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The Post-Partum Products I Can’t Live Without

Your post-partum experience can be a good one. Find the right products and it can take you a long way.

It’s a pretty popular thing to buy items that will help you have a more comfortable pregnancy experience, whether that’s clothes, undergarments, accessories, you name it! While many choose to invest in items during their pregnancy, which they can never use again, I chose the opposite. I wanted to ensure I could find items that would allow me to get back to myself. During my pregnancy, I tried really hard to stay present when it came to finding products to truly help me have a smooth post-partum transition after giving birth. After much research (and scrolling on Instagram), I packaged together the best items that have truly been helping me throughout my recovery period, going back as far as using in the hospital!

The Honey Pot Post-Partum Pads

This is my 2nd time giving birth, so nothing really came as a surprise to me – except these pads! The post-partum pads they give you in the hospital are an ode to everything you remember pads being back in the 1990’s. At least for me. The pads are large and in charge, but they do the job! So when I saw The Honey Pot offered post-partum pads and saw how small the packaging was, I was skeptical! HOW ON EARTH will these cute, thin pads hold anything?! And do they know how long they need to be to cover EVERYTHING? Nevertheless, I jumped in & bought em because I love the products, so I knew these couldn’t be an epic fail.

In the hospital, I used the big pads. I felt secure, safe, and as uncomfortable as they come. But I felt secure. Then I decided to grab one from the Honey Pot. OMG! The length was perfect. They were thin! It felt cool when you put it on – because childbirth can be a little rough on your lady parts. It was perfect! Since then, I’ve worn them every single day with no accidents, no mishaps, and dare I say – NO CHEMICALS! Please take it from me: these are a WINNER!!!

The Honey Pot Mommy-to-Be Wash

Of course I had to follow up with the wash, which is bomb! Skip the “Winter’s Adam” wash you buy at Target and grab this! I always feel so fresh and clean after each wash and it doesn’t take ten zillion hormone-altering chemicals to get you feel that way. This is also a WINNER!

Young Living’s Rose Ointment

Rose ointment: how I love thee? Let me count the ways! I am back on the breastfeeding train and I will never forget trying to get Laila going last go round. My nipples were cracked & bled for weeks and finally they turned pink from being so raw. Let that marinade for a second. RAW! My motto is “Lanolin is a lie.” That cream was always super thick and hurt like no other putting it on. Not to mention I had to add a child who latched like an alligator onto it. Enter: rose ointment.

This ointment is like a Swiss Army Knife for all things dry, chapped, cracked, etc. I used this on my nipples initially after every feeding and when she was asleep to give myself some relief. Today, I am 3 weeks post-partum with no cracking, bleeding, and matter of fact: latching on doesn’t feel like Jaws is clamping down each time. Winner!

Nature Bond Silicone Manual Breastmilk Saver

WHAT?! I owe dinner to whomever invented this contraption here! Girl! When I tell you this little pump has saved me so much milk while breastfeeding! It’s insane.

While nursing on one side, I use this pump to suction out the milk that’s letting down on the other. In the past, I would just let myself leak until it was time to nurse on that side. Today, I am pumping about 3 oz. of milk at a time while feeding. When nursing, every single drop counts!

The Bellefit Corset

One of the downsides of pregnancy is the fact that the muscles in your stomach can separate due to a growing belly. Since I knew I was having a c-section, not to mention a big baby, I decided to find a band this time that I can truly wear to help bring everything back together. Not to have a perfect waistline, but to make sure that everything goes back to its proper place.

I didn’t get a chance to wear mine until the 2nd week, post-partum, but it has been a gem to wear! Nothing crazy, easy to get off to use the bathroom, and it’s keeping everything tucked and tight, holding everything in place until it stays there. After spending way too much money after my first time giving birth, this corset is the one and only purchase I needed! It has been the best!

Glossier Cloud Paint & Boy Brow

Okay, I know – these aren’t post-partum products, but they sure have been essential in my post-partum journey. Anything that keeps me from looking like Michael Jackson in the middle of the street dancing with the rest of the ghouls and goblins in the graveyard is worth mentioning in my book.

I am in LOVE with both of these. I can easily put on my foundation, add a little cheek color and make sure my brows are filled in, all without any brushes or formal equipment. It’s the perfect toss on and go products that every cool girl needs in her bag!

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Open Access

The tyrosyl-tRNA synthetase like gene located in the tyramine biosynthesis cluster of Enterococcus duransis transcriptionally regulated by tyrosine concentration and extracellular pH

  • Daniel M Linares1,
  • Maria Fernández1,
  • Beatriz Del-Río1,
  • Victor Ladero1,
  • Maria Cruz Martin1 and
  • Miguel A Alvarez1Email author
BMC Microbiology201212:23

Received: 27 July 2011

Accepted: 14 February 2012

Published: 14 February 2012

The tyramine producer Enterococcus durans IPLA655 contains all the necessary genes for tyramine biosynthesis, grouped in the TDC cluster. This cluster includes tyrS, an aminoacyl-tRNA synthetase like gene.

This work shows that tyrS was maximally transcribed in absence of tyrosine at acidic pH, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Mapping of the tyrS transcriptional start site revealed an unusually long untranslated leader region of 322 bp, which displays the typical features of the T box transcriptional attenuation mechanism. The tyrosine concentration regulation of tyrS was found to be mediated by a transcription antitermination system, whereas the specific induction at acidic pH was regulated at transcription initiation level.

The expression of the tyrS gene present in the TDC cluster of E. durans is transcriptionally regulated by tyrosine concentration and extracelular pH. The regulation is mediated by both an antitermination system and the promoter itself.

Biogenic AmineTyramineLeader RegionTyrosine ConcentrationtyrS Gene

Biogenic amines (BA) are low molecular weight organic bases present in a wide range of food products where they become organoleptically undesirable [1]. It is also worth noting that several toxicological problems resulting from the ingestion of food containing large amounts of BA have been described [2]. Although there is no specific legislation regarding BA content in many food products, it is generally assumed that they should not be allowed to accumulate [35].

Fermented foods are likely to contain high levels of BA, mainly due to the decarboxylase activity of some lactic acid bacteria (LAB). BA are produced by the decarboxylation of a precursor amino acid by the enzymatic action of an amino acid decarboxylase [6, 7]. In these foods, the main BA are tyramine, histamine, cadaverine and putrescine, which are produced by decarboxylation of tyrosine, histidine, lysine and ornithine, respectively [8].

The presence of the genes encoding the amino acid decarboxylase and the amino acid-amine antiporter is a general feature observed in all the gene clusters involved in the biosynthesis of tyramine, histamine, putrescine and cadaverine [912]. We have found an open reading frame coding for a protein of 418 amino acids with a molar mass of 47.38 kDa located next to the tyrosine decarboxylase (tdcA) and the tyrosine-tyramine antiporter (tyrP) genes of Enterococcus durans IPLA655. The predicted amino acid sequence shares strong similarity to the tyrosyl-tRNA synthetase genes (tyrS) of gram positive bacteria. The aminoacyl-tRNA synthetases catalyze the covalent attachment of amino acids to their cognate tRNAs, a crucial reaction for the accuracy of protein synthesis. These enzymes are encoded by genes regulated strictly by antitermination systems; when the corresponding amino acid, tyrosine in this case, is at low concentration, it is not linked to the tRNA, and this uncharged tRNA interact with the antiterminator located between the promoter and the start codon, stabilizing it and allowing transcription. By contrast, when tyrosine is at high concentration, it is linked to the corresponding tRNA (charged tRNA) that cannot stabilize the antiterminator, and consequently the transcription stops [13]. Genes with high similarity to those encoding the corresponding aminoacyl-tRNA-synthetases are generally associated to the genes involved in BA biosynthesis in other species, in the case of tyramine (Enterococcus faecalis JH2-2 [14], Enterococcus faecalis V583 [15], Enterococcus faecium MV5 (GenBank HM921050), Enterococcus hirae (GenBank AY303667), Carnobacterium divergens 508 [16]), Lactobacillus brevis IOEB9809 [17], Lactobacillus brevis ATCC367 (GenBank NC_008497), and the histamine (Lactobacillus buchneri B301 [10], Lactobacillus hilgardii 0006 [18]).

