text:
- Research article
- Open Access
Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability
© Kort et al; licensee BioMed Central Ltd. 2008
- Received: 22 July 2008
- Accepted: 06 December 2008
- Published: 06 December 2008
In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis.
We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase.
The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.
- Colony Form Unit
- Heat Shock Response
- Competence Gene
- Subtilis Cell
- Colony Form Unit Count
Ever since the pioneering work by Louis Pasteur and Robert Koch at the end of the nineteenth century, the detection of viable bacteria has been carried out by cultivation and enumeration of colony forming units (CFU). Practically all judgments on hygiene, food safety, conservation treatments, drinking water quality, infections of pathogens, efficacy of disinfectants and antibiotics are based on growth on solid agar medium followed by CFU counts. However, the assessment of cell viability on agar plates is laborious, it requires at least an overnight incubation and the outcome yields little information on bacterial physiology. Besides, the assessment of CFU counts is limited to bacteria that are readily culturable under laboratory conditions and even when they are, the failure of bacteria to reproduce on an agar plate does not necessarily imply that they are metabolically inactive or were inactive at the time of sampling [1, 2].
To overcome the shortcomings of CFU enumeration pointed out above, a number of alternative, cultivation-independent methods have been applied over the years to get hold of indicators for bacterial cell viability. The most commonly employed methods include fluorescence-based assays for enzymatic activity, electron transport and membrane permeability and molecular approaches for the detection of rRNA or specific mRNA molecules. The fluorescence-based assays provide signals that can be related to known cell properties, which can be assessed at the cellular rather than population level by the use of fluorescence microscopy and flow cytometry. The molecular approaches include microarrays and real-time PCR to select and quantify specific RNA molecules. The latter methods have been generally looked upon as providing indicators of specific aspects of bacterial physiology rather than indirect measures for bacterial cell viability valid under a wide range of lethal stress conditions. However, the feasibility of cultivation-independent biomarkers under a number of well-defined conditions as indirect indicators for cell viability is still a matter of debate [1, 3, 4].
In this study, we evaluated a number of commonly applied cultivation-independent methods under accurately defined conditions by a gradual exposure of the Gram-positive model bacterium Bacillus subtilis to heat stress. We systematically determined the effects of heat exposure on two cultivation-dependent measures, the ability to form colonies on agar plates, expressed in CFU counts, and outgrowth of the heat-exposed bacteria diluted in liquid cultures, expressed in the time delay to reach mid log phase. Furthermore, we determined a number of cultivation-independent measures, including the permeability of the bacterial membrane for fluorescent compounds, the stability of ribosomal RNA molecules and the presence of specific gene transcripts, as identified with microarrays and quantitative PCR methods. The results presented here reveal clear correlations between viability and cultivation-independent indicators in B. subtilis exposed to lethal heat stress. Notably, the genome wide expression profiles of heat-exposed B. subtilis reveal a coherent transcriptional program including known heat shock responses [5, 6] and the rapid expression of sporulation and competence genes, the latter only known to be expressed in the stationary growth phase [7, 8]. This and other forms of coordinated gene expression in the bacterial cell continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce.
Strains and culture conditions
B. subtilis strains used in this study
B. subtilis strains
trpC2/parental strain in this study
Bacillus Genetic Stock Center
food product isolate
Stanley Brul/[9]
food product isolate
Stanley Brul/[10]
Daniel Kearns [31]
Chester Price/[32]
spoIIAC::erm (sigF)
Adriano Henriques
spoIIGB::erm (sigE)
Adriano Henriques/[33]
spoIVCB::erm (sigK)
Adriano Henriques
DS323 → 168 1A1 (sigD)
This study
PB344 → 168 1A1 (sigB)
This study
AH46 → 168 1A1 (sigF)
This study
AH74 → 168 1A1 (sigE)
This study
AH70 → 168 1A1 (sigK)
This study
Heat treatment
An overnight culture of B. subtilis was diluted 20× in TSB to a volume of 600 ml and incubated at 30°C at 100 rpm until OD600 reached 0.5. Subsequently, portions of 40 ml of the preculture of B. subtilis were exposed to seven different temperature regimes in 250-ml bottles in a reciprocal-shaking water bath at 65°C. Duplicate samples were taken when the temperature in the bottles reached 40, 50, 57, 58, 59, 60, and 62°C (at incubation times of approximately 100, 200, 300, 320, 380, 500 and 720 s after transfer, respectively). Immediate quenching of metabolic activity in the samples was performed with cold methanol, according to described methods [11]. Briefly, 40-ml culture samples were sprayed in 160-ml of a stirred solution of 60% methanol, 66.7 mM HEPES, pH 6.5 that was kept at – 45°C in an ethanol bath. Besides RNA-isolations, the heat-treated cultures were directly subjected to outgrowth analysis to monitor the recovery time and flow cytometric analysis, as further described below. The outgrowth was carried out in 10× dilution series by OD measurements in time at 37°C in TSB by use of the Microbiology Reader Bioscreen C (Oy Growth Curves Ab Ltd, Finland). The time delay in reaching the mid log phase (OD wideband = 0.5) compared to the 30°C-control sample was used as a measure for the increased recovery time from heat injury for each specific exposure.
RNA isolation
The RNA isolation protocol was adapted from an extraction method described previously [12]. In brief, cells were disrupted in a 2-ml screw-cap tube in the presence of 100-μm zirconium beads and SDS/phenol for 60 seconds in the Minibeadbeater 8™ (Biospec Products). After chloroform extraction, nucleic acids were precipitated, vacuum-dried, dissolved in distilled water and incubated for 15 min in the presence of DNase I (Qiagen). Finally, RNA was purified by phenol/chloroform extraction and ethanol precipitation as verified by absorption properties in the 200–300 nm region by the use of the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA). RNA MW profiles were verified with the 2100 Electrophoresis Bioanalyzer (Agilent Technologies). The RNA 6000 ladder (Ambion Inc, Austin TX, USA) consisting of 6 transcripts at a concentration of 20 ng/μl, ranging from 0.2 to 6.0 kb in length was used as a marker. In addition, a fast migrating "reference marker" compound was added to all samples for software alignment of all electropherograms within one LabChip run (Agilent Technologies).
Construction of B. subtilis microarrays
The B. subtilis microarrays were constructed by the Micro-Array Department (MAD), University of Amsterdam. An oligonucleotide library of 65-mers (Sigma-Genosys Ltd.), which covered 4100 open reading frames of the B. subtilis 168 genome [13], was spotted in duplicate on glass slides with a Generation III microarray spotter (Molecular Dynamics, Sunnyvale, California, USA) using Lucidea Spotting Pins (GE Healthcare) as described previously [14].
Labelling of cDNA
Fluorescently labelled cDNA was prepared from 12.5 μg of total RNA by random hexamer pd(N)6-primer (Roche, Mannheim, Germany) polymerization using Superscript II reverse transcriptase (Life Technologies). Concentrations of nucleotides in labelling reaction mixture were 0.4 mM dATP, dGTP, dCTP and 0.2 mM dTTP. The final concentration of Cy3-dUTP or Cy5-dUTP (GE Healthcare) was 0.1 mM. Unincorporated Cy-dye-labelled dUTP, dNTP's, primers and salts were removed by purification with AutoSeq G50 columns (GE Healthcare).
Hybridization and image analysis
Microarray slides were incubated for 45 min at 42°C with prehybridization solution (1% bovine serum albumin, 5× SCC and 0.1% SDS, filtered), washed three times with milliQ water, and dried by the use of a nitrogen flow. The Cy3-labelled cDNA from untreated cells sampled at 30°C, was mixed with Cy5-labelled cDNA from heat-treated cells for all hybridizations. After 2 min of denaturation at 95°C, hybridizations of microarray slides were performed overnight at 42°C in 40 μl of EasyHyb buffer (BioCat, Heidelberg, Germany) with both labelled cDNAs and 2.5 mg ml-1 yeast tRNA (final concentration). Microarray slides were washed at room temperature for 10 sec in 1× SSC/0.2% SDS at 37°C, 10 sec in 0.5× SSC at 37°C, and twice for 10 min in 0.2× SSC at room temperature. Slides were dried by the use of a nitrogen flow and scanned with a ScanArray 5000 laser scanner (Perkin Elmer Life Sciences). The TIFF-images were quantified with the software package ImaGene version 5.6.1 (BioDiscovery, El Segundo, CA, USA).
Data processing and normalization
The spot intensities of B. subtilis oligonucleotide microarrays were processed by the use of Microsoft Office Excel 2003 SP2. The intensities of 8200 spots were calculated by extraction of the median background (B) from the median signal (S) and values for S-B < 1 were set to 1. The raw Cy5/Cy3 ratios (r) were calculated from the remaining background-corrected median signals. The total intensity normalization was carried out by the normalization factor N, defined as the average Cy5 (S-B) divided by the average Cy3 (S-B)). The normalization factor was determined from spot intensities with S/B > 2 for both Cy5 and Cy3 channels in order to avoid bias resulting from the degradation of the major of the transcripts at temperatures from 57–62°C. The normalised Cy5/Cy3 ratios (R = r/N) with S/B > 2 for either the Cy5 or Cy3 channel were calculated. Ratios from duplicate spots on the array and 2 independent experiments were averaged and 2log transformed. Next, all gene IDs with less than 2 observations out of 8 conditions (30, 40, 50, 57, 58, 59, 60, 62°C) were removed from the data set, leaving 3850 out of 4100 gene IDs (94%). Expression profiles were analyzed with K-means clustering into 4 groups with the Pearson correlation as distance metric by the use of Multiple Experiment Viewer MeV v4.1 [15]. Subsequently, the 2log ratios of spot intensities were selected which showed 5 or more useful ratios out of 8 temperature conditions, comprising a total of 3562 ratios (87%). In addition, all determined transcription levels for each gene at each temperature condition were analyzed by the use of the T-profiler http://www.science.uva.nl/~boorsma/t-profiler-bacillusnew, a tool that uses the t-test to score changes in the average activity of predefined groups of genes [16, 17]. Group definitions were obtained from the DBTBS http://dbtbs.hgc.jp/ver4/, the database of transcriptional regulation in B. subtilis, release 4.1 [18].
Microarray data accession number
Microarray data were deposited in the Gene Expression Omnibus (GEO) database http://www.ncbi.nlm.nih.gov/geo/ under accession number GSE12372.
Flow cytometry
