pitched concepts to a panel of distinguished investors and consultants who provided professional advice.
Data is driving customer engagement, and there is a wealth of tools an enterprise can use to collect, assess and analyze data to enhance its interactive customer experience strategy.
Tastes and trends in food change and evolve rapidly. What may have been an innovative concept last year may be exactly the wrong one now.
Consumers of all kinds have a nearly endless array of options from which to choose, whether they're purchasing furniture, cars, housewares, or any good or service. The same holds true for where they spend their money when dining out.
Beverages are an important aspect of the consumer dining experience and play a critical part of a restaurant's branding efforts. Being on trend is key to success, say experts.
As any business owner knows the location decision can sink the business or be the foundation of initial success and growth. But it involves much more than negotiating a lease or property purchase.
Strategic and profitable growth for today's restaurants, quick service eateries and retailers is a comprehensive endeavor. But it's doable and the number one tip to getting a foundation built is to jump in and start making it happen.
The ICX Symposium in Atlanta later this month will feature a session featuring an Intel executive addressing the transformation of retail through responsive stores. ICX Association Executive Director Scott Slucher interviewed him about the interplay of tech and CX.
With the Interactive Customer Experience Association set to host 'Inflection Point: The Future Tech of Customer Experience,' aimed at getting B2C brands ready for the future of customer engagement tech, we talked to the Association's executive director about what to expect.
Design is a key aspect for the customer experience and plays directly into the "corporate soul" and building a relationship with customers.
The ICX Symposium in Atlanta later this month will feature a keynote address from three friends, executives at major brands, talking about the future of customer experience. ICX Association head Scott Slucher interviewed the trio about what to expect.
The ICX Symposium in Atlanta later this month will feature a session featuring two executives from MaxMedia addressing retail's emotional economy. ICX Association Executive Director Scott Slucher interviewed the duo about the emotional truths of customer engagement.
QSRWeb.com polled hundreds of operators about their business practices to compile the data.
Digital signage is becoming increasingly common in both quick service and fast casual restaurants, so for this month's "Ask the experts" feature, we asked Jeff Pinc, director of food services for Panasonic, 'Have digital menu boards reached critical mass?'
While bolstered consumer confidence is good news for restaurants, rising costs are perhaps their biggest challenge in today's business landscape.
The price for cheese increased by 6 cents, but wheat prices dropped 16 cents over the past week.
1000 Degrees moves into Deer Park
Technomic report says customers blame restaurants for third-party delivery service failures
PizzaRevs up in the Lone Star State
RSR reveals digital gift card winners
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'Littler' Little Caesars opens in North Carolina
What's it worth to dress like me? About $8 million if you're Chick-fil-A
ChowNow/Google partnership may be match made in heaven for restaurants
Massive Big Food study holds lessons for restaurant industry today


text:
The Covenant men's golf team shaved 12 strokes off their first round score, but it was not enough to advance to the third and final round of the NCCAA Men's Golf National Championships.
Covenant posted a round two score of 313 but the Scots could not climb into one of the top 12 spots to move on to tomorrow's final round.
Lee University currently holds a one shot lead over three teams with a 578 through two rounds. Zach Moore of Southern Wesleyan continues to lead all individuals as he is 8-under.
Sophomore James Van Nice once again led the Scots as he carded a 73 on Tuesday after a first round 77. Van Nice had two birdies in his round and he finished the day in a tie for 32nd, but missed the individual cut by one stroke.
Senior Cameron Lewis and freshman Alan Craig both had the biggest improvement among Covenant golfers on Tuesday as both trimmed 10 strokes off Monday's opening round. Lewis posted a 75 on Tuesday after an 85 in the first round, while Craig had a 77 after an 87 yesterday.
Junior Scott McKnight and freshman Nate Haney each scored 88s on Tuesday.
With Tuesday's round complete, the team has completed its fall portion of the 2013-14 schedule. Covenant will be back on the course in March.