The number of tyrS genes is heterogeneous within the genera Enterococcus. Some tyramine-producing species as E. faecium have a unique tyrS, whereas E. faecalis has two different tyrS genes [15]. So far, this aspect remains unknown for E. durans (no genomic data are available). Concerning the two different genes encoding tyrosyl-tRNA-sinthetases in E. faecalis V583, tyrS-2 is homologous to the tyrS of other bacteria with a unique tyrS, whereas tyrS-1 is located in the tyramine cluster. Database search revealed that tyrS-1 has higher similarity (> 80%) to other tyrS genes associated to tyramine biosynthesis clusters than to its own tyrS-2 gene located elsewhere in the genome (52%). The presence of two tyrS genes in the genome of a tyramine producing strain suggests that one (tyrS-2) would be implicated in protein biosynthesis, whereas the one linked to TDC cluster (tyrS-1) could be a sensor of the intracellular tyrosine pool to regulate tyrosine decarboxylation [9]. In addition, phylogenetic analyses of TyrS proteins associated to tyramine clusters, supported the hypothesis that these proteins made tight clusters and were clearly separated from their relatives encoded elsewhere in bacterial genomes. These results suggested a co-evolution of tyrS together with tdcA and tyrP. This fact prompted us to exhaustively investigate the transcriptional regulation mechanism of this gene and its putative role on the regulation of the tyramine operon.

tyrSexpression depends on the tyrosine concentration and extracellular pH

RNA from E. durans IPLA655 cultures grown in presence (10 mM) or absence of tyrosine at two different pH conditions (4.9 and 7.5), was analyzed by Northern blot hybridization with a tyrS specific probe (Figure 1A). Very low expression was detected in cells grown at pH 7.5 independently of the presence or absence of tyrosine. Noteworthy, at pH 4.9, an intense band corresponding to a transcript of 1.6 kb was observed. A policystronic mRNA including tyrS-tdcA was never detected [19]. The bigger band present in all lines with a low intensity would correspond to unspecific hybridization to the extremely abundant 23S rRNA molecules. This tyrS up-regulation was specially enhanced in absence of tyrosine, suggesting that the initiation of transcription or mRNA stability is controlled by pH and tyrosine.
Figure 1
Figure 1

Transcriptional analysis of the tyrS gene. A: Northern blot analysis with a tyrS-specific probe and B: RT-qPCR quantification of tyrS mRNA levels in presence (+Y) and absence (-Y) of 10 mM tyrosine and different pH conditions (pH 4.9 and pH 7.5). The asterisk indicates statistically significant difference (p ≤ 0.005; Student's t-test) in comparison to the other conditions. C: Effect of different tyrosine concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 2, 5 and 10 mM) on tyrS expression at pH 4.9

The strength of these environmental conditions on tyrS expression was quantified by RT-qPCR. Data in Figure 1B confirmed that tyrS is maximally transcribed in absence of tyrosine and at pH 4.9, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Even when tyrosine was not added to the media, no induction was detected at pH 7.5. These results confirmed that both conditions (acidic pH and absence of tyrosine) are needed for expression of tyrS gene.

Next, we examined whether intermediate tyrosine concentrations have an effect on tyrS expression. Therefore, we investigated at optimal pH 4.9, the effect that different tyrosine concentrations in the media (0, 0.01, 0.05, 0.1, 0.5, 1, 2, 5 and 10 mM) exert on gene expression by comparing RT-qPCR results obtained in each condition. As indicated in Figure 1C, tyrS expression showed an inverse correlation with the increased tyrosine concentration and exhibited a great sensitivity to very low tyrosine levels, since the maximal expression level was reached in absence of tyrosine and an increase of 0.01 mM tyrosine in the media was enough to reduce this level to the half. Not significant changes in transcription were observed above 2 mM tyrosine, probably because of saturating concentrations of tyrosine. Such concentrations were assayed because tyrosine can reach very high concentrations in some cheeses and even precipitate forming crystals [20].

Mapping of the tyrStranscription initiation site

To map the precise start point of the transcription of tyrS, primer extension was performed using RNA samples extracted under optimal conditions of expression (pH 4.9 and absence of tyrosine).

A single band of 322 bp was observed, indicating that the position +1 of the mRNA corresponds to a T residue located 322 nucleotides upstream of the ATG codon (Figure 2). Seven nucleotides upstream this point, it was localized the -10 sequence TATGAT spaced 17 nucleotides downstream of the -35 sequence TTGACA, that nearly matched the consensus sequence for LAB promoters [21]. In a position 9-14 nucleotides upstream the ATG codon of this gene, it was identified the Shine-Dalgarno region (CGGAGG) (bases fitting with the consensus sequences are underlined).
Figure 2
Figure 2

Primer extension identification of transcription start site (*) of tyrS and transcriptional regions -10 and -35 (boxes). Lines T, G, C and A correspond to DNA sequence reaction (note that the nucleotide sequences, 5' at the top and 3' at the bottom, represent the complementary DNA strand, so that it may be read directly)

Structural features of the tyrSleader region

In E. durans IPLA655, the region upstream of the start codon of tyrS showed a 322 bp noncoding sequence that was named the tyrS leader region. A hypothetical representation of the secondary structure of the tyrS leader region is plotted on Figure 3. This region exhibits the sequence features of the tRNA-mediated antitermination systems described by Grundy et al. [22]. It contains the typical T box sequence UGGGUGGUACCGCG (nucleotides 187-200) (bases fitting with the consensus are underlined), a tyrosine specifier UAC (nucleotides 104-106), and most of the other less conserved boxes (AGUA-I box [AGUA, nucleotides 34-37], GA box [AGAAAG, nucleotides 58-63], GNUG box [GCUG, nucleotides 73-76], and F box [GCGUUA, nucleotides 142-147]). In addition to these conserved sequences, the tyrS leader region may be folded into three stem-loop structures (I, II and III) preceding a factor-independent transcriptional terminator/antiterminator. However, the AGUA-II and GAAC boxes that can be found in similar antitermination systems are not present.
Figure 3
Figure 3

Primary-sequence and structural model of the E. durans IPLA655 tyrS mRNA leader region upstream the start of the coding region. The specifier (UAC), the Tbox sequence, and other highly-conserved motifs typical of genes regulated by tRNA-mediated antitermination appear highlighted in boxes. Sequence between arrows can adopt two alternative mutually exclusive structure conformations: terminator and antiterminator (stabilized by the cognate tRNA in absence of tyrosine). A transcriptional fusion of the tyrS promoter and the leader region with a deletion of the TBox-Terminator region (PtyrS Δ ) was made (dashed line) to probe the role of the Tbox in the mechanism of tyrosine sensing

Tyrosine concentration sensing is mediated by an antitermination system

We investigated whether the conserved primary sequence and structural motifs located upstream the start of the coding sequence play a role in the regulation of tyrS expression by a transcription antitermination system. For this purpose we compared the amount of mRNA specific of the leader region (mRNA-L) and the amount of mRNA corresponding to the coding part of the gene (mRNA-C) under optimal expression condition (pH 4.9), and in presence or absence of tyrosine. This region-specific transcriptional quantification was performed by RT-qPCR using specific primer pairs for each region (see Methods).

As shown in Figure 4A, level of mRNA-L was not affected by tyrosine concentration, whereas mRNA-C level did not follow the same profile. In presence of tyrosine, the ratio mRNA-L/mRNA-C was 4.2, whereas this value decreased to 1.2 in absence of tyrosine (optimal conditions for tyrS expression). The ratio close to 1 observed in absence of tyrosine indicates no transcription termination and consequently the expression of tyrS. The 4.2 ratio indicates higher levels of mRNA-L than mRNA-C, suggesting that the transcription in the presence of tyrosine was under the control of a termination system. These data are coherent with a tyrosine concentration regulation of tyrS mediated by a transcription antitermination system.
Figure 4
Figure 4

Regulatory effect of the Tbox on tyrS expression. A: Quantification of tyrS mRNA-C (in black) and mRNA-L (in white) levels at pH 4.9 in presence (+Y) and absence (-Y) of 10 mM tyrosine. Numbers above indicate the ratio mRNA-L/mRNA-C in the corresponding condition. B: Effect of Tbox deletion on β-Galactosidase activity of PtyrS Δ -lacZ fusions at different conditions of pH and presence/absence of 10 mM tyrosine (Y). Data represent the average of three independent experiments. The higher activity observed at pH 4.9 (asterisks) was statistically significant (p < 0.005; Student's t-test) in comparison to that at pH 7.5

Assessment of PtyrSΔactivity

The role of the T box in the mechanism of tyrosine sensing by tyrS was analyzed using a transcriptional fusion of lacZ reporter gene with the tyrS promoter and the leader region, but with a deletion of the T box-Terminator motif (PtyrS Δ ) (Figure 4B). The lacZ activities under the control of PtyrS Δ at pH 4.9 were similar in the absence (33.8 mmol/mg total protein/min) and presence (31.5 mmol/mg total protein/min) of tyrosine, confirming that tyrosine regulation is located on the T box region. On the other hand, independently of the presence of tyrosine, promoter activities at neutral pH were lower than 5 mmol/mg total protein/min, showing an 8-fold higher strength of PtyrS Δ under acidic pH than at neutral pH. These data indicate that the induction of tyrS expression by pH is transcriptionally regulated by the promoter.

Putative role of tyrSin tyramine cluster

To test the hypothesis that TyrS plays a physiological role on tyramine biosynthesis and/or in the regulation of the related genes (tdcA and tyrP), tyrS was over-expressed under the control of the nisin promoter. In all cases, the concentration of tyrS transcripts (assessed by RT-qPCR) was 80-fold over the physiological expression level. The presence of soluble translated TyrS was tested by Anti-HIS immunodetection. An intense band of expected size was observed under induction conditions. Next, we analyzed the in vivo effect of the over-expression of tyrS in cells grown on the aforementioned conditions, (pH 4.9 in GM17-Y and GM17 + Y media). Negative controls of uninduced cultures were carried in parallel. Under these experimental conditions, level of tdcA-specific mRNA (quantified by RT-qPCR) was not affected by the overexpression of tyrS (data not shown). In addition, the concentration of tyramine in supernatants was examined by HPLC. Only the expected differences depending on the tyrosine concentration in the media were observed (260 ± 40 μM and 3100 ± 80 μM in GM17-Y and GM17 + Y cultures, respectively), but no significant differences between tyrS-induced cultures and the negative control were observed.