Heat-treated B. subtilis cell suspensions (OD600 = 0.5) in the temperature range of 40–62°C were directly labelled with propidium iodide (Molecular Probes Inc, Eugene, OR, USA) at 2 μM final concentration and incubated for 2 min at room temperature. For calibration purposes a sample treated for 10 min at 96°C (100% PI-permeable) was mixed with an untreated sample in relative amounts of 0, 20, 40, 60, 80 and 100% PI-permeable cells. For flow-cytometric analysis the cell-suspensions were diluted 40-fold in filtered phosphate-buffered saline solution and directly measured on a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Mijdrecht, The Netherlands) operating an argon laser (488 nm). For each sample, at least 10,000 cells were analyzed. Fluorescent signal data were collected in the FL4 detector channel, and the photomultiplier voltage was set at 917 V for all measurements. Data were captured and analyzed using the EXPO32 analysis software (Beckman Coulter). Figures were prepared for publication using MicroCal Origin (version 7.5).
Fluorescence Microscopy
Labelling was carried out with fluorophores Syto9 and propidium iodide (PI) according to methods described in the LIVE/DEAD BacLight kit (Molecular Probes Inc.) [19]. Subsequently, 10-μl aliquots of the cultures were embedded in 1% (w/v) final concentration of agarose and deposited on the surface of a microscope slide. Samples were analyzed by the use of the BX61 TRF fluorescence microscope (Olympus Inc.).
Quantitative PCR
The extraction of RNA was carried out on a cell suspension of 750 μl of bacterial cells, which were quenched immediately after heat exposures as described above in detail. For cDNA synthesis, 1 μg of the isolated total RNA was vacuum dried and incubated in 5 μl Random primer mix, consisting of 3 μl 5U μl-1 RNAsin (Promega) and 2 μl 2 μg μl-1 pd(N)6 random primer (Roche) for 5 min at 70°C. After cooling down for 10 min, 8.5 μl RT mix was supplemented: 1 μl RNAsin (10 U μl-1, Promega); 2.5 μl First Strand Buffer 5× (Invitrogen); 1 μl 0,1 M DTT (Invitrogen); 3 μl 2 mM dNTP's (Invitrogen) and 1 μl Superscript II enzyme (200 U μl-1, Invitrogen). The RT reaction was carried out for 60 min at 42°C. Subsequently, to degrade the RNA, 2.5 μl of 2.5 M NaOH was added to the reaction mix and incubated for 15 min at 65°C. This was followed by addition of 2.5 μl 2.5 M NaAc and 10 μl milliQ. The cDNA was purified by the use of a G50 Autoseq column, dried in vacuum and resuspended in 25 μl prior to the Q-PCR reactions.
List of primer probe combinations for Q-PCR
Primer Type
Amplicon size
62 bp
B.sub gspA-R
61 bp
59 bp
63 bp
97 bp
B.sub 23-S-R
Construction of sigB, sigD, sigF, sigE and sigK mutants
The DNA recipient strain B. subtilis strain 1A1 was cultured overnight in Spizizen salts minimal medium, containing per liter 14 g K2HPO4, 6 g KH2PO4, 2 g (NH4)2SO4, 1 g Na3C6H5O7 (sodium citrate) and 0.4 μg MgSO4, 0.2 g casamino acids, 5 g glucose (Tritium Microbiology) and 80 μg L-tryptophan (Sigma Aldrich Inc.). The overnight cultures were diluted 10 times in Spizizen salts minimal medium, and grown for 3 more hours under the same conditions. Subsequently, one volume unit of starvation medium (same as Spizizen salts minimal medium, but without casamino acids and L-tryptophan) was supplemented and growth was continued for two more hours. Competent cells were incubated for 15 min at 37°C in the presence of isolated genomic DNA from sigma factor mutants as listed in Table 1 and plated on selective agar media. The transformed sigF, sigE and sigK strains were checked for the loss of ability to sporulate after overnight growth in nutrient agar (Tritium Microbiology) supplemented with 0.5% (w/v) CaCl2.H2O and 0.5% (w/v) MnSO4. The sigD strain was checked for loss of motility by microscopy.
Heat exposure regime
Colony forming units
Culture-dependent and independent viability indicators
2log ratios μ-array
2log ratios q-PCR
CFU( 10 log)
23S rRNA
Outgrowth in liquid cultures
The recovery of bacteria from each temperature exposure was determined in dilute liquid cultures in triplicate by monitoring the time delay in reaching the mid log phase (OD wideband = 0.5) compared to the 30°C-control sample. Small increases in the average lag time were observed (0.2 and 0.8 hours) for temperature exposures of 40 and 50°C (Table 3). However, their significance remains uncertain as standard deviations for observed lag times ranged from 0.2 to 1.5 hours. A more drastic and significant change in the lag time of approximately 4 hours occurred at a temperature of 57°C, while at this temperature no significant loss of CFU was observed (Figure 1B). Apparently, the cells are able to fully recover from temperature treatments up to 57°C during an extended incubation in growth medium at 37°C. For more stringent temperature treatments in the range of 58 to 62°C the lag time ranged from 8 to 12 hours (Table 3). The relatively large increase in lag time from 4 to 8 hours for temperature treatments of 57 and 58°C coincides with the first loss of CFU at the latter temperature. Thus, for temperatures from 40 to 57°C the increase in lag time exclusively results from cellular recovery processes, while the increase in lag time at 58°C and higher results from both cellular recovery processes as well as the loss of viable cells in the culture. These results clearly show that the time delay to reach the mid log phase is a more sensitive measure to assess the effect of a heat exposure than CFU counts.
Membrane integrity
The effect of heat on B. subtilis cells was also studied with a number of cultivation-independent methods, including the fluorescence-based BacLight kit (Molecular Probes Inc.). This kit employs two nucleic acid stains, the green-fluorescent SYTO9 and the red-fluorescent propidium iodide stain, which differ in their ability to permeate bacterial cells. The cellular membrane is in general permeable to SYTO9, while propidium iodide penetrates only bacteria with damaged membranes. Propidium iodide reduces SYTO9 fluorescence when both dyes are present together. Thus, bacteria with intact membranes fluoresce green, while bacteria with damaged membranes fluoresce red [19]. Microscopic inspection confirmed that this system can be applied to stain heat-exposed B. subtilis cells (data not shown). However, we found that the fluorescence of the SYTO9 fluorescent dye was relatively unstable under the conditions used, as the green fluorescence signal slowly decayed, while the fluorescence of the propidium iodide dye remained stable. Therefore, we subjected bacteria that were stained only with propidium iodide to flow-cytometric analysis. A calibration curve of mixtures of living and heat-killed bacteria (10 min at 96°C) showed a perfectly linear correlation between the percentage of heat-exposed bacterial cells (0, 20, 40, 60, 80 and 100%) and the number of events with relatively high red fluorescence intensity (data not shown). The heat-exposed bacterial cell samples were stained with propidium iodide and analyzed by flow-cytometry. The three-dimensional representation in Figure 1C shows the number of cells plotted as a function of fluorescence intensity in the red FL4 channel for each temperature treatment. Clearly, the heat-induced decrease of non-fluorescent cells coincides with an increase of membrane-permeable red-fluorescent cells all with Gaussian-shaped population distributions. The effect is most prominent at 62°C (apart from the control experiment at 96°C), at which 25% of the cell population is permeable to the propidium iodide dye. A comparison of CFU counts with data from propidium iodide stained bacteria (Figure 1B, C and Table 3) shows that the membrane integrity is a much more insensitive measure for the applied heat regime than the ability to form colonies. In the range of 59–62°C the majority of bacteria do still contain an intact membrane, while more than 99.9% of these bacteria lost their ability to form colonies. Apparently, heat-induced cell death precedes the loss of membrane integrity.
Ribosomal RNA stability
Genome-wide expression profiles
Heat-induced expression of gene clusters
Heat effects on transcriptional regulation
Besides the classical heat shock responses, we identified a number of novel responses to heat stress. Notably, the response induced at the lowest temperature, already maximal at 40°C, includes the expression of genes under control of σD, the transcription factor for motility. This response is only present at 40–50°C; it involves among others the fla/che operon, the lytA-C genes for autolysis and genes encoding methyl accepting chemotaxis proteins mcpA-B. Surprisingly, the heat-induced transcriptional response also includes a number of sporulation and competence genes, described in more detail below. One of the most prominent responses at high temperature was the derepression of large regulons involved in carbon metabolism, controlled by the CcpA (average of 109 genes), AraR (regulator of the arabinose operon; average of 12 genes) and IolR (regulator of the inositol metabolism; average of 13 genes) transcription factors. Note that both the inositol and arabinose operons are controlled by CcpA, a global regulator that directs the carbon flow (Figure 6).
We observed a comparable partial activation for genes involved in competence development. Competence is another stationary-phase differentiation process leading a cell-type to allow uptake of foreign DNA. The transcription factor for competence is ComK, which is post-translationally activated by the product of the comS gene, which is part of large operon that encodes surfactin biosynthesis and is in turn under control of the ComP/ComA two component system [7]. Again only a minor sub-set of competence related genes was involved, including comS, srfAB, srfAC, srfAD, phrA and rapA under control of ComA and addA, addB, gidA, gidB, comFA, comGG, thdF, yyaA controlled by ComK (Figures 5 and 7).
Validation of transcript levels by quantitative PCR
Microarray results have been validated by the use of quantitative PCR. We selected the 23S rRNA gene as an internal standard based on its stability under the experimental conditions used in this study (Figure 2). In addition, we selected the cotJA gene, which encodes a spore coat protein, under control of transcription factor σE, which is known to be active during sporulation in the mother cell. Furthermore, the dnaK gene, which encodes a class I heat shock gene, a chaperone, under control of the transcriptional repressor HrcA. Finally, the gspA gene, which encodes the general stress protein GspA and is a general reporter gene for activity of the general stress response transcription factor σB [25]. The Q-PCR analysis shows a good match between levels of heat-induction resulting from microarray and Q-PCR analysis of three typical heat-induction profiles (compare Figures 5, 7 and Table 3). The Q-PCR results of gspA transcripts confirm the transient induction of the σB-controlled genes at 40 and 50°C, the more stable induction of dnaK transcripts at elevated temperatures and the gradually increasing levels of cotJA transcripts as a representative of the σE regulon.
Assessment of cotJA transcript levels in σ-factor mutants
Heat-induced expression levels of cotJA in B. subtilis σ-factor mutants
EL30°C (pg)
EL60°C (pg)
2log R
168 1A1 (wt)
TNO2007.164 (sigB)
TNO2007.160 (sigD)
TNO2007.166 (sigF)
TNO2007.167 (sigE)
TNO2007.169 (sigK)