August 25, 2016
JACKSONVILLE, Fla. - The Chattanooga Lookouts broke a 3-3 tie with a five-run seventh inning on the way to a 9-3 win over the Jacksonville Suns in front of 3,154 at the Baseball Grounds of Jacksonville ... (click for more)
The Covenant women's basketball team, under fourth-year head coach Sarah Harris, has announced its schedule for the 2016-17 season. The Scots will play 24 regular season games, including 14 USA ... (click for more)
University of Tennessee at Chattanooga senior Derrick Craine is one of 40 players from across the Football Championship Subdivision (FCS) named to the College Football Performance Awards National ... (click for more)
JACKSONVILLE, Fla. - The Chattanooga Lookouts broke a 3-3 tie with a five-run seventh inning on the way to a 9-3 win over the Jacksonville Suns in front of 3,154 at the Baseball Grounds of Jacksonville Wednesday night. After falling to Jacksonville Tuesday, Chattanooga bounced back with a big win Wednesday. The win moved the Lookouts to just four games behind division leading ... (click for more)
The Covenant women's basketball team, under fourth-year head coach Sarah Harris, has announced its schedule for the 2016-17 season. The Scots will play 24 regular season games, including 14 USA South games and 10 non-league contests. Covenant, which went 10-15 in 2015-16, opens the year at home on Nov. 15 against regional foe Sewanee at 5:30 p.m., inside Barnes Physical Education ... (click for more)
A Chancery Court jury on Wednesday gave a verdict totaling $562,000 for 25 city police personnel who filed suit almost five years ago alleging that the city never lived up to terms of a 2010 pay plan. Attorney Stevie Phillips, who tried the case along with Janie Parks Varnell, said, "The jury awarded each officer what we had asked for to the penny." She stated, "These 25 members ... (click for more)
The Tennessee Department of Environment and Conservation (TDEC) defended the operation of its septic permit office in Bradley County, including the work of manager Hank Thompson. The Bradley County Commission is studying whether to take over the operation of the office, saying there have been a number of complaints against Mr. Thompson and that he recently kicked Commissioner ... (click for more)
Re: Alexander's remarks about Tennesseans not affording "Affordable Health Care Act" prices Mr. Alexander, Your message stated, Tennesseans can't afford a 44 percent to 62 percent increase in the Affordable Care prices. I simply ask, as Senate Health Committee chairman, why don't you do something about it? Don't tell us something most know already. ... (click for more)
No. 1 – Did you know that one in every five of the 42,000 children in the Hamilton County school district is what the experts call "food deprived?" No. 2 – Can you get your arms around the fact that a huge percentage of those 8,400 children get little, if nothing, to eat from Friday at lunch until they return to our schools on Monday? No. 3 -- Did you know that you – just ... (click for more)


text:
Book your room at Hotel Scott House Rome
Get an overview of this hotel3-star hotel, walk to Basilica di Santa Maria Maggiore
Located in the heart of Rome, this hotel is within a 5-minute walk of Santa Prassede and Basilica di Santa Maria Maggiore. Roman Forum and Teatro dell'Opera di Roma are also within 1 mi (2 km).
Hotel Features
Dry cleaning, a 24-hour front desk, and a computer station are available at this smoke-free hotel. Free buffet breakfast and free WiFi in public areas are also provided. Additionally, express check-out, tour/ticket assistance, and multilingual staff are onsite.
Room Amenities
All 34 soundproofed rooms feature free WiFi and Select Comfort beds. For a bit of entertainment, guests will find LCD TVs with satellite channels, and other amenities include free minibar items and coffee makers.Would you like to give us feedback? Share it! Opens in a new window
Hotel Scott House RomeVia Gioberti 30 Rome RM 00185
Hotel Amenities
Hotel Amenities
Guests can enjoy a complimentary breakfast each morning. A computer station is located on site and high-speed wired and wireless Internet access is complimentary. For a surcharge, the property provides a roundtrip airport shuttle (available on request) and a cruise ship terminal shuttle. This Rome hotel also offers tour/ticket assistance, multilingual staff, and dry cleaning/laundry services. A total renovation of this property was completed in December 2012.
Hotel Scott House Rome is a smoke-free property.