The E. durans IPLA655 tyrS gene present in the tyramine cluster, which encodes a protein with high similarity to tyrosyl-tRNA-synthetases, is controlled by the intracellular concentration of tyrosine in a similar way than aminoacyl-tRNA synthetases are regulated by their substrate amino acid. The analysis of TyrS sequence revealed the typical HIGH and KMSKS domains of class I aminoacyl tRNA synthetases, being the HIGH motif perfectly conserved, and the KMSKS motif is represented by the KFGKT sequence, as in E. coli [23], Bacillus subtilis [24], and E. faecalis [14]. Mapping of the transcriptional start site revealed a long untranslated leader region of 322 bp with a highly conserved set of primary-sequence and secondary structure elements. These elements include three stem-loop structures, a highly conserved 14-bp sequence designated the T box, and a factor-independent transcriptional terminator (Figure 3). These features are also present in other genes of gram positive bacteria, mainly genes encoding aminoacyl-tRNA synthetases, but also amino acid biosynthetic genes and transporters [2527]. Several studies have revealed a crucial role for conserved leader region motifs in regulation of gene expression at the level of premature termination of transcription [28]. In order to test whether this mechanism regulates the tyrS gene of E. durans TDC cluster, the levels of mRNA were quantified using specific primers for the leader and coding region of tyrS. When E. durans was starved for tyrosine, the predominant transcript was a 1.6 kb mRNA fragment, which is the expected size for full-length mRNA (mRNA-C). Interestingly, when tyrosine was present in excess, full-length mRNA was dramatically depleted, whereas the truncated mRNA-L species kept almost constant. Thus, tyrosine had no effect on the total number of mRNA-L molecules but caused a stoichiometric replacement of full-length mRNA by truncated RNA molecules. These data are consistent with the idea that tyrosine controls tyrS expression by promoting the premature termination of transcription rather than by inhibiting the initiation of transcription.

Experiments involving transcriptional fusions of the tyrS promoter with ß-galactosidase provided evidence for this mechanism. We showed that deletion of the T box-Terminator domain of the leader region originates a complete lost of regulation by tyrosine. Early termination at pH 4.9 in presence of tyrosine observed in vivo in the leader tyrS mRNA (which shows that this sequence promotes terminator formation specifically in presence of tyrosine) was not observed for the PtyrS Δ promoter. This effect can be expected because the T box sequence is present in a side bulge of the antiterminator overlapping the terminator-antiterminator structures.

In addition to the tyrosine regulation, transcription of tyrS is under strict pH control in E. durans, being expressed mostly at acidic growth conditions. The aminoacyl-tRNA synthetases catalyze the covalent attachment of amino acids to their cognate tRNAs. Incorporation of tyrosine to proteins is an essential function for cell life, which should not be restricted to acidic conditions. One explanation for this would be the presence of two different tyrosyl-tRNA synthetases, one of which would be induced under stress conditions (acidic pH and extremely low tyrosine concentration). In general, there is only one aminoacyl-tRNA-synthetase for each amino acid in most bacteria, however, several exceptions are known. Indeed, two very similar lysyl-tRNA synthetases, lysS (constitutive) and lysU (heat inducible) have been described in Escherichia coli [29]. In gram-positives, in addition to the aforementioned case of the two tyrS of E. faecalis, there are two distinct histidyl-tRNA synthetase genes in Lactococcus lactis [30], and two tyrosyl-tRNA synthetase genes (tyrS and tyrZ) and two threonyl-tRNA synthetase genes (thrS and thrZ) in Bacillus subtilis [31, 32]. In this last case, the normally silent thrZ gene is induced during threonine starvation or by reducing the intracellular concentration of ThrS, which is the housekeeping threonyl-tRNA synthetase sufficient for normal cell growth [33].

The location of genes encoding an aminoacyl-tRNA-synthetase associated to the gene clusters involved in tyramine and histamine biosynthesis is a general feature [9, 10, 14, 1618, 34]. One of the reasons to study the expression of tyrS in E. durans is to find out whether this protein could have a role on the genetic regulation of the tyramine cluster, being activated under limiting levels of tyrosine to prevent massive decarboxylation of this amino acid, ensuring its availability for protein synthesis. Consistent with this idea would be 1) the common location of genes encoding aminoacyl-tRNA sinthetases next to the operon of decarboxylation (BA-biosynthesis) of the corresponding aminoacid [9, 10, 14, 1618, 34], 2) the expression of this gene only under acidic pH, which is the condition regulating positively the biosynthesis and accumulation of tyramine [19, 35] and 3) the fact that tyrS and the genes of the tyramine biosynthesis pathway (tdcA and tyrP) require opposite conditions of tyrosine concentration for optimal expression (Figure 5) [19]. Altogether, these data raise the question whether TyrS could act as a negative regulator. However, overexpression of tyrS on multicopy plasmid during growth of the E. durans strain carrying the wild-type allele had no observable effect on the expression profile of the decarboxylating gene tdcA or on the tyramine concentration observed in the supernatant.
Figure 5
Figure 5

Genetic organization and transcriptional profile of the TDC cluster in E. durans IPLA655. Promoters (P) and termination regions are indicated. The different mRNA are represented by wavy lines. Numbers indicate the size of the corresponding gene in base pairs (bp). Regulation of the genes by tyrosine and pH is indicated below. Acidic pH is required for optimal expression of the three genes. In addition, tdcA and tyrP are positively regulated by 10 mM tyrosine (+Y), whereas expression of tyrS is negatively affected by tyrosine (+: positively regulated; -: negatively regulated; tyrS: tyrosyl-tRNA synthetase; tdcA: tyrosine decarboxylase; tyrP: tyrosine-tyramine antiporter). Structures upstream tyrS represent the stems I, II, III and terminator of the leader region. The terminator/antiterminator mechanism that regulates the tyrS gene is also indicated: readthrough of the leader region is induced by limitation of tyrosine. Uncharged tyrtRNA stabilize formation of antiterminator structure in the mRNA, which prevents terminator formation (SD: Shine-Dalgarno; ORF: open reading frame of tyrS)

Computational three-dimensional modelling of E. durans TyrS protein revealed nucleic acids-binding domains that might suggest a role as transcriptional regulator. However, the same domains have been identified in the highly similar TyrS structure of Thermus thermophilus (Protein Data Bank: 1H3E), and predicted to interact with tRNA (Figure 6). This data is consistent with the electrophoretic mobility shift (EMSA) assays carried to test TyrS binding to the promoters of the TDC operon. Under the wide range of conditions studied (different pH, salt concentration, presence or absence of tyrosine...) no specific binding of TyrS was observed (data not shown). These data, together with the finding of tyramine clusters without a tyrS gene in Tetragenococcus halophilus (GenBank AB059363) and histamine biosynthesis clusters without a hisS gene [36], would suggest a non critical biological function of these genes in the modulation of the contiguous decarboxylation operon. In any case, it can not be discarded that tyrS could exert a post-transcriptional regulation of tyramine biosynthesis. In fact, both enzymes -TyrS and TdcA- share tyrosine as substrate.
Figure 6
Figure 6

TyrS structural model achieved using Swiss-Pdb Viewer v. 4.04 software and structure superposition onto the highly similar Thermus terhmophilus tyrosyl-tRNA synthetase. (Protein Data Bank: 1H3E). 1H3E is shown in green, and TyrS model is shown in magenta and yellow. Analysis of the two aligned structures indicates that all of the DNA/RNA binding sites are in regions that interact with tRNA in the 1H3E structure (shown in blue).

Consequently, two are the possibilities that can be considered: i) there are two tyrS genes in E. durans -as described for E. faecalis- and the one ligated to TDC would be a stress-related gene to ensure sufficient charged TyrtRNA for protein biosynthesis in those conditions that tyrosine is being decarboxylated, or ii) this is the unique tyrS gene and the low expression levels observed under neutral pH conditions are enough to assure protein synthesis for general metabolism and the increased expression at acidic pH would guarantee protein biosynthesis when tyrosine is being decarboxylated. The presence of a second tyrS gene was investigated by Southern hybridizations of E. durans IPLA655 total DNA using as probes the tyrS gene of its own tyramine cluster and also tyrS2 from E. faecalis V583 (the one that is not linked to the tyramine cluster), and no additional tyrS genes were found (data not shown). Consistent with this would be the impossibility to knockout the tyrS gene, which would support the hypothesis of a single tyrS, therefore essential. Then, the most plausible model is the presence of a unique tyrS which exhibits a severe induction under acidic pH and low expression levels under neutral pH. It is important to highlight that the transcription quantification is relative, all the expression values were standardized to the reference condition, in this case pH 7.5 (Figure 1B). In fact, the signal observed by Northern blot (Figure 1A) is not zero in any of tested conditions. The tyrS basal expression that takes place at neutral pH would be enough to assure protein synthesis.

In this paper, we provide evidence that transcription of the E. durans IPLA655 tyrS gene is controlled at two levels, initiation and elongation. The initiation of transcription was shown to be enhanced in acidic environments, whereas elongation of transcription is subject to a tyrosine-dependent attenuation mechanism related to the T box system. This dual mechanism has thus never been described, since previously reported genes induced by tRNA-mediated antitermination, were not found to be pH dependent.