In this study we subjected the Gram-positive bacterium B. subtilis to a lethal temperature regime and analyzed the effects of the treatment in a comparative analysis using a number of cultivation-dependent and independent methods. The application of cultivation-independent methods as an alternative to assess bacterial viability is highly valuable for a number of reasons. A rapid measurement of the physiological state of the bacterium indicating whether it survived a certain processing condition (heat, high pressure, antimicrobial compounds) would save enormous amount of time and labour, currently lost in the classical routine of plating and counting. The timely and laborious plating method renders high-throughput screenings an almost impossible mission, although the recently reported miniaturization of the culture plate to a micro-Petri dish holds great promise for the future [26].
Any cultivation-based method results by definition in an estimate of the survivors after a treatment under predefined growth conditions, which are usually very different from those present during the actual processing conditions. A rapid measurement of the physiological state of the bacterium would be beneficial, as it reflects the direct cellular response towards processing conditions and could yield information on the successful survival strategy of the bacterium. This could allow a rational design for preferred adaptations in processing conditions aimed at a more efficient bacterial inactivation for harmful microbes or survival for beneficial ones.
The need for cultivation-independent methods to assess bacterial cell viability is most evident when microbes only grow in their natural habitat and colony formation is absent or extremely slow in any of the available growth media. Likewise, the enumeration of colonies is not applicable for bacteria present in dense populations of multiple species for which no selective media are available. In those cases the highly selective PCR-based assessment of specific gene transcripts, as further discussed below, can be applied to assess cell viability of one bacterial species in a complex population.
The workflow in this study starts from the assessment of bacterial viability upon exposure to a lethal heat treatment with conventional methods, including the enumeration of colonies, the determination of outgrowth curves and the widely used dead/live stain in the Bac Light kit [19]. These experiments set a clear reference for molecular studies discussed below and roughly establish the 40 to 57°C temperature regime as inhibitory and sub-lethal, as judged from the extension in the lag phase and CFU counts (Table 3). The membrane integrity as judged from the dead/live stain is clearly a less sensitive indicator for the subtle heat regime applied here than colony enumeration or lag phase extension (Table 3), but more stringent temperature treatments do lead to a 100% dead (propidium-iodide permeable) cell population (Figure 1C).
The electrophoretic analysis of total RNA extracts clearly indicates a much higher heat tolerance of 23S rRNA molecule for the applied heat regime than 16S rRNA molecule, as one can assume that constant level of 23S rRNA cannot exclusively result from higher 23S rRNA synthesis rates (Figure 2). The difference in heat susceptibility of 23S and 16S rRNA molecules is commonly observed among prokaryotes, including B. subtilis, Staphylococcus aureus and Salmonella typhimurium [27–29]. The results clearly show that B. subtilis is able to fully recover from the observed heat-induced rRNA degradation, as significant cell death only occurs from 58°C exposures, while a major part of 16S rRNA has been degraded at 57°C (Table 3). Therefore, the level of 16S rRNA is not a very good indicator for cell viability under these conditions.
Additional file 1: Time-lapse movie of heat-induced expression profiles. The time-lapse movie of expression profiles was constructed by the use of Windows Movie Maker version 5.1 (Microsoft Inc.). All genes were sorted in the gene order present on the B. subtilis genome and presented in 32 columns of 96 genes each. Only those genes that contained a detectable transcription level at 30°C were analyzed. This filter led to a total of 75% of the genes (3072 out of 4100 genes). The 29 frames used for the movie consist of 8 measurements from 0–12 minutes and 21 interpolations. The parameters time, temperature and CFU counts were inserted in the microarray images, which were generated with the TIGR MultiExperimentViewer version 4.0.01. The 2log ratios were colour-coded, ranging from -3 (green) to +3 (red). Note that there is one region throughout the whole series of profiles with nearly identical expression levels to the reference profile at 30°C. This region contains 67 ORFs from rplK to rpsL and encodes at least 47 different ribosomal proteins. (WMV 2 MB)
In addition, gene clusters were identified containing the classic heat shock operons class I and class III (h, a, Figure 5), while the class II heat shock or σB regulon was only identified in the T-profile analysis as a result of the widespread distribution of genes from this regulon over the genome. The cluster of genes known to be highly induced upon stress is the mobile genetic element ICEbs1, which up regulation occurs upon global DNA damage (cluster d) [30]. This fact and the up regulation of the cluster of pyrimidine genes (cluster g) may be part of the cell's response towards heat-induced DNA damage. The gene clusters (e, i, j) which are all involved in catabolic carbon metabolism may indicate the cell's attempt to deal with energy stress and generate ATP. Most surprising is the up regulation of a number of genes, known to be involved in stationary phase processes sporulation (cluster f) and competence development (cluster c). As their activation is usually strictly controlled by dedicated transcription regulators, we tested whether the observed up regulation of inner spore coat genes (cotJ operon) is dependent on sporulation-specific transcription factors. We confirmed that the heat-induced expression of the cotJ operon is dependent on sigF and sigE, but not on sigK genes, in agreement with the current model of the sporulation signal transduction cascade (Table 4). The activation of the sporulation sigma factors is normally coupled to the course of morphogenesis during spore differentiation. It is not clear at this point what mechanism causes the heat-induced short-cut in part of the sporulation program and whether the expressed gene products play an alternative role in heat resistance of the mother cell.
The observed cellular responses suggest that heat stress causes a wide variety of physiological stress. Which type of damage leads to cell death is not clear, but the microarray data indicate that the transcriptional machinery is at least still intact in cells that completely lost the ability to reproduce. This can be concluded from the results that show increasing transcript levels of most genes in the clusters a-j at temperatures from 58–62°C, whereas the majority of the cells died at these temperatures (compare Table 3 and Figure 5).
Next, we defined a number of criteria to identify transcripts that could act as biomarkers for cell viability: (i) high expression levels in living bacteria, (ii) relatively low expression levels in heat-inactivated bacteria, and (iii) genes that are widely conserved throughout the bacterial kingdom. We selected a set of 4 genes, all under the control of the HrcA regulator, which matched these criteria; they include grpE, dnaK, groES and groEL (see Figure 7). We observed that the above criteria for expression levels of these genes also matched in microarray studies on other bacterial species, where the cause of death was different: Lactobacillus plantarum exposed to ultra high-pressure treatments and Staphylococcus aureus exposed to biocides (data not shown).
Both dnak and groE operons are preceded by a SigA-type promoter, which ensures transcription of these genes under growth conditions without stress (in line with the dnaK expression levels in table 3), as protein folding is also needed under these conditions. A fascinating question involves the mechanism for the reproducible rapid decrease of these transcripts in bacteria that lost the ability to grow. In living bacteria, the CIRCE element (controlling inverted repeat of chaperone expression), located between the transcriptional and translational start sites of both dnaK and groESL operons [22], is involved in the regulation of both operons. In the current model the GroE chaperonin, the activator of the HrcA repressor, which binds to CIRCE, is titrated away in the presence of unfolded proteins in an ATP-dependent process, leading to inactivation of HrcA and enhancement of transcription of dnaK and groESL operons [21]. Thus, the down regulation or degradation of these transcripts in excess of unfolded proteins asks for an extension of this model. No matter what regulatory mechanisms, the identification of 4 transcripts of chaperone genes as biomarker for viability needs to be further validated for a wide range of environmental conditions to determine the lethal conditions for which they are bona fide.
This study contains a systematic comparison between cultivation-based assessment of cell viability and cultivation-independent indicators for cell viability by sampling and analyzing heat-exposed B. subtilis cells going through the transition from culturable to unculturable cells. Accordingly, some viability indicators appear more sensitive than the conventional enumeration of colony forming units (CFU's), while others appear less sensitive measures: during heat stress the extension of lag time for outgrowth, the degradation of 16S rRNA and the induction of classic heat shock genes precede the loss of CFU's, while the loss of membrane permeability and the degradation of 23S rRNA occur after the loss of CFU's. The genome-wide transcriptional response indicates that heat stress causes a wide variety of physiological stresses. Which type or types of cell damage lead to cell death is not clear, but the microarray data indicate that the transcriptional machinery is at least still intact, as a subset of genes is still expressed in cells that lost the ability to reproduce.
The heat-induced transcriptional program includes known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Further research needs to assess whether the activation of a small number of genes in these well-studied developmental programs is a result of deregulation due to heat damage or a meaningful, physiological event fitting in the cell's strategy to cope with heat stress. Notably, the transcription of a limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead – include those of essential genes encoding chaperones of the protein folding machinery and may serve as molecular biomarkers for bacterial cell viability.
The authors thank Stanley Brul, Adriano Henriques and Daniel Kearns for generous strain donations. Tiemen van Eijndthoven, Michel Ossendrijver and Jojet Staal are gratefully acknowledged for their experimental contributions to this work. We thank the 4 anonymous referees for reviewing the manuscript.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Is it possible to edit after how much time you're automatically logged out?
I think you're automatically logged out after five minutes or so, but that's kind of short. When I'm typing a pm I take more time than that and am logged out. So when I want to submit the message I have to copy the text to keep it, log in again, copy the text and then submit it.
Maybe it's a sign that I 'talk' too much, but it is kind of annoying to keep logging in again