- 24-hour front desk
- Airport transportation (surcharge)
- Total number of rooms - 34
- Number of floors - 2
- Free breakfast
- Dry cleaning/laundry service
- Elevator/lift
- Express check-out
- Cruise terminal shuttle (surcharge)
- Ferry terminal shuttle (surcharge)
- Computer station
- Tours/ticket assistance
- Free WiFi
- Free wired high-speed Internet
- Luggage storage
- Multilingual staff
- Offsite parking reservations required
- Smoke-free property
- Television in common areas
Family Friendly Amenities
- Family Hotel
- Free Wi-Fi
- Soundproofed rooms
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi , Free wired high-speed Internet
Offsite parking reservations required
Room Amenities
- Pay movies
- Soundproofed rooms
- Air conditioning
- Phone
- Coffee/tea maker
- Minibar (stocked, free items)
- Daily housekeeping
- Private bathroom
- Shower only
- Bidet
- Desk
- In-room safe
- Free WiFi
- Blackout drapes/curtains
- In-room climate control (air conditioning)
- Free toiletries
- Hair dryer
- Free cribs/infant beds
- Satellite TV service
- Sleep Number by Select Comfort mattress
Where to Eat
Guests are offered a complimentary buffet breakfast each morning.
Start your morning with a free buffet breakfast, available each day of your stay.
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- In-room accessibility
- Accessible bathroom
Hotel Policies
Check-in time starts at 2 PM
Special check-in instructions:
Check-out time is 11 AM
Payment types
- Pets not allowed
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- No onsite parking is available.
No rollaway/extra beds available
- A tax is imposed by the city: EUR 4 per person, per night, up to 10 nights. This tax does not apply to children under 10 years of age.
Optional extras
- Airport shuttle fee: EUR 60.00 per vehicle (one way)
Hotel Name
- Hotel Scott House
- Hotel Scott House Rome
- Hotel Scott Rome
- Scott Hotel Rome
- Scott House
- Scott House Hotel
- Scott House Hotel Rome
- Scott House Rome
- Scott House Rome Hotel
We should mention
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation. This property offers transfers from the cruise terminal, ferry terminal, and airport (surcharges may apply). Guests must contact the property with arrival details before travel, using the contact information on the booking confirmation. Children 6 years old and younger stay free when occupying the parent or guardian's room, using existing bedding. Only registered guests are allowed in the guestrooms.


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A police pursuit ended with a motorcycle crashing into a Grayson County deputy's car and a fight between the suspect and an officer.
Around 12:30 a.m. Wednesday, Grayson County Sheriff's Office deputies were involved in a chase with a subject on a motorcycle that began on Delhart Road. The pursuit ended on Justice Road in the Oldtown community near Galax.
The suspect reportedly crashed his motorcycle in front of a Grayson patrol car, which then struck it. The suspect then fought with an officer.
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The Galax Gazette is your source for local news, sports, events and information in Galax, Grayson, Carroll, and surrounding areas.


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Written on: 14/07/2014 by King154 (1 review written)
It's got everything you need for sport and plenty more. A watch for adventures
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You should listen to this https://play...
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convention members jam sign up here https://surf...
38 days ago
Today I learned MySpace is still a thing.
35 days ago
Who knew? I clicked on an old link to something on MySpace recently and it didn't work.
33 days ago
Satan's Pilgrims and Boss Martians this Saturday at Doug Fir, Portland, OR!!!!
31 days ago
wake up!
29 days ago
i've had 'the oasis' by the madeira stuck in my head for two days now...........and it is welcome to stay there a while longer
23 days ago
Where is the live feed of the convention?
14 days ago
Sorry Ted, there was no live feed!
11 days ago
Par Avion announces California Mini Tour Aug. 30-Sept. 3 Details: On Thursday, September 1st, Par Avion will be Making a rare landing on the mysterious atoll of the Forbidden Island Tiki Lounge in Alameda, CA. Bonus: Ferenc of Frankie & The Pool Boys is joining us onstage that night. Rebecca, Richie and I look forward to meeting you! Sample us here: http://bit.l... and note that this is part of a late summer mini-tour. Should you be elsewhere in California, take a peek at our itinerary. Perhaps we have a gig near you. Details here: http://bit.l...
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Simon Fowler is editor of the genealogy magazine Ancestors and writes widely on family and social history topics. Previous books include the successful Family Skeletons with Ruth Paley, as well as The Joys of Family History and several of the National Archives' prestigious Pocket Guide history series. His articles have been published widely in popular history magazines including BBC History and History Today.