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in Table 1. Escherichia coli TOP10 (Invitrogen A/S, Taastrup, Denmark) and Lactococcus lactis NZ9000 were used for plasmid propagation. E. coli cells were grown in aeration in Luria-Bertani medium at 37°C. L. lactis and E. durans strains were grown at 30°C on M17 medium (Oxoid, Hampshire, United Kingdom) supplemented with 0.5% glucose (GM17) containing 10 mM tyrosine (GM17 + Y), or on GM17 without tyrosine (GM17-Y). The media was previously adjusted to the corresponding pH condition (pH 4.9 or pH 7.5). When needed, erythromycin (5 μg ml-1 for E. durans and L. lactis), ampicillin (100 μg ml-1 for E. coli) or chloramphenicol (5 μg ml-1 for E. durans and L. lactis) were added to the culture media.
Table 1

Strains and plasmids used in this study

Characteristics *

E. coli TOP10

L. lactis NZ9000

Plasmid-free strain

[37, 38]

E. durans IPLA655

Isolated from artisanal cheese. Tyramine producer

IPLA Collection

EryR, nisR-nisK

EryR, lacZ promoterless gene

CmR, expression vector

AmpR, pUC18 with PtyrS cloned in SmaI

This work

AmpR, pUC18 with PtyrS Δ cloned in SmaI

This work

EryR, pILORI4 including PtyrS Δ -lacZ fusion

This work

CmR, pNZcLIC including tyrS

This work

* AmpR, ampicillin resistant; EryR, erythromycin resistant; CmR, chloramphenicol resistant

DNA manipulation procedures

Procedures for DNA manipulation, transformation of E. coli cells, and recombinant techniques used were essentially those described by Sambrook et al. [43]. Large-scale isolation of E. coli plasmids for nucleotide sequence analysis was performed with the Plasmid Midi Kit (Qiagen Ltd., Crawley, United Kingdom) according to the manufacturer's instructions. Constructions for E. durans were achieved using L. lactis NZ9000 as intermediate host. Plasmid and chromosomal DNA of E. durans and L. lactis were isolated and transformed as described previously [44]. All enzymes for DNA technology were used according to the manufacturer's specifications. DNA hybridizations were performed using the non-radioactive DNA Labelling and Detection Kit (Roche Molecular Biochemicals) following the manufacturer's instructions.

RNA manipulation and northern blot analysis of tyrStranscripts

Total RNA was isolated from cells of E. durans IPLA655 grown in GM17-Y and GM17 + Y at pH 4.9 and pH 7.5 to exponential phase (optical density at 600 nm [OD600] of 0.6). Purified RNAs were resuspended in DEPC 0.1% (diethyl pyrocarbonate) treated water, and total concentration and yield were determined by UV spectrophotometry by measuring absorbance at 260 nm using a BioPhotometer (Eppendorf, NY).

After extraction, RNA samples were treated with DNase (Fermentas, Vilnius, Lithuania), as described by the manufacturer, to eliminate any genomic contaminations. 20 μg of each sample were subjected to electrophoresis through a 1.5% agarose gel containing 5% formaldehyde and 1X MOPS buffer [20 mM 3-N-morfolino-propanesulfonic acid (MOPS), 1 mM EDTA, 5 mM sodium acetate; pH 7.0]. Transfers and hybridizations were performed as described by Sambrook et al. [43]. DNA probes were labeled with [α-32P]dATP by nick translation with the DNA polimerase/DNase I (Invitrogen A/S, Taastrup, Denmark). Primers used in the PCR-amplification of the probes are summarized in Table 2.
Table 2

Oligonucleotides used in this study

Sequence (5' to 3')

tyrS probe amplification (F)

tyrS probe amplification (R)

TyrS prom (F)

tyrS prom (R)

mRNA-C quantification (F)

mRNA-C quantification (R)

mRNA-L quantification (F)

mRNA-L quantification (R)

PtyrS mapping (R)

PtyrS Δ cloning and sequencing (F)

PtyrS Δ cloning and sequencing (R)

tyrS overexpression (F)

tyrS overexpression (R)

*F, forward; R, reverse

Quantification of tyrSexpression by reverse transcription quantitative PCR (RT-qPCR)

Gene expression analysis was carried out by RT-q PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using SYBR® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA). cDNA samples were synthesized from total RNA using the iScript™ cDNA Synthesis kit (Bio-Rad, Hercules, CA). After dilution of cDNA, 5 μl were added to 20 μl of PCR mixture (12.5 μl of SYBR Green Supermix, 1 μL of each primer at 7 μM, 5.5 μl of RNAse free water). Amplifications were performed with specific primers (Table 2) designed using Primer Express software (Applied Biosystems, Carlsbad, CA), using primers for 16S ribosomal RNA as an internal control to normalize RNA concentration [19]. Cycling settings were those default-established by Applied Biosystems, Carlsbad, CA. For each condition, RT-qPCR analysis was performed on RNA purified from three independently grown cultures.

Primer extension and DNA sequencing

Total RNA isolated from E. durans IPLA655 grown in GM17-Y pH 4.9 was used as template for primer extension. The reaction was done mixing 15 μg of RNA with 1 pmol of oligonucleotide 5'end-labeled with [γ-32P]-dATP using T4 polynucleotide kinase (New England Biolabs Inc. Ipswich, MA) and a mixture containing deoxynucleotides triphosphate (1 mM each), RNase inhibitor (Gibco, BRL), and 20 U of Avian Myeloblastosis Virus reverse transcriptase (Promega, Madison, USA). The control sequence used as size standard was determined with the same reverse primer using as template 2 μg of the double-stranded recombinant plasmid pDA12 (Table 1). Each sequence reaction was performed according to the dideoxynucleotide chain termination sequencing method using 5 μCi of [α-32P]dCTP and the T7 Sequencing™ Mixes Kit (Pharmacia Biotech Inc. Piscataway) under manufacturer instructions. Finally, the samples were resolved in parallel on a denaturing 8% polyacrylamide gel containing 7 M urea to determine the endpoints of the extension products.

Construction of PtyrSΔ-lacZtranscriptional fusion

A fragment including the tyrS promoter and leader region with a deletion of the T-box-Terminator domain (PtyrS Δ ) (Figure 3), was amplified using primers TDC123 and TDC130 (Table 2) and cloned in the SmaI site of pUC18, resulting pDA15 plasmid. A 450 bp EcoRI-PstI fragment including PtyrS Δ was cloned into the low copy number vector pILORI4 [41] for fusion to a promoterless lacZ reporter gene, yielding the pDA16 plasmid. All clones were sequenced.

Plasmid pDA16 was firstly constructed in L. lactis NZ9000 strain, and subsequently transformed into E. durans IPLA655, and lacZ activity was tested under specific pH and in presence or absence of tyrosine in the culture media. For promoter assay, a negative control with the pILORI4 plasmid was carried.

Determination of β-galactosidase activity

In order to determine β-galactosidase activity, cells grown in the corresponding conditions of pH and tyrosine concentration at OD600 = 0.6 were harvested, resuspended in 1 ml of 10 mM Na2HPO4, pH 7.0, and then disrupted with 0.2 g of glass beads (Sigma). After centrifugation at 12000 g for 10 min at 4°C, different dilutions of the resulting supernatants (CFE) were immediately assayed in a final volume of 1 ml, which included 750 μl of 10 mM sodium phosphate buffer pH 7.0, 200 μl of CFE and 50 μl of 20 mM o-nitrophenilgalactopiranoside (ONPG). The mixture was immediately incubated at 37°C and absorbance was measured (λ = 420 nm). Each condition was assayed independently by triplicate and the values were standardized to protein contents of cell extracts, determined by using the BCA Protein Assay Reagent Kit (Pierce, Rockford, Ill.).

Overexpression of tyrSand immunodetection

The gene encoding for TyrS was amplified using primers TYSF and TYSR (Table 2) and cloned into a pNZcLIC expression vector using the VBEx system [45], yielding the corresponding derivative pNZcTyrS. For detection purposes, a decaHis-tag was added to the C-terminal of the target protein.

tyrS expression was carried using the NICE system [46]. The genes encoding nisR and nisK were introduced in E. durans IPLA655 in the low copy number plasmid pNZ9530 [40]. After induction with 2 μg L-1 nisin, expression of the protein was confirmed by Western blotting analysis of cell lysates by 10% SDS-PAGE electrophoresis gels, subsequently electroblotted and immunodetected with an anti-His-tag antibody (Amersham Pharmacia Biotech Inc. Piscataway). Chemiluminescence detection was done using the Western-Light kit (Tropix Inc. Bedford, MA) and quantified using the Fujifilm LAS-3000 imaging system (Fuji Photo Film Co. Ltd; Tokyo).

Analysis of tyramine by HPLC

The quantitative analysis of tyramine production was undertaken by reverse-phase high performance liquid chromatography (RP-HPLC) using a Waters liquid chromatograph controlled by Millenium 32 Software (Waters, Milford, MA, USA). The samples were prepared by centrifugation at 8,000 × g for 10 min. The resulting supernatants were filtered using Millipore 0.2 μm filters and derivatized using dabsyl chloride, as described by Krause et al. [47]. Separations were performed using a Waters Nova-pack C18 column (150 × 3.9 mm). Usually, 10 μl of the derivatized sample was injected and detection performed at 436 nm. The solvent gradient and detection conditions were similar to those described by Krause et al. [47].

This research was performed with financial support from the Ministry of Science and Innovation, Spain (AGL2010-18430) and the European Community's Seventh Framework Programme (BIAMFOOD-211441). We are grateful to Paloma López for technical assistance with Primer Extension experiments, and Begoña Redruello for experienced support provided for protein modelling and structure alignment. Strain L. lactis NZ9000 and plasmid pNZ9530 were kindly provided by NIZO food research, and plasmids pILORI4 and pNZcLIC were kindly provided by Oscar Kuipers and Bert Poolman, respectively. D. M. Linares is the recipient of a contract from Gobierno del Principado de Asturias. B. del Río is beneficiary of a JAE DOC contract (CSIC).