subset: commoncrawl


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Last Sunday I had a dream of a day. It started with taking a trip to the Saatchi gallery in London where the Little Black Jacket exhibition by Karl Lagerfeld and Carine Roitfeld arrived from Tokyo on Thursday. The opening on Thursday evening included the likes of both Lagerfeld and Roitfeld sharing cocktails (OK, I'm not actually sure if there were cocktails but they help set the scene and it seems like the sort of event that would have cocktails but Karl, when you read this, correct me if I'm wrong) with the likes of Keira Knightley and Edie Campbell. I can't believe they forgot to invite me! As my invite for last Thursday obviously got lost in the post I decided to go on Sunday instead. I can't believe I have never been to the Saatchi gallery before. It's fabulous. You are greeted by a fountain and a tall column saying 'Saatchi gallery'
(take a minute to get over my excellent Paint skills) As an interpretation of this (a photograph that I LOVE and saw today by the way)
I'm going to practice some drawing and maybe post it on here at some point. Don't laugh! The whole place just oozed an artistic vibe that was both hippie and sophisticated. By the time we got to gallery 4 we had climbed up the stairs and been faced with a dimly lit room with perhaps the most wonderful photographs of them all. Surrounded by beautiful people in even more beautiful jackets I proceeded to take notes on the best stylings. Alexa went for denim on denim in that chic bohemian way of hers whilst Carine Roitfeld looked like a modern day Chanel with a hat, pearls, an LBD, white bangles on each wrist and a pair of metal scissors. It is not just an exhibition of sum peeple in sum jakettes. It is about identity and finding yourself through a sartorial medium. You could analyse each outfit like you would analyse a novel in an English class. Take Elisa Sednaoui for example. She paired her classic Chanel piece with a big white tutu; an attention grabbing piece that could suggest her success as she is a model, actress and director. However, the balletic tone reminds us that she is still only twenty four although she has accomplished a lot in her not so long life so far. Tild Swinton went for a more conservative look with her buttoned up little black jacket over a black poloneck.
How would you wear your little black jacket?
Another great thing about this exhibition is that it is not exclusive. Although situated in a posher part of London, the gallery is admission free. Although the little black jackets worn by the celebrities are Chanel anyone can pick up a look-a-like from somewhere as economically friendly as Primark and style it to show their personality. It was not an exhibition filled with an array of snobs looked down their noses at couture gowns but a celebration of an iconic piece that can be enjoyed by pretty much anyone. Chanel originally designed the little black jacket in 1916 (almost one hundred years ago!) and it rose to popularity when Chanel reopened after the Second World War in the '50s.
Natalia Vodianova: Little Black Jacket by styleicon97 featuring chanel
Dolce Gabbana silk blouse
H m
Boucle jacket
£22 - target.com
£22 - target.com
Chanel vintage jewelry
Matthew williamson
H m
Pearl Edged Cream Corsage
Leopard print top
Leopard top
Fringe jacket
£22 - target.com
£22 - target.com
Chanel tweed jacket
Azzedine Alaia black skirt
£2,055 - montaignemarket.com
£2,055 - montaignemarket.com
River Island black skirt
Feather hair accessory
Hair clip accessory
£8.71 - etsy.com
£8.71 - etsy.com
Dakota Fanning: Little Black Jacket by styleicon97 featuring chanel jacket
Valentino lace sweater
£1,230 - net-a-porter.com
£1,230 - net-a-porter.com
Floral shirt
£19 - chicwish.com
£19 - chicwish.com
Plus size jacket
£22 - target.com
£22 - target.com
Chanel jacket
Lanvin long skirt
£2,650 - net-a-porter.com
£2,650 - net-a-porter.com
Long pleated skirt
£21 - sheinside.com
£21 - sheinside.com
Marni studded bag
£850 - marni.com
£850 - marni.com
Vintage handbag
£22 - modcloth.com
£22 - modcloth.com


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Matthew 10:39
"Whoever finds his life will lose it, and whoever loses his life for my sake will find it."
"...whoever loses his life for my sake will find it."
Obedience. The Lord taught me this word and how important this word. The important is not losing your life and all of your possessions but turning your focus away from those possessions. This world is so inviting that we forget this word (OBEDIENCE).
It is like you are committing into a relationship that is everlasting, perfect, pure and contended. Everyday, I always pray and surrender everything to Him. It is so hard to face the day using your own strength, it is draining. In my past life, I have been using my own strength, knowledge and skills. When you look at my life, I look like I am obeying God but...no. I have been on clinging on my own life and keep doing things without quality and sense.
Let us always remind ourselves with this word. Obedience.
It will be hard at fist but the process and the journey of obeying God is worth it.
Personally, I am enjoying it. It is hard, everyday I feel tired but the Lord will always be my strength.
Terno-ing mode with my sister. Check out her blog! The Queen Says It All.
The shirt that we are wearing are for sale for only 250 pesos. We are selling this as our fundraising for our upcoming summer camp on May 2014. We have other designs but this one is our favorite and best seller. :)
Hope you enjoyed!


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A stellar high school comedy with an A+ cast, a brilliant script loaded with witty dialogue, eye-catching cinematography, swift editing, and a danceable soundtrack.
A stellar high school comedy with an A+ cast, a brilliant script loaded with witty dialogue, eye-catching cinematography, swift editing, and a danceable soundtrack.
Rim of the World is not going to inspire young viewers to look up at the stars, pretend to run from alien monsters, or continue…
Roger Ebert on James Ivory's "Howards End".
"The Ballad of Narayama" is a Japanese film of great beauty and elegant artifice, telling a story of startling cruelty. What a space it opens…
An article about the reissue of "Two Weeks in the Midday Sun."
An article about Champ Clark's one-act play,"Wild Son: The Testimony of Christian Brando," currently running at Santa Monica Playhouse through May 26th, performed by John…
An article about the reissue of "Two Weeks in the Midday Sun."
Return now to those thrilling days of yesteryear.
One of our foreign correspondents looks at Cold War.
A Far Flung Correspondent takes a look at the Paul Newman classic The Hustler.
Bong Joon-ho's class satire is the first South Korean film to win the Palme d'Or.
Reviews from the Cannes Film Festival of the latest by Abdellatif Kechiche, Justine Triet, and Elia Suleiman.
I'd heard absolutely nothing in advance about "Foolin' Around," but I was eager to see it anyway just because its stars were Gary Busey and Annette O'Toole, two of my favorite new movie actors.
Busey was responsible for a wonderful, Academy Award nominated 1978 performance in "The Buddy Holly Story," and O'Toole co-starred in one of 1978's overlooked treasures, "One on One," a college basketball movie with Robby Benson. They were both so individual, so talented, so clearly intelligent, that I figured, "Foolin' Around" would almost have to be an interesting movie. It wouldn't be a hit, I knew - hit movies don't open at discount admission prices - but it might be a small, valuable discovery.
It was not. It's a dimwitted, old-fashioned comedy that's sort of inspired by old Disney movies and halfway similar to 1940s rah-rah college comedies, and directed and edited with such a puzzling lack of tempo that none of the material ever quite seems in focus. If good new actors are hard to find, good roles are even harder, and a few more movies like this could wreck Busey and O'Toole before they really get to develop their genuine talents.
Busey stars as a country boy who enrolls as a freshman at the University of Minnesota and has to work his way through school. O'Toole, the heiress of a gigantic local corporation, is a psych major who meets Busey during a cockeyed experiment he volunteers for. O'Toole's mother (Cloris Leachman) is president of the corporation, her grandfather (Eddie Albert) is the founder and chairman, and her boyfriend (John Calvin) is in line to marry her and become vice president.
The Calvin character is the most old-fashioned element in a movie full of retreads. He's one of those stuck-up, spoiled rich kids who used to be stock characters in college comedies - insufferably egotistical, convinced the world's his for the asking. The movie's story inevitably revolves around, O'Toole's decision to jilt Calvin and fall in love with Busey. And the last scene inevitably involves a big society wedding, with the bride changing her mind halfway down the aisle.
All of this is very, very old stuff. But if it were kidded just a little bit, or approached with the slightest bit of freshness, it might still work. "Foolin' Around" seems to believe it has discovered all this material for the first time (have the people who finance these movies ever seen old movies?), and treats it with dreary comic reverence.
The characters never really come alive, and the movie's construction and editing are so bad that obligatory conversations are never held, important plot points are left hanging indefinitely, and - worst of all - the comic momentum is interrupted by difficult but unnecessary and distracting stunts. Two big sequences involving walking on a high-rise girder, and floating over Minneapolis with a hang glider must have been tricky and expensive to film, but they bring the story and the comedy to a grinding halt. The movie's material might have been marginally successful in other hands, but this movie wasn't directed or edited with a knack for comedy.
This message came to me from a reader named Peter Svensland. He and a fr...
A review of the newest film by Quentin Tarantino.
A review of CBS All Access' The Twilight Zone.