Workhouse: People Places Life Behind Doorby Simon Fowler
Workhouses cast deep shadows over Victorian Britain and terrified the poor for generations. Known for their soul-numbing routines, deprivation and cruelty, they were after 1834 almost the sole source of relief for paupers across the land. For old couples and orphans, starving families and single mothers, these institutional monsters became what the New Poor Law intended—the last resort of the desperate.
Workhouse takes you behind the stern doors to reveal the reality behind the legend. It explores all aspects of institutional life, from everyday details of food, uniform and the daily grind to the scandals that shocked society and were an impetus for change. The narrative traces the experiences of guardians and staff who governed inmates' fates, drawing on dramatic personal accounts from the anonymous 'Indoor Pauper' to famous figures such as Charlie Chaplin. It shows how the grim buildings fascinated Charles Dickens and George Orwell, were condemned by celebrities such as Florence Nightingale, and yet became deeply rooted in their communities.
Product Details
- 9781905615285
- Publisher:
- National Archives (PRO), The
- Publication date:
- 09/15/2008
- Pages:
- 288
- Product dimensions:
- 5.10(w) x 7.70(h) x 1.00(d)
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Weather News
Jakarta floods leave 50,000 homelessBy Indonesia correspondent George Roberts, Monday January 21, 2013 - 08:37 EDT
Twenty people are confirmed dead and 50,000 are homeless after days of severe flooding in the Indonesian capital Jakarta.
A weekend of relatively fine weather has helped the city recover in some parts, but there are still areas that are completely under water.
On Saturday, the number of people relying on temporary shelters was 20,000, but that has now jumped to 50,000 homeless.
Among the dead are a baby girl and a small boy.
While some people have been able to begin cleaning and patching up, the weather bureau is forecasting more heavy rain to hit in the next few days.
It is expected to keep up into early February.
A quarter of a million people have already been affected by the flooding and authorities say they are prepared to evacuate hundreds of thousands of people if necessary.
More breaking news
ABC News
Sydney Morning Herald
National Nine News
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Weather News
First spring soaking under way
Rain and thunderstorms are spreading across southern and central Australia today as the first big system of spring pushes east.
Wine industry soon to have smartphone app advising on irrigation and harvesting
The wine industry will soon have another tool at its fingertips to help navigate important on-farm decisions.
Plenty of fuel this bushfire season
One of Australia's wettest winters on record has primed parts of the country for above-average bushfire potential this season.


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Europe faces data protection vacancy
Europe could be without a data protection tzar from next week as none of the candidates applying for the job are suitably qualified.
Europe could be without a data protection tzar from next week as none of the candidates applying for the job are suitably qualified.
The U.S. House of Representatives is moving toward votes on two bills intended to put a spotlight on the inner workings of HealthCare.gov, the health insurance shopping site recovering from a rocky rollout in late 2013.
A U.S. surveillance court has renewed its approval of a U.S. National Security Agency program that collects U.S. residents' telephone records in bulk.
The U.S. Department of Justice will appeal a district judge's opinion saying a phone records collection program at the National Security Agency likely violates the U.S. Constitution.
The U.S. National Security Agency is attempting to build a new breed of supercomputer that theoretically could make short work of cracking most keys used for encrypted communications.
The American Civil Liberties Union will appeal a judge's decision to throw out the civil liberties group's lawsuit challenging U.S. National Security Agency surveillance.
The U.S. National Security Agency was developing in 2008 a software implant for Apple iPhones that allowed the agency to take almost total control of the device, including retrieving text messages and voicemail and remotely turning on its microphone ...
French government and police officials requested 6,145 phone and data taps in 2012, fewer than in 2011, according to figures released by the French National Commission for the Control of Security Interceptions (CNCIS) earlier this week.
"A child born today will grow up with no conception of privacy at all," Edward Snowden warned Wednesday in a message broadcast to U.K. television viewers.
Security researcher Mikko Hypponen has canceled his talk at a RSA security conference in San Francisco, reacting to a report that the security division of EMC allegedly received US$10 million from the U.S. National Security Agency to use a flawed ran...
A prominent venture capitalist proposed on Monday a plan to split California into six new states, including one called "Silicon Valley" that would stretch from San Francisco to San Jose and include the entire region where many of the biggest tech com...