Authors’ Affiliations

Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Spain

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Daniel and Slavica Trajković. (on wall) Branko Filipović-Filo, ’Painting’, 1986

Interview with Daniel & Slavica Trajkovic

Kolekcija Trajković – Belgrade, Serbia

What was the first piece of artwork you purchased, and when was this?
During the 1970s and 1980s, before we started collecting art, we were regular visitors of museums, galleries and other cultural events in Belgrade during the transition period in Serbia. As is often the case, the step you take from admiring art to the purchasing of the first painting is not a big one. A much bigger step or mystery is how the first artwork, that decorates the house, can become the first of many in your collection. Our first painting was acquired at the end of the 1980s – it was a painting of one of the representatives of the new Belgrade figuration.

Why do you collect?
Whenever someone asks us how we started buying paintings or how we ever decided to create a collection, I would make up an interesting story depending on who asked and what the occasion was. The truth is is that we never knew the real reasons. Of course, all other questions and issues related to the collection are fairly easy to answer – explaining the events that preceded and what and whom had the impact on making our collection and it's conception, why these authors were selected and everything else that has happened over the past thirty years.

Does your collection follow a concept or a specific theme?
From the first moment, our collection had a clearly defined concept with us opting for Serbian art from the second half of the twentieth century. The collection begins with artworks by artists from the 1960s to the end of the twentieth century. Some of the most significant Serbian artists from the new figuration of the 1960s are represented in the collection, as well as representatives of new Conceptual Art from the 1970s and new Post Modernism from the 1980s. The 1990s were represented by artistic practices of artists who created art in a closed society with a group of colleagues from the new Belgrade sculpture. If we consider the group of artists we have, as well as which of their artworks are in our collection, we are certain that our collection transcends the conventional understanding of the meaning of a “private collection” and as a result, is currently the largest private collection in Serbia.

Who are the artists you are currently following?
Dušan Otašević, the painter and member of the Serbian Academy of Science and Art. Over the past years our Foundation has organized three solo exhibitions by this artist, loaned artworks for two group exhibitions abroad and published a large monograph of his entire opus with reproductions and texts from some of our most distinguished art historians. Some of these exhibitions include "Kitchen And Other Affairs" at the Heritage House Belgrade, "Ludwig Goes Pop + The East Side Story" at the Ludwig museum, Budapest and "Monuments Should Not Be Trusted" at Nottingham Contemporary, UK.

Do you have a personal relationship with the artist you collect?
It is not possible to create a large collection without a personal connection to the artists. We loan artworks to many of artists, mostly for their exhibitions and of course help them in any way possible.

Why did you decide to make your collection publicly accessible?
From the first moment our collection was created we had an awareness of the importance of national art as an unprofitable business. We believe that the collection only makes sense if it's accessible to everyone. With that in mind, for several years the main activity of our Foundation is to find an adequate space for the future museum. Despite all the problems we are having we are hopeful for a successful end.

Which publicly accessible private collection would you recommend visiting?
Ours, of course!

More Information On  Kolekcija Trajković

Daniel and Slavica Trajković. (on wall) Branko Filipović-Filo, ’Painting’, 1986
(on wall) Bora Iljovski, ’Personal Escort’, 1981; (on table) Dušan Otašević, ’Drink’, 1967; (on wall, top) Tomislav Kauzlarić, ’Research with a newspaper’, 1979; (on wall, bottom) Kosta Bogdanović, ’Relief from the cycle Vizanteme’, 1989
(l–r) Tomislav Kauzlarić, ’Research with a Newspaper’, 1979; Djordje Ivačković, ’22.II 71’, 1971; Bozica Radjenović, ’Flyer’, 1990; Marija Dragojlović, ’Green Box’, 1988; Zoran Grebenarović, ’Shadow of Golden Totem’, 1988; Dušan Otašević, ’Solving the Puzzle’, 1990; ’Construction’, 1990
(l–r) Radomir Damnjanović, ’Cabins on the Sandy Coast’, 1962; Radomir Damnjanović, ’Quadro’, 1982; Djordje Ivačković, ’11.IV 1983’, 1983; Neša Paripović, ’Angle’, 1969; Bora Iljovski, ’Semi Boot’, 1970
(l–r) Miodrag B. Protić, ’Constellation’, 1976; Branko Filipović-Filo, ’, Painting 48’, 1986; Dušan Otašević, ’Yes sir’, 1966; Petar Omčikus, ’In a Barber Shop’, 1965; ’In a Barber Shop’, 1977
(l–r) Dušan Otašević, ’Liberated Belgrade’, 1996-1997; Predrag Nešković, ’Birds and Man’, 1973; ’Trainer’, 1973; Božica Rađenović, ’Dreamer’, 1990; Dušan Otašević, ’Double Double Coil and Trouble’, 2012; Božica Rađenović, ’Acrophobia’, 1990; Dušan Otašević, ’Eat’, 1967
(l–r) Božica Rađenović, ’Revealing’, 1989; Slobodan Trajković, ’Flag’, 1983; Dušan Otašević, ’Pissing happily, or, Blue Flux Flooding’, 1966

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Sunday, 23 June 2013

Showcase Sunday (#38)

Showcase Sunday is a new weekly meme hosted by Vicky @ Books Biscuits and Tea. We will now be taking part in Showcase Sunday instead of In My Mailbox. If you'd like to join see HERE for more details.

Donna's Showcase:

For Review:

eBooks for Review/Free audio book:
An Endless Summer (Summer, #2) The Raven Boys (Raven Cycle, #1)

Freebies/Bought Kindle books:
A Beautiful Struggle (Beautiful #1) Now or Never Never Too Far (Too Far, #2) Forever Too Far (Too Far, #3)

Thank you to HarperCollins, Mira Ink, Sourcebook Fire, Book Nerd Tours & C.J Duggan

Jess's Showcase:

It's been a while since I had a showcase, so I have a little more than expected! Below is the build up of weeks, not just one week.

e-books downloaded
Forever Too Far (Too Far, #3) Belong To You (Cole, #1) Shadowed (Fated, #3) The Impact of You Forever My Girl (Beaumont, #1) My Everything (Beaumont, #1.5) Entwined with You (Crossfire, #3) Obsession Lost and Found (Lost and Found, #1) Going Under

Books for review:
Camp Boyfriend (Camp Boyfriend, #1) Eversea (Eversea, #1) This Time Around The Trouble With Goodbye (Fairhope, #1)

Phew! That's a lot of books. Hope you've all had a great week too!

1 comment:

Ner (Cup of Coffee) said...

I've been wanting to read the Study series for such a long time. Hope you'll enjoy it :)

In the meantime, I've wanted to let you know that I've nominated your blog for the Super Sweet Blogging Award ( on my blog. Hope you guys don't mind ^_^

XX Ner


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Bottom Line Inc

Barefoot Running

There’s a lot of controversy about barefoot running, says Dr. Johanna Youner, board-certified podiatric surgeon and a Fellow of the American College of Foot and Ankle Surgeons. Barefoot running, or running using a minimalist shoe, may be beneficial for some people, but it changes the foot’s biomechanics, so it may not be appropriate for heavier runners. The optimal running footwear depends upon the runner’s physiology and biomechanics. Walking barefoot also changes whic muscles are used and can cause injuries. Additionally, the foot’s natural padding changes with age. Pain should be your guide—if it hurts to walk or run barefoot, try traditional shoes with padding.

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Critical vs. creative Thinking

Is critical thinking more important than creative thinking or is creative thinking more important than critical thinking? Take a stand on one side of this question and present a response that will support your stand. In your response, identify and explain metaphorical thinking and the importance of this.

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Critical & Creative Thinking

Critical thinking is about the application of logic and a proactive way of analysis, critique, conceptualization, synthesis and evaluation of any problem or information for the purpose of arriving at a well-thought out decision or position. When critically thinking, elements of thought are implicated - purpose, assumptions, reasoning, frame of reference, context and ...

Solution Summary

How is critical thinking and creative thinking related? Is one more important than the other? This concise solution explores these questions. Further references are listed for expansion.


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IG Sindh urges women to report incidents of domestic violence

IG Sindh A.D.Khawaja has formally inaugurated the women and children protection desk in Karachi’s West zone on Thursday.

“The objective of establishing the Desk is to stop the incidents of harassment of women and children and initiate action against the culprits,” IG Sindh said.

The IG urged the women to report the incidents of domestic violence to police.

Mr. Khawaja called upon the representatives of media to create awareness among masses about the importance and role of women and children in the society.

DIG West Amir Farooqi, SSP West Omar Shahid Hamid and SSP Central Dr. Muhammad Rizwan Khan were also present on the occasion, said a statement.

Story first published: 17th May 2018

Originally Posted on SamaaNews

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Nawaz blamed non-state actors for Mumbai attacks: Rana Sanaullah

Punjab Law Minister Rana Sanaullah defended the PML-N Quaid by saying that Nawaz Sharif had revealed nothing new in his recent interview.

“Pakistan as a state was not involved in Mumbai attacks,” Rana Sanaullah said, clarifying Sharif’s statement on SAMAA’s talk show Awaaz.

“Nawaz Sharif said non-state actors were behind the Mumbai attacks, which meant the state was not involved.”

Security Analyst Lt Gen (retd) Amjad Shoaib said Nawaz Sharif’s statement was uncalled for and portrayed a negative image of Pakistan across the world.