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If multiple applicants (and existing employees) look the same on paper, how do you distinguish between them? Whether you are making a hiring decision or investing in training, you need to accurately evaluate a person's skills, knowledge and behaviours. This is readily done with Drake on-line assessment solutions, helping to predict and shape on-the-job performance.
In this world of accelerated change, knowledge is everything. Yet assessing skills and behaviours are equally important for hiring, onboarding, training and promotion. Drake links modern technologies with the human touch, in order to attract and assess top performers to suit your company and the job at hand.
DrakeWize provides administrative and technical skills evaluations with a suite of assessments covering over 100 Windows based applications and 50 MacIntosh programs. DrakeWize can accurately identify an individual's skill level in both general computer skills and a wide variety of specific computer applications. DrakeWize offers a broad selection of job-specific skills tests, including:
- Accounting skills
- Legal skills
- Industrial skills
- Clerical skills
- Call centre skills
- Medical skills
- PC skills
- IT skills
Virtual Customer Contact (VCC) Skills
80% of sales representatives fail within their first year. For positions that require employees to work directly with customers, Drake offers VCC assessments. This is a multi-media assessment that simulates the work of customer contact personnel in a multi-tasking environment.
Behavioural Assessments
"Did you know that 87% of people who leave their organisation or their team do so because of Personality Conflicts and NOT Capability?" (Dr. Kurt Einstein)
Behavioural assessment solutions can help remove the subjectivity in the selection process, and identify the traits best suited to your corporate culture, and to the specific position.
What behavioural assessment technology makes this possible?
Drake P3, installed in over 2,000 sites worldwide, is an advanced behavioural and personality profiling solution that identifies the performance characteristics of successful employees.
Drake Picasso clarifies personality profiles and individual capabilities. It is a powerful psychometric assessment tool used to determine leadership and operating styles, reasoning skills, conflict handling styles, personality traits, the role one plays within a team, occupational preferences, and behavioural competencies.
What is the end result?
With behavioural assessments, you receive:
- Assistance in eliminating the guesswork out of attracting and selecting the right people for your position
- Accurate assessments of individual's skills, knowledge and behaviours
- Assistance integrating testing into your HR processes.
How is it done?
Drake has a library of existing tests that organisations can simply choose from if needed. If customised testing is needed, a Drake consultant can help tailor an assessment solution to suit your unique needs. All assessments are web-based, so no special hardware or IT skills are required.
What is the payoff?
The immediate payoff comes from preventing bad hires. Sometimes candidates sound good in an interview but don't actually have the needed skills or behavioural styles. Drake assessment solutions ensure candidates really do have the skills and behaviours you need, and at the levels desired. Further savings come from ensuring training dollars are not wasted teaching skills at too low or too high a level.
Contact the Drake Solutions team to find out more:
Theodore Moudakis firstname.lastname@example.com
Tom Key firstname.lastname@example.com
or call 13 14 48


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Viewing and Editing Project Properties
Running Projects in the Emulator
Searching the WURFL Device Database
Finding Files in the Multiple User Environment
CLDC Emulation on a Windows Mobile Device
Installing CLDC Emulation on a Windows Mobile Emulator (Windows Only)
JSR 82: Bluetooth and OBEX Support
JSR 135: Mobile Media API Support
JSR 177: Smart Card Security (SATSA)
JSR 205: Wireless Messaging API (WMA) Support
JSR 211: Content Handler API (CHAPI)
JSR 226: Scalable 2D Vector Graphics
JSR 238: Mobile Internationalization API (MIA)
JSR 256: Mobile Sensor API Support
This documentation sometimes uses user.home to represent the root location of user files. For a list of user file locations, see File Locations. The default locations are as follows:
C:\Documents and Settings\user\Application Data
On Windows, if you do not remember the location you specified, select Help > About in the main window.
Mac OS
/Users/uname/Library/Application Support/


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Based on the hit PlayStation 3 game. Use your imagination to create detailed levels in the whimsical LittleBigPlanet universe, then share them with the world, and race through them with your friends. The critically acclaimed game LittleBigPlanet makes its way to the PSP, offering new SackBoy adventures with the same play, create, share experience for gamers on-the-go, in addition to brand new levels and environments specifically created for the platform.
Sony Computer Entertainment Europe's SVP of Worldwide Studios Michael Denny welcomed the creators of LittleBigPlanet to the PlayStation family today, announcing that SCE has acquired Media Molecule. Terms of the deal were not disclosed, but Denny said that the UK developer is now a wholly owned part of SCE. Read More »
One of the undeniable draws of LittleBigPlanet is its style. A big part the game's appeal is the charming look of a tiny stuffed character working its way through a big, homespun world. But discounting its substance would be a grave error, because beneath this cute, quirky, polished exterior lurks a very clever puzzle-based platformer that strikes just the right balance of challenge and reward. This is a game charming enough to lure in casual players while still being complex enough to give experienced gamers a way to exercise those finely honed skills.
Of course, if you've played the masterful PS3 version, you know this already. And if so, I'll warn you now: This is not a remarkably different game. Read More »
Also known as: Little Big Planet, LBP PSP
Release Region: United States
Release Date: November 17, 2009
Publisher: Sony Computer Entertainment
Release Region: United Kingdom
Release Date: November 20, 2009
Publisher: Sony Computer Entertainment
Release Region: Australia
Release Date: November 19, 2009
Publisher: Sony Computer Entertainment
LittleBigPlanet at IGN
LittleBigPlanet at GameSpy
LittleBigPlanet at GameStats


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Hello everyone! These pictures were taken after a rainy week when the weather was not so warm and the sky was full of clouds. I hope you like the post. See you soon!
Jeans: ZARA
Shirt: Atmosphere
Scarf: Pimkie
Bag: ZARA
Hello Sinzy,
I'm Valencia, the community manager of WhatIWear. I checked out your blog, and I think your style is awesome! It should be seen by more people! That's why I'd like to invite you to WhaIWear.com. With 900.000 page views a month, WhatIWear.com is the leading street style website in Asia. Plus, we're still growing, so it's perfect for rising fashionistas to have their chance to SHINE at the spotlight.
Maybe you can post these pics as your first looks on WhatIWear? I bet our members will be rooting for you as well!
We cannot wait to see your awesome style.
Click www.whatiwear.com to get started.
See you soon,
Community manager


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One thing I love about Christmas is the amazing boxes of goodies stores have in stock. although Christmas is long gone I'm still to get through this box of 12 goodies from Lush! If you've been following my blog for a while you may have noticed that I do post a lot of Lush reviews. I love their products and ethos and generally I just think they're a great place to buy bath and body items. So when I received this box on Christmas I was ecstatic!
The box contains: Snow Angel Bath Melt, Golden Wonder Ballistic, Hot Toddy Shower Gel, The Olive Branch Shower Gel, Celebrate Body Lotion, Star Light Star Bright Bath Melt, Cinders Bath Bomb, Dashing Santa Ballistic, Holly Golightly bubble bar, Lord of Misrule Bath Bomb, Karma Soap & Honey I Washed the Kids Soap.
As you can tell the box is a great mix of bath bombs, melts, shower gels and soaps. It features some of Lush's best selling products and all around i'm super happy with the box which retails at £49.99. What I would say though is that the more glittery items can mess up your bath big time! The glitter gets into places you never would have imagined… even your kitchen… I don't know how but it got there!
Are you a fan of any lush products? Let me know so I can try them out!
Reader Interactions
17/01/2015 at 13:31
Ohh this Box has a couple of my favourite Christmas products by Lush! ^ ^
17/01/2015 at 13:52
Divine! I love Lush stuff so much.. I want all of the new lovely Valentines Day Lush stuff!!
AMANDA LOVES | UK Beauty and Style Blog
17/01/2015 at 18:25
Love LUSH stuff, by far my favourite brand! I was bought the dashing santa but still haven't used it! Oops. I got my hands on some of the Valentines day range the day it was released, can't wait to use them 🙂
Hannah x | hannatalks
17/01/2015 at 20:42
This looks great! I'm still working my way through my Christmas LUSH haul haha
Megan x
London Callings
19/01/2015 at 22:46
I'm so sad we don't have lush in Jersey anymore. It's one of my favourite shops! x