A U.S. senator has called on the Federal Trade Commission to investigate Target's security practices after the large retailer reported a data breach affecting 40 million customer credit and debit cards.
AT&T has joined Verizon Communications in promising to report on government requests for information about its customers, setting the stage for semiannual disclosures from both the dominant U.S. wireline carriers starting early next year.
LGS Innovations, a company formed during the Alcatel-Lucent merger to supply technology to the U.S. government, has been sold to a pair of U.S.-based investment companies for about US$200 million.
The U.S. National Security Agency defended its foreign intelligence surveillance programs Friday after news reports that the agency has targeted foreign leaders and business executives.
The latest business news, reviews, features and whitepapers
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Comprehensive buying guides, features, and step-by-step articles
First impression on unpacking the Q702 test unit was the solid feel and clean, minimalist styling.
For work use, Microsoft Word and Excel programs pre-installed on the device are adequate for preparing short documents.
The Fujitsu LifeBook UH574 allowed for great mobility without being obnoxiously heavy or clunky. Its twelve hours of battery life did not disappoint.
The screen was particularly good. It is bright and visible from most angles, however heat is an issue, particularly around the Windows button on the front, and on the back where the battery housing is located.
My first impression after unboxing the Q702 is that it is a nice looking unit. Styling is somewhat minimalist but very effective. The tablet part, once detached, has a nice weight, and no buttons or switches are located in awkward or intrusive positions.


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The 2012–2013 VIVA! & Gala season launches on Friday, October 26 with a high-energy, interactive performance by Inca Son (Sound of the Inca).
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Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
Should I do this experiment?
The question is whether DNA uptake can provide bacteria with enough nutrients to make a difference to their growth rates. I've always argued that getting some nutrients from the DNA is inevitable, so so demonstrating that this increases growth in a lab culture would not be good evidence that selection for these nutrients is why cells take up DNA. But maybe I was wrong.
In the past we have tried to show that H. influenzae can use nucleotides from DNA for growth, but the experiments have had lots of problems, I think in part because H. influenzae is quite fastidious in its nutrient requirements, and in part because we didn't devote a full-press of our time and brain-power to it.
Our colleague Steve Finkel has shown that E. coli can use nutrients probably acquired by DNA uptake for growth, but the effects are modest. This is probably because the cells are only taking up very small amounts of DNA because their competence genes are not induced under normal culture conditions.
Rather than now trying again with H. influenzae, I'm considering trying to demonstrate nutritional benefits of DNA uptake using Bacillus subtilis. B. subtilis is a soil bacterium, very easy to grow and not at all fastidious. I've worked with it before and one of the post-docs did her PhD work on it. B. subtilis takes up lots of DNA under lab conditions, though not during exponential growth. Competence is induced under conditions referred to as 'post-exponential growth', meaning after cell growth has stalled because nutrients are depleted.
Keeping cells competent over a long period might be complicated, because B. subtilis competence and sporulation are induced simultaneously (though apparently in different sub-populations of the culture). I'd first have to read up on the latest work on the regulation of DNA uptake, and try to find the best conditions for observing a growth effect. But if I can show that B. subtilis cultures grow better when they can take up DNA, I think many other biologists would find the result much more compelling than I do. This would be good, because they are not at all convinced by the results that I think are compelling.
3 comments:
- firstname.lastname@example.com 12, 2007 at 9:02 AM
I'm not sure how helpful this will be, but when I saw this post I thought "flux balance analysis".ReplyDelete
Using the H.influenzae metabolic reconstruction, it would be possible to simulate it's growth rate on a variety of media. For example theoretically increase the amount of nucleotide influx into the cytosol and see by how the model predicts a growth rate increase.
I appreciate this might sound a bit far fetched, but it was an idea I had when I read your post.