“Nobody should be declared a traitor,” Shoaib said. “I personally believe this is not treason, only an irresponsible statement.”

“Ajmal Kasab was arrested in Nepal before the Mumbai attacks,” said Shoaib, quoting a German scholar’s book.

He said Kulbhushan Jadhav was involved in terror activities in Pakistan and Indian PM Narendra Modi proudly claimed that he is the son of the soil.

Story first published: 14th May 2018

Originally Posted on SamaaNews

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Posted On: Feb 6, 2018

Roadmap to the Music Industry:

Five Things Musicians Should Know About Music Grant Applications by Nat Jay

As a recording artist in Canada, you are fortunate to have strong financial support from a number of funding bodies to help further your recording, marketing, and touring efforts. But what do you need to know before spending time filling in every application you see?

Recording artist and multi-grant recipient Nat Jay outlines five things everyone should do before applying for a music grant….

The first thing to do when starting a grant application is READ THE GUIDELINES. It tells you about the grant, who is eligible, what costs are eligible, what to include in your application, how much you can ask for, when to expect your results, how your application is being evaluated, etc. It basically tells you everything you need to know to create a strong (and hopefully successful) application. It’s also great to refer back to throughout the application process to make sure you’re on the right track. This is a great habit to develop right off the bat as it becomes even more vital if you receive the grant and have to adhere to the very specific completion guidelines.

True Stories:

a. A band I work with wanted to apply for a travel grant, but 5 minutes of reading the guidelines revealed they were not eligible, as they had already received this grant within the fiscal year. This saved a lot of time and effort for no reason and saved them a grant writer fee.

b. A band I wrote an application for received the grant, but they failed to read the completion guidelines about how much they could pay themselves, and ended up having to pay back the majority of the grant after the money was already spent.

Grant writing is time consuming, especially good grant writing. Whether you’re going it alone or working with a grant writer, make sure you allow lots of time to get a good understanding of the requirements of the application and get everything together with some time to spare. If you’re a grant virgin, give yourself even more time, as you’ll be creating all the supporting documents from scratch. Depending on the complexity of the grant, allow 1-2 months in advance of the deadline. Grant writers may need even more notice (2-3 months) so they can meet the needs of several different clients for the same deadline.

The most important part of your application is, well, every part. That means you need to put real effort into your supporting documents. Making sure you have an understanding of what supporting documents are required by doing #1 (Reading the Guidelines), will also help you do #2 (Planning). Funny how that works, isn’t it? While each application may vary slightly, some of the most common supporting documents include: artist bio, professional photo, comprehensive marketing plan, press quotes, awards and achievements, 1-2 letters of support from significant industry pros, goals statement, videos, 1-2 demos. Recording grants may also ask for a producer bio and travel grants will want show confirmations and an itinerary. Many applications require you to complete a budget as well. Sound like lots of work? It is! But it’s completely manageable with the right info and timeline.

Most grants in Canada are meant to support artists who are already putting some capital into their project. This means the grant is considered a contribution to your project and you are expected to contribute a percentage as well in order to receive those funds. For example, FACTOR (The Foundation Assisting Canadian Talent On Recordings) will contribute a maximum of 75% to your project, so if they give you $1,500, you are required to contribute $500 as well. And they will check this, as you’ll be required to submit both an invoice and proof of payment for every transaction you make in relation to the project.

One of the first things jurors do when reviewing an application is look at an artist’s website, as it can immediately demonstrate several things: Does the artist take their career seriously? Are they an active artist? Do they have interesting things going on? Do they have a release history? With myriad site building tools out there (Wix, Squarespace, Weebly, Wordpress, etc.), and some even specifically for musicians (Bandzoogle), there really isn’t an excuse for someone not to have even the simplest of websites. If you feel like you don’t have enough of the basic info to fill a website (bio, photos, demos, live video), the reality is you likely aren’t ready to apply for a grant. But if you do have the info, go forth and build! And if you’re applying for a grant, make sure to include links to your social media and keep all info on the site up-to-date, especially during those evaluation months where jurors will be nosing around.

In addition to these five tips from Nat Jay, make sure that you’re a member of your provincial Music Industry Association! Not only do they facilitate a number of funding programs within your province, but are also your resource to answer questions and provide knowledge to help you achieve success.

Nat Jay is a Canadian singer/songwriter, recording artist, and consultant who has written many successful grant applications for herself and other artists in Sound Recording, Touring, Showcasing, and Marketing.


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The Dog Next Door

The Dog Next Door

And Other Stories of the Dogs We Love

eBook - 2011
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Baker Publishing Group
Dogs are truly one of God's finest creatures, a marvelous gift for us humans. In the perfect follow-up to A Prince among Dogs, Callie Smith Grant compiles a delightful collection of true stories that celebrate the dogs in our lives. These stories will touch our hearts, renew our spirits, and show us how God made these wonderful creatures for unique purposes.Readers will love these uplifting glimpses into the lives of ordinary and extraordinary dogs and the people who love them. The stories are warm, captivating, and ideal for a good curl-up-and-read or a perfect gift for any dog lover.
A heartwarming collection of true stories about the beautiful relationship between people and their dogs.

Publisher: Grand Rapids, MI : Revell, c2011
Edition: E-book ed
ISBN: 9781441237330
Characteristics: 1 online resource (180 p.) : ill
Additional Contributors: Grant, Callie Smith

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Use Insults to Your Long-term Benefit - BrianaDragon Creations

Use Insults to Your Long-term Benefit

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I just wrote about not feeding trolls, and it made me think that there are trolls offline as well as on. In real life you’ll encounter a great number of damaged people who will try to bring you down. Much like internet trolls, these real life trolls give you a chance to make yourself a better person.

When someone insults you, there are actually ways that you can use it to your benefit. One of the main ways is when you learn not to react to insults. When you remain calm and think before speaking rather than becoming reactionary, you become a bit better of a person. You learn calm, restraint and respect. And yes, even people who treat you badly deserve your respect. Lack of respect and proper social interaction are why they’re broken in the first place. If you treat them well, at least they’ll be seeing an example of the kind of person they could become and how good people act.

Often when people insult me, I say thank you. It doesn’t work in every case, but in many it does. When people call me a freak, nerd, weird or fashion victim, I thank them. It sounds odd, but it makes me feel good and it throws them off their game. I can turn most insults into something positive. I perceive most insults as compliments. And let me tell you, there’s nothing quite like the look on someone’s face when they insult you and instead of reacting poorly, you light up with a big smile and say thank you.

Insults can also teach you about yourself and others. Let’s say someone calls you fat. Instead of speaking or reacting, think about it. Are you overweight? If so, maybe you should take better care of your body. If not, think about why they said it to you. Are the insecure and looking to hurt someone so they feel better? Have they come from a bad situation where people insulted them, so they do it to others instinctively? You learn a lot about yourself and others when you think about insults rather than having a knee-jerk reaction to them.

When you learn these things, insults stop hurting as much. You realize that they don’t matter. You learn to love yourself and be more confident. You also learn not to insult and be judgemental of others. You comprehend the pain it can cause, and also the reasons people say such hurtful words. you come to be a far better person in the long run.

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Is Murphy Brown one re-boot too many?

There should be a rule against re-booting sitcoms that already lasted seven years after they stopped being funny. Murphy Brown ran for 10 seasons on CBS. The 1988 – 98 sitcom had a talented cast, led by Candice Bergen, and featuring strong supporting players, including Faith Ford, Charles Kimbrough, Robert Pastorelli, Joe Regalbuto and Grant Shaud. Buffalo,… Read on


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Babywearing: Awemama

Have you heard of Awemama yet? They are a new babywearing subscription program that I am extremely excited about! They just launched and I had the privilege of trying the service out in advanced. I was asked about what  carrier type I preferred, what size I like, the fabric, allergy information, and a few more questions. After that, Awemama handpicked a carrier to send me.

I did not know in advanced what was being sent so I watched tracking like a hawk. When it arrived, I immediately opened it and WOW WOW WOW inside was a new wrap from one of my favorite babywearing companies! Bijou Wear Plaidass Bravo!! Bravo is an Awemama exclusive and to my surprise, I was sent my base size–9!!

For $29.95 per month, you get to try out a carrier for up to 28 days and you can either buy the carrier (minus the initial $29.95 since that was already paid) or send it back to try another. This is such a convenient way to try all the carriers! When you buy a wrap from a retailer and it isn’t love, you usually cannot return it. This is an excellent alternative! The carrier comes washed and ready for wear. Awemama also keeps up on carrier maintenance so you don’t have to worry about that either.

Bijou Wear Plaidass Bravo is such a stellar wrap! It is a double weave 100% cotton wrap in a gorgeous merlot and gray. This was a really comfortable wrap with Minerva (20 lbs) AND Maxine (~40 lbs) so that was a plus. I comfortably did a single layer carry with Max which really blew me away. Bravo also helped my double hammock be rock solid with Minerva. This wrap great for heavy babies and toddlers.

Thank you so much Awemama for letting me be a part of your launch!! You all are going to go far!!

Be sure to follow Awemama on Facebook and Instagram! Also, join their Facebook group.

Cloth Diapers, review

Review: Ah-Dee-Eh-Dee Cloth Diaper

Woah hey! This is my first cloth diaper review. As much as I’ve talked about cloth diapers over on Instagram, I recently realized that I’ve never written a cloth diaper review!