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Rama (Comic)
(Amar Chira Katha)
The story of Rama for children, told in comic book form.
- 32 pages, Indian paperback comic
- (retail only)
- DescriptionDetails
- Reviews
One of the most important avatars of Vishnu, Rama is best known for his role in the great Hindu Epic Ramayana. This children's book is based the Ram-Charit-Manas of Tulsidas.
Rama's story is told in this Amar Chitra Katha comic book that appeals to children and adults with a young heart.
Add your review of this product
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"A Health Promoter is an agent of change in their community"– Juan de Dios Blandón, Health Promoter of San José de la Mula, Matagalpa
In rural Nicaragua, women and children often suffer from preventable disease. AMOS helps prevent death and disease by equipping local communities with the tools they need to reach out to their most vulnerable neighbors through the model of Community-Based Primary Health Care. We train and accompany local volunteers, called health promoters, to carry out activities that improve community health.
Health promoters are people who are selected by their community's health committee, which is made up of community leaders who care about improving the health outcomes in their community. The health promoters are trained in the basic provision of health care services, in the prevention of disease, and in the promotion of health in their communities. They minister to people in their communities to improve physical, spiritual, and emotional health.
Each promoter offers primary health services to their community in several ways:
- Attending to patients two days a week at a community clinic the promoter manages.
- By taking a community census, the health promoters and health committee determine who is the most vulnerable to sickness and death. This often includes children under five years old, pregnant women, persons with chronic illnesses, and the elderly.
- As part of their work, health promoters do home visits to people as defined as vulnerable to promote health, provide follow-up care, and prevent illness.
- In the case of a health emergency, all of the AMOS health promoters are certified as first responders and are formally recognized by the Nicaraguan Ministry of Health (MINSA) to arrange for immediate help in their communities.
At AMOS, we strive to work holistically with the communities to facilitate the process of empowerment, so that in the end, it is the people themselves who can take action in a way that improves the health of every member of their community — especially the most vulnerable.
AMOS Health Promoters receive ongoing education and training in the following areas in order to improve health outcomes in their communities:
- Developing a plan with pregnant mothers in order to ensure better birth outcomes and decrease maternal and neonatal mortality.
- Lay epidemiology and anemia
- Rational use of medications
- Integrated management of childhood illnesses (AIEPI)
- Nutritional counseling
- Evaluation techniques and procedures that will guarantee that the health promoters and AMOS staff know how they are improving and what areas they need to work on improving.


text:
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TOPSHAM — With a state Project Canopy grant in hand, the town is planning this year to conduct an inventory of its trees.
The Board of Selectmen on Jan. 3 unanimously accepted the funding, according to Rod Melanson, Topsham's natural resource/assistant planner.
The program recently aided 21 projects in 19 communities with more than $100,000 in an effort to help community forestry endeavors, according to Maine's Project Canopy Facebook page. A grant from the U.S. Department of Agriculture Forest Service Urban and Community Forestry Assistance Program funds the Project Canopy Assistance Program.
Project Canopy, which is administering federal program funds, is part of the Maine Department of Agriculture, Conservation and Forestry, Melanson said.
Topsham is using the funds to conduct a street tree inventory within the town's growth area, covering parts of town like Main Street, Route 24, Route 196, Route 201, Topsham Fair Mall Road, and Winter, Bridge and Elm streets, Melanson said.
The town received $4,650, which it is matching with $800 and in-kind work, such as people doing tree inventories and the planning office covering mapping services, report writing and database managemt from the planning office.
The town's tree warden, Kyle Rosenberg, with the help of the planning office, plans to coordinate volunteers and students to work with the Tree Committee on the project, which will create a new tree inventory database for the town.
"The database will help us … in our mapping," Melanson said. "We're going to have a … geographic information database (and will) have a report that will get into tree distribution," with elements like condition, size, value, species and location.
The next piece of the project will be development of a management strategy, according to Melanson, who said it will ask "are we meeting the goals of having trees along our streets, and how do we take care of those better than we currently do? Basically, the inventory will help us plan better for trees that we have, and trees that we'll be planting in the future."
The town plans to conclude its reporting by early autumn.


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|Dolphins Owner Stephen Ross Needs To Do More Than Just Bring In Tennis Players
If you look at the nine-year period before the most recent nine years (from 1993-2001), Miami made the playoffs seven out of those nine seasons.
A big reason for that lack of success has been the Dolphins quarterback search. In just the last eight years Miami has gone through seven different starting quarterbacks, and I certain that we will need another quarterback for next season.
It has been a never-ending cycle of finding a new quarterback, that quarterback not doing well, and then dumping him at the end of the season. What Miami must do is get their new starter from the first round of the draft. Joey Harrington was the only first-round quarterback that Miami has had in the last decade, and the only reason Miami got him was because it was the Lions who drafted him that early and he wasn't good enough for them to hang on to.
The Dolphins MUST use their 15th overall draft pick to either get a quarterback or use it to trade up and get an even better quarterback. I, personally, am not planning on buying any tickets to a game next season if they don't fix their quarterback situation. I know I am not the only one who feels that way, too, and we all know how much Stephen Ross hates empty seats.
Ross has tried way too hard to bring glamour into the franchise and has not focused enough on bringing in wins. After that Head Coach debacle that Ross put us through these past few days, he better be ready to do something really good during the offseason to make us forget this week.
I for one have gotten tired of the mediocrity. It is time to make something big happen. Ross did a good job last offseason by bringing in Brandon Marshall, but now he needs to do an even better job this offseason by bringing in a good quarterback and a good offensive coordinator that will actually be able to score points.
We are all waiting Mr. Ross. It is time to see what you can do.
I encourage you guys to write letters to Stephen Ross with your opinions on what he has done and what you think he should do. Here is the address you can write to to reach him:
Miami Dolphins
ATTN: Stephen Ross
7500 SW 30th Street
Davie, Florida, 33314.
Thanks for reading, and let me know what your impressions of the current state of the Miami Dolphins organization is.
Subscribe to DolphinShout


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Thursday, 25 July 2013
Hip Hip Hooray.... We are 4 today!!!
What a wonderful four years it has been since we opened the doors to Milly & Dottie's Emporium. We have welcomed visitors from far and wide through those doors and made some lovely friends along the way. We would like to thank you all for coming to visit us in our busy emporium and for keeping us company over the last four years; and to join us in our celebrations, by raising a glass of something fizzy and feasting on some of our favourite custard creams!
Hip Hip Hooray Milly & Dottie are four today!
Posted by Milly and Dottie at 11:12 5 comments:
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Мисс Мира Казахстан 2014 Мадина Давлетова 24 years old. PHOTOS
Madina Davletova appointed "Miss World Kazakhstan 2014", by Miss Kazakhstan Organization after the original winner Aiday Isaeva did not qualify for Miss World because of age. However Aiday Isaeva will compete at Miss Universe 2014. Madina was the 2nd Runner-up at the Miss Kazakhstan 2014 pageant.


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Dr. Carolyn Chambers Clark, Award-Winning Author and Wellness Nurse Practitioner
To receive a $10 discount on your first purchase at www.iherb.com, sign in/register and add your purchases to your cart. Prior to paying put in this code BNN951.
Think positive and you'll be positive!
Google has placed ads on many pages to help me pay for this web site.They collect information about what pages you visit.
If you want to know Google's privacy policy, please go to http://www.google.com/privacy_ads.html


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Mobile Commerce Minute Episode #356:
Notifications are emerging as the new mobile operating system. As mobile users find themselves buried in apps that once held promise but have somehow found their way off the main screen, notifications can help bring the value back if they are done right. A recent study by GroupM looks at the willingness and concerns consumers have with notifications and the results should leave everyone feeling opportunistic. Bottom line? Do notifications properly and there will be prosperity. However, the opposite is also true.
With Rob Woodbridge and Chuck Martin


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We believe that the best environment for the raising and training of children is in a godly home with a father and mother. ABBA Fund provides interest-free covenant loans to Christian couples who are called by God to expand their families through adoption and who need help with the "cash flow crunch" posed by adoption expenses.
Abba is an Aramaic word typically used by a child to address his father, expressing complete confidence that the father would provide every need. The Bible proclaims that God is the Father, Helper and Defender of the fatherless (Psalm 10:14; Psalm 68:5; Proverbs 23:10-11). ABBA Fund exists to glorify God as one of His instruments in helping the fatherless (James 1:27).
Email: firstname.lastname@example.com


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Here you'll find lists of document suppliers, booksellers, as well as lists of libraries to help you bolster your custom holdings lists and groups.
Document Suppliers — List of fee-based services that supply documents such as articles, patents, government documents, and dissertations.
Booksellers and Publishers — In some cases it might be easier and more cost-effective to purchase books rather than borrow them.
Sue's AV Lenders List— Lists of libraries (by OCLC symbol) that will lend Audiovisual materials.
Kate's Mid-America List – Custom groups for MALA, COKAMO, MOBIUS, and MALA-AMIGOS libraries
LVIS-10-2016 – Custom holdings list of Libraries Very Interested in Sharing (LVIS)


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TLKE offers high watch-making expertise from creation & manufacturing through to supply chain.
Expertise in transcription of Brand DNA, codes & seasonal themes to build outstanding and creative collections conveying global Brand Identity with accurate price positioning.
TLKE bring facilities of a major player, thanks to strong & historical links with the best manufacturers in term of infrastructure & quality.
Supply Chain
Direct supervision of manufacturing, quality & fill rate in Asia.