Steve's group at USC has done really nice work on long-lived cultures. He has a lot of great ideas. I think that you should definitely do the experiment. DNA is a perfectly viable c,n, energy source, and it seems likely that many bugs (B. subtilis being a great example) can use it that way. An interesting thought occurs- a few recent studies (from the Holden lab at UCSB, and at least one other) have shown that DNA is a significant component of at least some biofilm matrices. Therefore, it seems plausible that DNA could be an environmentally relevant nutrient source. Was one of those studies in H. influenzae? I thought it was (I recall a really cool J.Bact cover, with DAPI stained thick cords of DNA in a biofilm matrix). Anyway, I love that you put your research out here in real time, and wish you luck with it!ReplyDelete
"Using the H. influenzae metabolic reconstruction..."? I didn't even know this existed! (I am so out of touch...) I've found the 1999 H. influenzae reconstruction paper and will now start going through it and the papers that cite it.ReplyDelete
We've known for about 15 years that there's tons of DNA in many (most?) bacterial environments. There's about 300 ug/ml in healthy resiratory mucus (where H. influenzae lives) and more than 1 mg/ml in people with respiratory diseases. The hard part is getting people to consider that nutrients from DNA might be more valuable to bacteria than rare and random changes in genotype.
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I Love Lucy TV Show Watchlist
Season 6, Episode 21 Lucy's Night in Town
Too bad about Lucy's evening at the theater: her tickets were for the matinee. Ricky sort of saves the day, but he's able to buy only two box seats, so they decide that the women will see the first act and the men the second. It doesn't work out that way, of course, and soon more eyes are focused on the Ricardo-Mertz box than on the stage. Theater Manager: Joseph Kearns.
Guest Cast
John Eldredge John Eldredge Man in Box
Joseph Kearns Joseph Kearns Theater Manager
Doris Packer Doris Packer Woman in Box
Gladys Hurlbut Gladys Hurlbut Woman with Purse
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- Rating: TV-G
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Book your room at Hotel Sahara
Get an overview of this hotel3-star hotel with restaurant and outdoor pool
Situated by the sea, this hotel is 1.2 mi (1.9 km) from Adriatic Golf Club Cervia and within 3 mi (5 km) of Casa delle Farfalle and Cervia Thermal Baths. CerviAvventura Adventure Park and Mirabilandia are also within 9 mi (15 km).
Hotel Features
A restaurant, an outdoor pool, and a bar/lounge are available at this hotel. Free WiFi in public areas and free self parking are also provided. Additionally, a spa tub, a children's pool, and tour/ticket assistance are onsite.
Room Amenities
All 63 rooms offer free WiFi, balconies, and LCD TVs with digital channels. Refrigerators, room service, and ceiling fans are among the other amenities available to guests.Would you like to give us feedback? Share it! Opens in a new window
Hotel Amenities
Hotel Amenities
In addition to an outdoor pool, Hotel Sahara provides a spa tub and a children's pool. The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. Public areas are equipped with complimentary high-speed wireless Internet access.
This Cervia hotel also offers tour/ticket assistance, a garden, and room service (during limited hours). Limited complimentary onsite parking is available on a first-come, first-served basis.
- Pool sun loungers
- Pool umbrellas
- Children's pool
- Total number of rooms - 63
- Number of floors - 5
- Bar/lounge
- Elevator/lift
- Tours/ticket assistance
- Free WiFi
- Parking (limited spaces)
- Garden
- Restaurant
- Safe-deposit box at front desk
- Free self parking
- Number of buildings/towers - 1
- Spa tub
- Outdoor pool
Family Friendly Amenities
- Outdoor pool
- Children's pool
- Free Wi-Fi
- Refrigerator
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking, Parking (limited spaces)
Room Amenities
- Private bathroom
- Free WiFi
- Balcony
- Ceiling fan
- Air conditioning
- Phone
- Refrigerator
- Hair dryer
- Shower only
- Desk
- In-room safe
- Digital TV service
- Room service (limited hours)
Where to Eat
Hotel Sahara has a restaurant on site.
Room service (during limited hours) is available.
Nearby Things to Do
An outdoor pool, a children's pool, and a spa tub are on site.
- Golfing nearby
Hotel Policies
Check-in time starts at noon
Check-out time is 10 AM
Payment types
- Pets allowed
- Up to 8 kg
- Restrictions apply
- For more information contact the property at the number on the booking confirmation
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No rollaway/extra beds available
Optional extras
- Crib (infant bed) fee: EUR 18 per night
- Rollaway bed fee: EUR 25 per night
Hotel Name
- Hotel Sahara Cervia
- Sahara Cervia
We should mention
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation. Guests can arrange to bring pets by contacting the property directly, using the contact information on the booking confirmation.