Over the summer, Ah Dee Eh Dee sent me a really cute all-in-one (AIO) diaper to try out with Minerva. I got it prepped right away and this diaper is now a staple in our diaper arsenal. The diaper has stay-dry Alova Suedecloth and the layers are 40% hemp, 30% bamboo, and 30% organic cotton. The diaper fits kids approximately 15-48 pounds. I did let Maxine wear it a couple times to see if it fit her (38ish lbs) and it does fit her!

We tried out the diaper for daytime use first. The diaper had no leaks and held in the destruction that Min decided to do to the diaper (hahaha!) I was shocked that it didn’t come out the diaper because it was a ton of crap. Color me impressed!!

The next time that we used the diaper, it was overnight. Minerva nursed the entire night it seemed and she peed a ton so there was a little leakage and her jammies were damp. The next time we used it overnight, I added a bamboo insert under the layers in the diaper and she did the nurse all night thing again (ugh) and woke up with no leaks! Her jammies weren’t damp at all.

   This diaper has rises on the front to fit many different sized kids. It even was forgiving with Min’s constant pulling the front of her diaper down. She does that with everything!  I look forward to adding more Ah Dee Eh Dee to my collection.

Check out Ah Dee Eh Dee on Facebook and Instagram!

babywearing, review

Review: Easy Feel Products SSC

Easy Feel Products is a company out of Gurgaon, India that I was introduced to through Tandem Trouble!  During my time as an ambassador for the brand, I’ve had a great amount of time to use this carrier and it turns out that I like the carrier a lot! Even after my ambassadorship is over, I will be using this carrier. Now onto the review!

I first received this carrier in the Terrracotta colorway in February when Minerva was eight months old and she was just big enough to fit it comfortably! Then it was super easy to carry her comfortably. As she’s grown, she has continued to feel weightless in this carrier. She is currently 20 lbs and about 29 inches long.

Minerva hooks brittany's mouth with her finger while Brittany winces. Minerva is being worn in a burgundy ikat print carrier

The padding in this carrier is phenomenal! It is firm and molds to my shoulders. The  position of the shoulder strap adjuster makes this extremely easy to adjust in front and back carriers which is part of why I like this carrier so much. This carrier also has perfect fit adjusters (PFAs) which help the carrier fit more snug and also makes nursing in the carrier easy for me.

Brittany looks down at minerva smiling while wearing minerva in a burgundy Ikat print buckle carrier

Minerva could ride arms in and out in this carrier. This carrier does not allow you to cross straps or hip carry.  The weight  range is 15-45 lbs (7-20 kg) and it comes in a wide variety of  beautiful designs. It is made of 100% handwoven heavy bottom-weight cotton Ikat which is very light weight yet very sturdy and comfortable. It is excellent in warm weather. It is also easily machine washable.  I recommend this carrier to wearers of all skill level.

Brittany is nursing Minerva in a burgundy carrier. The sun glows behind them.

Be sure to check out Easy Feel Products on Facebook and Instagram! Also, join their Facebook Group, Cuddles & Snuggles with Easy Feel!

babywearing, Photography, Picture of Me, review

Review: Ethos Babywearing Toile Bleu

Have you heard of Ethos Babywearing ? They’ve been around for a couple of years and have some innovative blends for their wraps! They also have a pretty cool convertible soft structured carrier. Today, I’m going to tell you about my time with this beauty–Toile Bleu.

When I first grabbed this budget BreatheBlend wrap out of the bag, I was shocked by how thin but dense it felt! It is a whopping 325 GSM. Toile Bleu needed some work and I was up to the challenge! Maxine (~40 lbs) and Minerva (~20lbs) both had plenty of time up in this beauty.  This wrap breathes so well and I really enjoyed it on our humid days especially. It was very grippy which helped me keep my seat popping queen (Minerva) in place once I tied off a carry.

This wrap was in my base size, so I got to try all my favorite base carries! I double hammocked almost every day that this wrap visited. Toile Bleu is ideal for me with double hammock because though it was super grippy, the passes moved easily for me and stuck in place once I moved onto the next step. I also enjoyed this with front wrap cross carry with the passes spread. I rarely do FWCC with spread passes, but this wrap made it a need for me! This wrap had some amazing shoulder pleats as well. It was flat and comfortable on my shoulders.

I recommend this wrap to more experienced wearers and also to those with heavy kiddos! Also, if you love grip you will LOVE Ethos Toile Bleu! This budget wrap is $145 for a size 9 which is great compared to size 9’s elsewhere.

Be sure to check out Ethos on Facebook and Instagram. Also, check out their Facebook group, Wear Ethos

Extra special THANK YOU to Ethos for your generous donation of this wrap to the Babywearing International of Hampton Roads lending library! Thank you, thank you, thank you!

Slings for All

I got to try out a Wildbird ring sling recently while I was teaching at a babywearing meeting. I was super excited when the meeting attendee brought it over because I was dying to try it out since I heard that our group added it to our library. Usually when teaching in person, I thread the ring sling on so that I can make sure that it does not twist on my back. When I attempted to thread the Wildbird sling, I could not get it back through the rings because there was not enough fabric. Without skipping a beat, I took off the sling and put it on the meeting attendee to  get her situated. What I wanted to do was cry. I was panicking on the inside. I felt so angry and embarrassed that  yet again, a ring sling did not fit me.

Later, I checked the Wildbird website and found that the slings are only 74-77 inches long. That is about a size small/medium. I am a fat* woman with broad shoulders.  My sling size is an extra large (XL). I can work with a long large. I prefer to use a 2XL.  Short one size slings are not  inclusive to the wide range of babywearers in the community.

I did write to Wildbird on their Facebook page and they stated “Hey Brittany! So glad you wrote us, we actually have longer slings in the works. Watch for news soon!” Soon. They do not currently have any longer slings in stock.  In their chatter group, the owner expanded on this statement and blamed growing pains on their inability to make longer slings right away. When asked what length the longer slings would be (after suggestions as well) Wildbird only stated that they would give everyone details soon.  Once again, fat babywearers are an afterthought.

Really, I should have known that their slings would not fit me. Their social media and marketing are filled with thin people wearing their slings. I am sure that some fat wearers can wear Wildbird slings since  not everyone is the same shape, but myself and several other wearers cannot. I truly hope that they do make good on their statement.

There are several companies that do offer long or sized slings. Sleeping Baby Productions, True North Ring Slings, Soul, and Maya Wrap are just a few of the many companies that offer long slings.

Fat me and baby Minerva comfortably in an XL Maya Wrap ring sling

*Fat is the term I use to describe myself because I am fat. I describe fat people as fat. It is not a dirty word. If you are uncomfortable with that, I don’t know what to tell you.

babywearing, review

Review: Kol Kol Baby Carrier Onbuhimo

Kol Kol Baby Carrier is a company out of India that makes affordable and comfortable baby carriers. Fun fact: The company is named after the first words that Bayiravi’s (owner) son spoke! So sweet!!  They will soon be releasing an onbunimo and I had the pleasure of trying it out .

Minerva is about 20 lbs and 28 inches tall.  She fit great in the carrier. With onbuhimos, all the weight is carried in your shoulders, so one of the most important aspects of the carrier for me  is the padding. Kol Kol’s padding was so soft! It reminded me of a pillow. It was very comfortable on my shoulders.  I also like their hood which is rare because I usually can do without a hood on most carriers.

 The carrier even fit Maxine comfortably! She will be four years old soon and is in 4T clothes. The onbuhimo fit great in my diaper bag which is a plus since I pack so much stuff when we leave the house and since there is so little fabric, it is  ideal for warmer weather as well.  The fabric breathed very well.  I think that this will be on my list of carriers to buy!

Be sure to check Kol Kol out on Facebook  and Instagram, and  also Join their group Kol Koling on Facebook!

Family, fun, Parenting, Playtime

Playtime: Windsor Castle Park Natural Playscape

Smithfield, Virginia is my hometown and such a beautiful place! Maxine is at an age where she can enjoy playing on her own so I decided to take her to check out the new Windsor Castle Park Natural Playscape.

Maxine has her back facing everyone. She is at the top of the slide and hovering before letting go. The background is very green.

She loved it! I loved that they had a side for younger children. We stayed over there today.

Maxine is walking across small logs in the ground. The background is green and trees

The entire area was beautifully shaded by trees. It didn’t feel as miserably hot as the thermometer read.

Maxine is playing in a large sandbox

Maxine is flying down the slide.

This slide was the perfect size for Maxine and she went on it several times. The other slide on the big kid size is 30 feet!

Maxine is walking in a wide green park

Maxine is climbing a wall.

Max had a blast and we will definitely be back soon!