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Hi, my name is Markus Krötzsch (Kroetzsch if you're short of umlauts), and I am a researcher at FZI/AIFB Karlsruhe, where I am also working on my doctoral thesis. I am also a member of RIF WG. My current semantic research is slightly schizophrenic: * On the one hand, I am investigating tractable description logics and conjunctive query answering in various DLs. In this role, I expect this WG to fix some useful definitions for commonly used tractable OWL fragments. * On the other hand, I am the maintainer and main developer of Semantic MediaWiki, which uses very lightweight ontologies, but which exports data that is OWL-DL compatible. In this role, I would especially like to see lightweight meta-modelling (punning), and some syntactical RDF compatibility. == What I hope/expect to contribute == If all goes well, Semantic MediaWiki can become an early adopter of "OWL 1.1" (or however we will call it), which would probably lead to a rather large number of OWL1.1 files on the web. We are also willing to contribute to the specification of tractable fragments. More information and contact details can be found at my homepage http://korrekt.org. Cheers, Markus P.S. As already indicated, I will join F2F1. On Dienstag, 9. Oktober 2007, Alan Ruttenberg wrote: > This is a good time for members to start introducing themselves to > the other members of working group. > The topics generally covered are: > > -- a bio summarising experience you have that's relevant to the > work of this group > > -- as much contact info as you care to share on this public list > > -- what you expect to get out of this WG > > -- what you hope/expect to contribute. > > As we will be using a wiki to record information about the working > group, it would be great if you could create an account on it and put > your introduction there as well, or link to your post. Here's how: Go > to http://www.w3.org/2007/OWL/wiki/. At the top right click "Log in/ > create account". On the next page click "Create an account". Use a > "wiki name" as your login, a concatenation of your captialized first > and last names, e.g. IanHorrocks. Once you have an account, click on > your home page, which will be linked to on the topmost line of each > page, and introduce yourself. Instructions on basic editing within > the wiki are available at [1]. You can also add your page to the wiki > participants page [2]. > > Also, in your email, please indicate your intentions/expectations > w.r.t. the first F2F meeting [3] by stating "Attending F2F" or > "Regrets for F2F". > > Thanks! I'm looking forward to talking to you on Wednesday and to > meeting many of you in Manchester in December. > > [1] http://meta.wikimedia.org/wiki/Help:Wikitext_examples > [2] http://www.w3.org/2007/OWL/wiki/Participants > [3] http://lists.w3.org/Archives/Public/public-owl-wg/2007Oct/0004.html > > Regards, > Alan -- Markus Krötzsch Institut AIFB, Universät Karlsruhe (TH), 76128 Karlsruhe phone +49 (0)721 608 7362 fax +49 (0)721 608 5998 email@example.com www http://korrekt.orgReceived on Monday, 22 October 2007 08:40:16 UTC
This archive was generated by hypermail 2.3.1 : Tuesday, 6 January 2015 21:41:59 UTC


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The UP welcomed the 4000th international student
Wednesday, 8 November, 2017
By this time the University of Pécs has more than 4000 international students, which is an important milestone in the history of the university.
A good indicator of progress is the fact that in 2010 the number of incoming students barely number exceeded 1500. The 4000th international student, Ide Kanako, a first year student of the Medical School from Japan was greeted by József Bódis, rector of the University of Pécs with a diploma at the Stipendium Hungaricum Gala.
The growth of the number of the international students was a strategic goal of the university management since 2010, naturally in line with the development of the infrastructure and the professors' team. As far as the former condition is concerned, the Modern Cities Programme is a great benefit for the university as it provides financial resources for the development and modernization.
One of the performing artists of the Stipendium Hungaricum Gala was Mino Rabenjafimanantsoa from Madagascar and her band, who was the Voice of the UP in spring. The international students had the opportunity to learn a Hungarian folk song with the help of the Eszterlánc Band and with an outstanding talent, Noémi Sturz, student of folk singing at the Academy of Music.


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Christoph Päper <email@example.com>: > > I don't see the point where this whole thing is better for search > > engines. If they wanted, they could have followed the values of the > > src attributes of the frame elements already as if they were hrefs > > of as. Some may do so, dunno. "Ian Hickson" <email@example.com>: > I agree with this... I don't see how XFrames particularly help with > search engines really. It does make it easier for them to return > results to framesets, but that seems to be all. "That's all"? That's already quite a lot. Search engines cannot currently return pages in the context that they are designed in. So you get a page with a lot of context missing. XFrames allows them to return the page in the context you would normally see it in. It doesn't help search engines *find* results, it helps them *return* them. Steven PembertonReceived on Tuesday, 1 October 2002 04:51:01 UTC
This archive was generated by hypermail 2.3.1 : Wednesday, 7 January 2015 15:08:47 UTC


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Equilar's Peer Group Submission Window Open Until December 31, 2016
Equilar announced that companies may now submit their peer groups through December 31, 2016. Equilar recommends companies that will file their proxies between January 15, 2017 and July 15, 2017 submit their peer groups. The company peer groups will be used to create the company's 2017 Equilar Market Peers™.
As a reminder, the Equilar Market Peers are used by Glass Lewis when it generates its say on pay vote recommendations.
Companies may submit their updated peer groups at: https://insight.equilar.com/app/peer_update/
Equilar's FAQs on its peer group submission process and how the Equilar Market Peers may differ from the Glass Lewis Market Peers powered by Equilar can be found at: https://insight.equilar.com/app/peer_update/peer_update_faq.jsp


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Kenyana 6598
Zero Dean @ Zerography.com
Pacifica Pier LTE 3
Pacifica Pier LTE 3
Pacifica, California
October 8, 2011
Pacifica Pier LTE
Taken in Pacifica, CA
October 8, 2011
A long-term exposure taken under the pier in Pacifica, Ca.
"Pacifica Pier LTE" is available as cards, prints & posters in my storefront on Redbubble
Abandoned Church. New Mexico.
Taken in Taiban, New Mexico
October 22, 2011
The last light of the day falls on an abandoned church in New Mexico.
Driving along some road in New Mexico — during my cross-country adventure — I noticed light falling in an appealing way on an old abandoned building. I wasn't in picture taking mode, but I'm always scanning for interesting lighting and compositions in the off chance I see something I like.
I wasn't sure if this building was worth turning around for. And I thought about it for another mile or so before I convinced myself that I should turn around and go back. I don't see that many abandoned buildings I like — especially when the lighting is "just so".
I pulled into the dusty lot and took a few snapshots with my Canon Powershot. I left my "good" camera in its bag — which I am kicking myself for now.
Anyway, it turns out this abandoned Presbyterian church is kind of famous and has been photographed quite a bit. I had no idea. It was just a chance encounter.
"Abandoned Church. New Mexico." is available as cards, prints & posters in my storefront on Redbubble.


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- Features
- High-intensity massage
Dual pivoting massage heads operate at up to 3100 pulses per minute.
- Variable intensity control
Variable 2-speed control allows you to go from relaxing to invigorating massage at the touch of a button.
- Includes 4 custom attachments
Swap massage heads for softer or firmer massage. Includes attachments for soft, medium, firm, or heated operation.
- Comfortable to use
Lightweight, contoured design that's easy to hold and use. An extra-long power cord gives you increased mobility.
- Optional heat*
- High-intensity massage
- Customer Reviews
- Support
Online Support Information


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Another week for Baltimore at the one-murder-per-day pace. Oldest victim: 97. There were also 18 non-fatal shootings this week and 12 gun arrests.
Monday, July 17
7:00 p.m. Maurice Finney, a 22-year-old African-American man of the 800 block of Stoll Street, was on the 3200 block of W. Baltimore Street when someone shot him and another man. A patrol officer found both men and summoned medics. Finney was shot in the upper body, and he died at a hospital. The other man is expected to survive. This was the second murder on that block in three days.
Tuesday, July 18
1:41 p.m. A man who police have not yet identified publicly was on the 1000 block of Harlem Avenue in Harlem Park when someone shot him and another man. One man died and the other was taken to a hospital for treatment.
1:45 p.m. Rashaw Scott, a 26-year-old African-American man, was parked in a car with a 20-year-old man on the 500 block of N. Fremont Avenue when someone shot him multiple times in the body. He tried to drive off but crashed the car. He died at a hospital. Scott lived on the 1000 block of Edmondson Avenue. He made news in 2013 when he escaped an assassination attempt in Cherry Hill that took the life of his 16-month-old son, Carter Scott.
Wednesday, July 19
11:15 p.m. Police called to the 100 block of W. 25th Street found a man who was dead, who they have not yet identified publicly, with "trauma to the neck."
Friday, July 21
6:50 p.m. Waddell Tate, a 97-year-old African-American man, was in his home on the 1700 block of Darley Avenue when someone broke in and beat him to death. Family members found him and called police, who were unable to determine if anything was taken other than Tate's life.
7:51 p.m. Ray Woodland, Jr., 45-year-old African-American man who lived on the unit block of S. Monastery Avenue, was on the 4500 block of Edmondson Avenue when someone stabbed him. He died soon after at a hospital. Police have published photographs of several persons of interest, and are looking for the publics help to identify them.
Saturday, July 22
11:35 p.m. Malone Sanders, a 21-year-old African-American woman, was on the 500 block of Brunswick Street when someone shot her multiple times in the head and body. She died. Hers was the second murder on that block this year; Sherman Smith was shot to death there on April 2, and Carlton Beachum was charged with that murder. There were no other murders recorded in the Millhill neighborhood during the past decade. Sanders had resided on the 1100 block of N. Gilmore Street.
Sunday, July 23
2:34 a.m. Vincent Waters, a 24-year-old African-American man, was on the 1100 block of Whitelock Street when someone shot him multiple times in the torso. Police found him there, medics got him to a hospital where he died. He had resided on the 1300 block of E. Biddle Street.
The person who was shot to death on July 14 in the 100 block of Reedbird Avenue in Cherry Hill was Melvin Allen Truesdale Jr. a 24-year-old African-American man, of the 3900 block of Whispering Meadow Drive.
The person killed on the 3200 block of W. Baltimore Street on July 14 was Christopher Hockaday, a 31-year-old African-American man. He lived on the unit block of South Hilton Street.
This week: 8
This year: 195


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Fracking In Balcombe
On 23rd July after months of applications, audits and public opposition Cuadrilla started moving their drilling equipment onto the Balcombe site, with the aim of starting exploratory drilling starting on Saturday 27th July. But the Balcombe residents aren't going to stop fighting.
The Balcombe villagers have repeatedly shown their opposition with Cuadrilla's plans for drilling in the area and on the 25th July are hosting the Great Gas Gala! The Great Gas Gala is a community led carnival and a clear demonstration of the local community's resistance to the threats to health and to the environment posed by the fracking industry.
The residents of Balcombe oppose drilling for a multitude of reasons, from the local fears over potential water and air contamination and the noise and traffic from the construction, to the national and global issues of what a dash for gas in the UK will represent. The UK's commitment to make a fair contribution to reduce emissions, in line with keeping global warming below a 2°C rise, gives a very clear global carbon budget and we already have an insufficient budget for the carbon that we are emitting. With Osborne pushing for fracking in the UK, offering the 'most generous tax breaks in world', we will not be able to meet these targets.
Visit the website for more information on the Great Gas Gala and how to get involved.


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1. Why is the appliance leaking?
2. How do you remove the lead in tap water?
3. What should I do if there is no cartridge replacement notice even after a year?
4. The upper flexible pipe leaks acidic water when I set the appliance on beauty care water. What can I do to stop the leaking?
1) Scales: Ca, Mg, Na, Ka and other mineral clusters are produced when water is electrolyzed. These clusters are called scales and are not harmful even if consumed. 2) Electrode plate: A metal board that conducts electricity during electrolysis. It is made of titanium and plated in platinum. It has superb electrolysis capacity and high durability. 3) Reversing the voltage: In order to electrolyze water, the plus and minus of the direct currents must be clear. When mineral clusters get attached to the electrode plate, there is a need to reverse the plus and minus of the current to remove the clusters. This basically sets the appliance on a cleaning function, similar to when it is placed on the "beauty water" setting.
5. What does cleaning consist of?
Changing the setting to "beauty water" once a day for 30 seconds is another way to clean the electrolysis cell. The cleaning function is necessary to maximize the life span of the electrolysis cell. 4) Minerals: Ca (calcium), Mg (Magnesium), Na (Sodium), Ka (Potassium) and other nutrients found in food and water. Minerals found in water are said to be especially good for the health in optimum amounts.
6. What should I do if I notice a build-up of white, floating clusters in my water?
These clusters are not harmful even if consumed, so please do not worry. Furthermore, if you set the machine on beauty water (acidic water) , and let the water run for two to three minutes, you can decrease the amount of minerals significantly. Please clean containers that you put Kangen Water™ in at least once every three months by the abovementioned method. 1) Citric acid: Citric acid is extracted from apples. It has the ability to dissolve clusters of minerals such as Ca and Mg. An ideal and safe way of cleaning water production appliances is to circulate citric acid through the appliance.
7. What is calcium addition?
1) Astringent effect: Acidic water contains a small amount of chlorine ions and has an astringent effect. This water is good for toning your skin. 2) Calcium glycerophosphate (Calcium powder): If the pH level does not increase even after electrolyzing water (especially when using well water or tap water in areas toward the Sea of Japan), adding calcium powder will increases the pH level.
8. How many years does the electrolysis cell remain effective?
3) Control PCB (Print Circuit Board): A board comprising print circuits that manage various data related to the appliance. Hi-tech components/condenser also built in.
4) ORP (Oxidation Reduction Potential): Electrical potential needed to reduce or slow down rusting and decay (oxidization). This process is also known as deoxidization. ORP is measured in terms of negative mVs. The ideal ORP level for the human body is said to be between -150mV and -400mV.
9. How do you use the reset switch?
10. What is beauty water (acidic water) and strong acidic water?
3) Astringent: Acidic water works as an astringent for toning you skin.
4) Astringent water: Refers to acidic beauty water. Has an astringent effect.
11. How does the automatic draining system work?
12. Some white bits are floating in the Kangen Water™. What are they?
1) Caustic lime: Okinawa is said to exist on top of coral. As a result, the water in Okinawa has a high mineral content.
2) Pre-filter: A filter used in situations where the original water supply contains a high percentage of well water or chemical substance. The transparent filter makes it easy to tell when the cartridge needs changing.
3) Anodized aluminum: An aluminum alloy often used in household pots.
13. Can the LeveLuk be used overseas?
14. How do I use the spout stand?
15. What does the five-year warranty specifically cover?
16. Why does the SD-501 only have one filter while some of the other top brand have multiple filters?
17. How many electrodes are installed in the SD-501, and what is the measurement of the surface area of these electrodes?
18. What is the expected electrode effective life after which the ionizer cannot attain the expected pH & ORP levels?
19. What is the cleaning cycle frequency and duration for the SD-501?
4) Minerals: Ca (calcium), Mg (Magnesium), Na (Sodium), Ka (Potassium) and other nutrients found in food and water. Minerals found in water are said to be especially good for the health in optimum amounts.
20. Is the flow control on the SD 501 electronic or manual?
21. In the event of a SD-501 requires servicing, can the ionizer be fully serviced and repaired in the U.S.?
22. Is the service and/or repair done at the dealer's expense?
23. What are the electrodes of each unit composed of?
24. Is the LeveLuk electrolyzing tank different from competing models?
See Which machine is right for you?


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Tuesday, July 26, 2011
A good start
I am getting enough hexies made now that I can start putting them together. I decided to use #5 pearl cooton and the daisy stitch for my filler hexies. The backstitch took too long and didn't stand out enough for me. I am having so much fun with this and do enjoy the photos and posts.
5 comments:
- Sue Pickwell said...
These look lovely, great job. When its all together it'll look lovely! I like the fillers using the Daisy stitch....
- July 26, 2011 at 4:51 AM
- Maria said...
What a wonderful start and the fillers look great
- July 26, 2011 at 10:00 AM
Looking great and I love the daisy stitch for the fillers too
- July 27, 2011 at 6:47 AM
- Samelia's Mum said...
So lovely! I"m yet to begin stitching my hexies. I like the idea of lazy daisy flowers.
- July 27, 2011 at 2:33 PM
- Deb R said...
I havent as yet started to stitch my filler flowers..but Im wiht you, I think your way will be quicker and stand out more..
they look great!!!
- July 28, 2011 at 12:08 PM
Subscribe to: Post Comments (Atom)


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Executive Chef Todd Lesakowski
Todd Lesakowski, Executive Chef of William K's Restaurant is a native of Buffalo who recently returned to the WNY area after residing on the east coast, working under many of the great chef's of New York City, Boston, MA and Providence, Rhode Island.
He began his love for cooking at an early age, working at his family's restaurant. He left Buffalo to further his career and moved to NYC. His portfolio includes working at the Gotham Bar and Grille under Executive Chef Alfred Portale as well as notable chef's Marco Morrisera, Ming Tsai, Ruth Rogers, Tony Maws, Chris Coombs, Adrienne Mosier and Oliver Maillard. His passion for food is evident in this creative cuisine and his many talents are reflected in his dishes- both in flavors and his presentation; each dish a work of art.


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|"Welcome and Thank You for stopping by my website.
My name is Mark A'Brial. I'm the owner of A'Brial's
Antiques and have been in business since 1993. We are
always looking to purchase quality items. We are
interested in antiques, classic furniture, sterling,
glassware, pottery, porcelain, old toys, jewelry, art
work, and sets of china. In addition, items from the Arts
& Crafts Movement, Art Deco Period, and various
collectibles are desirable. We will buy one item or an
entire estate.


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Horsemeat in Tesco burgers case 'may lead to prosecutions'
Criminal prosecutions may take place after horse and pig meat were found in beefburgers, a minister has said.
Food minister David Heath told MPs that the contamination was "almost certainly a matter of criminality".
Horse and pig DNA has been found in some burgers sold by Tesco, Iceland, Lidl and Aldi and Dunnes.
On Thursday, Tesco took out adverts in British newspapers apologising for the matter and promising to investigate.
In its advertisement, which has the headline "We Apologise", Tesco says: "We and our supplier have let you down and we apologise.
"So here's our promise. We will find out exactly what happened and, when we do, we'll come back and tell you.
"And we will work harder than ever with all our suppliers to make sure this never happens again."
'Culturally taboo'
Mr Heath made a statement in response to an emergency question to the Commons by shadow environment secretary Mary Creagh.
He told the house the Food Standards Agency had set up a four-point action plan in response to the contamination, which was originally discovered in Ireland earlier this week.
The agency was to carry out an urgent review of the traceability of food products, which was to be completed by Friday he said.
It was also looking into the methodology of detecting contaminants, reviewing whether legal action was appropriate and taking an overview of how it worked alongside other agencies.
Ms Creagh said there was "understandable" consumer anger.
"Consumers who avoid pork for religious reasons will be upset they may have unwittingly eaten it and eating horse is strongly culturally taboo in the United Kingdom.
"The food industry lobbies vigorously for a light-touch regulation system from government. Testing, tracking and tracing ingredients is expensive but not testing will cost retailers, processors, British farmers and consumers much more."
The shadow minister said the axing of 700 trading standards officers in job cuts and a £12m reduction in meat inspection budgets could make food fraud more widespread.
Labour MPs called on the government to reverse the policy.
Mr Heath told Ms Creagh: "It is very important neither you, nor anyone else in this House, talks down the British food industry at a time when the standards in that industry are of a very high level.
"Because something has been discovered in Ireland, which is serious, which may lead to criminal proceedings, does not undermine the very serious efforts which are taken by retailers, by processors and by producers in this country to ensure traceability and ensure standards of food that are available to consumers."
He added that there manufacturers had a responsibility to establish the provenance of the ingredients in the food they offered.
"This [the beefburger case] is probably a matter of criminality, it's been detected and it's being dealt with, but it's quite wrong to extrapolate from that and say such matters are commonplace within the food industry when they are not."
Cuts to the Food Standards Agency should not compromise its work, he continued.
On Tuesday, it emerged Irish food inspectors had found almost 30% horsemeat in one brand sold by Tesco. Smaller amounts of horsemeat as well as pig DNA were found in other products.
A total of 27 burger products were analysed, with 10 of them containing traces of horse DNA and 23 containing pig DNA.
In addition, 31 beef meal products, including cottage pie, beef curry pie and lasagne, were analysed, of which 21 tested positive for pig DNA.
Irish food inspectors have identified two plants in the Republic and one in Yorkshire where the contaminated meat was processed.
Mr Heath said it had probably originated in a "third party country".
The products identified, which pose no risk to public health, have been withdrawn from supermarket shelves.