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Elephant Journal

Campaign Supporters Include:

  • Better Boulder
  • BoCHA, The Boulder Community Housing Association
  • Daily Camera
  • Elephant Journal
  • FIDOS, Friends Interested in Dogs + Open Space
  • Open Boulder
  • Sierra Club
  • South Boulder Creek Action Group
  • yellowscene magazine
  • KC Becker, State Representative
  • Elise Jones, Boulder County Commissioner
  • Stan Garnett, District Attorney
  • Matt Appelbaum, Boulder Mayor
  • Macon Cowles, Boulder City Councilperson
  • Suzanne Jones, Boulder City Councilperson
  • George Karakehian, Boulder City Councilperson
  • Tim Plass, Boulder City Councilperson
  • Andrew Shoemaker, Boulder City Councilperson
  • Mary Young, Boulder City Councilperson
  • Linda Shoemaker, CU Regent, Former BVSD Board Chair
  • Laurie Albright, Boulder Valley Board of Education President
  • Sam Fuqua, Boulder Valley Board of Education Member
  • Lesley Smith, Former Boulder Valley Board of Education Member
  • Steve Fenberg, State Senate Candidate, Director of New Era Colorado
  • Claire Levy, Former State Representative
  • Will Toor, Former Boulder Mayor, Former Boulder County Commissioner
  • Leslie Durgin, Former Boulder Mayor
  • Susan Osborne, Former Boulder Mayor
  • Mark Ruzzin, Former Boulder Mayor, Current BHP Commission Member
  • Suzy Ageton, Former Boulder City Councilperson
  • DeAnne Butterfield, Former Boulder City Councilperson
  • Angelique Espinoza, Former Boulder City Councilperson
  • Gary Myre, Former Boulder City Councilperson
  • Françoise Poinsatte, Former Boulder City Councilperson
  • Richard Polk, Former Boulder City Councilperson
  • Andy Schultheiss, Former Boulder City Councilperson
  • Ken Wilson, Former Boulder City Councilperson
  • Bryan Bowen, Planning Board Member
  • John Putnam, Planning Board Member
  • Bill Holicky, Former Planning Board Chair
  • Angela McCormick, BHP Commission Chair
  • Valerie Soraci, BHP Commission Member
  • Shelley Dunbar, Open Space Board of Trustees Member
  • Tom Isaacson, Open Space Board of Trustees Chair
  • Kevin Bracy Knight, Open Space Board of Trustees Member
  • Ann Moss, Arts Commission Member
  • Richard Turbiak, Arts Commission Chair
  • Nikhil Mankekar, Human Relations Commission Member
  • Zane Selvans, Transportation Advisory Board Chair
  • Andria Bilich, Transportation Advisory Board Member
  • Daniel Stellar, Transportation Advisory Board Member
  • Masala Housing Co-operative
  • Dennis Arfmann, Civic and Non-Profit Volunteer
  • Nancy Blackwood, Urban Designer, Member of the Executive Committee for the University Hill Neighborhood Association
  • Becky Boone, Political Activist
  • Buzz Burrell, Political Activist
  • Ed Byrne, Political Activist
  • Michelle Estrella, Political Activist
  • Leslie Glustrom, Research Director, Clean Energy Action
  • Clif Harald, Executive Director of the Boulder Economic Council
  • Landon Hilliard, Alternative Transportation Coordinator for BVSD
  • Michael Leccese, Executive Director of ULI Colorado
  • Neil Lurie, President of the Center for ReSource Conservation
  • Deborah Malden, Arts Liaison & Advisor at the Boulder Chamber
  • Lincoln Miller, Executive Director of Boulder Housing Coalition
  • Bob Morehouse, Founder of Vermillion Design, Longtime Civic and Non-Profit Volunteer
  • Elisabeth Patterson, Public Affairs Associate at the Boulder Chamber
  • Dan Powers, Executive Director of Boulder Tomorrow
  • Sue Prant, Executive Director of Community Cycles
  • Steve Welter, Political Chair of Sierra Club Indian Peaks Group
  • Karen Worminghaus, Executive Director of eGo Carshare
  • Bob Yates, City Council Candidate, Civic and Non-Profit Volunteer
Questions? Comments? I’d love to hear from you! Drop me a line at aaronboulder@gmail.com
Paid for by Aaron Brockett for City Council, Dale Deegan Treasurer
A copy of our report is filed with the City Clerk of the City of Boulder, Colorado.


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Top VQA wine promoters honoured at Experts Tasting

More than 125 wine writers, educators and industry professionals gathered at Brock Wednesday, May 2 for the 29th annual Experts Tasting.

Organized by Brock’s Cool Climate Oenology and Viticulture Institute (CCOVI), the tasting promotes Ontario VQA wine and celebrates individuals who have made contributions to growing and promoting the grape and wine industry.

The 2018 tasting focused on Cabernet Franc and examined the 2013 to 2016 vintages. Five flights showcased a total of 33 wines with industry experts guiding the attendees through the tasting.

Wine writers, educators and industry professionals gathered at Brock University for the 29th Experts Tasting Wednesday, May 2.

“The Experts Tasting has become a pivotal educational opportunity for guests to taste and learn alongside their peers in the industry,” said Barb Tatarnic, organizer of the event and Manager of Outreach and Continuing Education at CCOVI. “Cabernet Franc is considered one of the core varietals for this region and having the tasting showcase multiple vintages clearly showed what this variety can bring to the Ontario industry year after year.”

The event also honours individuals who help raise the profile of Ontario wine with VQA Promoter Awards, which acknowledge individuals who celebrate, through promotion and/or education, the Ontario VQA wine industry with unselfish enthusiasm, constructive input and unsolicited promotion.

The lifetime achievement award was presented posthumously to Ontario wine industry pioneer and winemaker Karl Kaiser by CCOVI Director Debbie Inglis.

“There would be few in this room who would not recognize the pivotal contributions that Karl made to our VQA wine industry,” Inglis said. “From putting Canada on the map as a wine-producing nation to changing the landscape of agriculture in Niagara, he showed us what could be crafted with the grapes that we grow here. He helped drive this industry forward to be what it is today.”

Kaiser’s daughters Andrea and Magdalena accepted the award on behalf of his family. Magdalena was also recognized for her own contributions to promoting and raising the profile of VQA wines in the promoter-at-large category, marking the first time a father and daughter have been recognized in the same year.

When Brock grad Ivone de Marchi (BSc ’72) learned of the passing of his former lab partner Kaiser (BSc ’74) last fall, it struck a chord with him. He wanted to honour Kaiser with a portrait, and on Wednesday the painting was unveiled and then gifted to Brock to hang in Inniskillin Hall.

CCOVI Viticulturist Jim Willwerth was recognized in the education category for the opportunities he provides grape growers and winemakers in support of Ontario VQA wines through his outreach and research work.

2018 VQA Promoters award winners (from left): Tim Coons, retail; Andrea Kaiser on behalf of Karl Kaiser’s family, lifetime achievement; Jim Willwerth, education; Magdalena Kaiser, promoter-at-large; Brittany Gregory, LCBO; Sarah Scott, hospitality. Absent from the photo are André Proulx, media, and Ian D’Agata, out-of-Ontario promoter.

The 2018 VQA Promoter Award winners in each category are:

  • Education: Jim Willwerth, CCOVI Scientist, Brock University
  • Hospitality: Sarah Scott, Manager of Dining Operations, Benchmark Restaurant
  • LCBO: Brittany Gregory, Product Consultant, LCBO Store #381, Kitchener
  • Media: André Proulx, Radio Producer and Wine Writer, Two Guys Talking Wine
  • Out-of-Ontario: Ian D’Agata, Wine Writer and Director of the International Wine Academy
  • Promoter-at-Large: Magdalena Kaiser, Director of Public Relations – Tourism and Marketing, Wine Marketing Association of Ontario
  • Retail: Tim Coons, Estate Manager, Trius Winery and Restaurant and Wayne Gretzky Estates
  • Lifetime Achievement: Awarded posthumously to Karl Kaiser, winemaker and wine industry pioneer

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Date Tags google

On Monday, I discovered pancake breakfast is pretty standard at the Moma Cafe and oh so delicious. Tuesday I had an awesome time playing volleyball. Lots happened Wednesday. I had my first experience with a modern bidet and I believe it went something like the following.

I went into the bathroom to deuce it up, and upon opening the stall I uttered, “what the fuck” which fortunately no one was there to hear. I was baffled because the back of the toilet seat had some sort of contraption, and there was a control box affixed to the side of the stall. Proceeding with caution I put the seat down, put a piece of the butt paper down and I sat upon the toilet. At this moment I let out a faint pleasurable “oooh” due to warm comforting feel of the seat upon my butt. Yes it was heated.

While I was doing my business I was checking out the controls to the side. I was quite intrigued by the button that read “rear cleaning” so after dropping the kids off at the pool I pressed the button. Next came the noise of the servo followed by a warm stream of water. I then proceeded to try the drier button but decided against the last button as I didn’t think a front cleaning would do me much good. Bidets are awesome.

Wednesday continued when three of my college friends, A, E, and M came to visit me for lunch. They were quite impressed by Google and seemingly determined that they picked the wrong choice of college major. I reminded them that Google hires non-computer science students as well, but they were skeptical.

Finally Wednesday at work ended amazingly as I had my free massage. Let me just say it was incredible and much needed after the soreness from Tuesday’s volleyball. Following the massage I met up with A, E and M that visited for lunch for dinner at a Chicago style pizza place called Patxi’s which was quite delicious. Damn, Wednesday was awesome.

Not too much exciting happened on Tuesday, I was just finishing up stuff for the long weekend, which the weekend was pretty awesome. Along with the A, E, and M I went into the city for the 4th. Because it was a pretty nice afternoon, and for other possibly dumb reasons, we decided to walk from basically the Bay Bridge to the Golden Gate Bridge. It was a long, but enjoyable 6 mile walk. After reaching our destination I played a little flip cup and then the four of us headed toward Berkeley for fireworks and pizza. Saturday I worked on priceTrackr, and today I did a hike/walk of the Stanford Dish area with E.

Finally this evening I went to dinner in Newark with part of my second family. We had some delicious sushi of which I am still full, then went back and celebrated an early birthday followed by some wii Guitar Hero. Finally it’s bed time. Night.

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