text:
What is Artificial Intelligence (AI)?
When computers and machines learn from collected data and derive an action from it, one speaks of Artificial Intelligence (AI). The term machine learning is also used in the machine sector.
In private life, one example is that of users of music streaming services. Here an algorithm constantly learns from the behaviour of the user:
- What music does he listen to?
- How often does he hear it?
- Which of the resulting recommendations does he accept?
As a result of this AI, the user hears his favourite music.
An example from the industry shows the economic potential of this technology: For an energy chain, the manufacturer predicts the expected service life under specified conditions of use. Sensors in the energy chain send real-time usage data to a computer around the clock, which may also process environmental parameters such as temperature and humidity. This data automatically synchronises a software and then issues an always current expected operating time of the energy chain. If a critical value is reached for the system, it automatically triggers an alarm or an action, e.g. by shutting down systems. The software decides what to do from the data it gets.
Despite all technology, it is still the case that a system can only be as intelligent as the human being who initially programmed its algorithms. And with all further development through permanent learning, scenarios of machines dominating the world emerge only in the imagination of science fiction writers.


text:
- Research article
- Open Access
- Published:
YY1 is autoregulated through its own DNA-binding sites
BMC Molecular Biology volume 10, Article number: 85 (2009)
5462 Accesses
23 Citations
The transcription factor Yin Yang 1 (YY1) is a ubiquitously expressed, multifunctional protein that controls a large number of genes and biological processes in vertebrates. As a general transcription factor, the proper levels of YY1 protein need to be maintained for the normal function of cells and organisms. However, the mechanism for the YY1 homeostasis is currently unknown.
The current study reports that the YY1 gene locus of all vertebrates contains a cluster of its own DNA-binding sites within the 1st intron. The intact structure of these DNA-binding sites is absolutely necessary for transcriptional activity of the YY1 promoter. In an inducible cell line system that over-expresses an exogenous YY1 gene, the overall increased levels of YY1 protein caused a reduction in transcription levels of the endogenous YY1 gene. Reversion to the normal levels of YY1 protein restored the transcriptional levels of the endogenous YY1 to normal levels. This homeostatic response was also mediated through its cluster of YY1 binding sites.
Taken together, the transcriptional level of YY1 is self-regulated through its internal DNA-binding sites. This study identifies YY1 as the first known autoregulating transcription factor in mammalian genomes.
The Gli-Kruppel-type transcription factor Yin Yang 1 (YY1) is a ubiquitously expressed, multifunctional protein that can function as an activator, repressor, or initiator binding protein depending on its promoter context, chromatin structure, and interacting proteins [1, 2]. YY1 is also known to undergo various post-translational modifications, causing different outcomes on the functions of YY1. YY1 interacts directly or indirectly with many key proteins including the components of 1) RNA polymerase II transcription machineries, such as RNA polymerase II, TFIIB, TBP and TAFII55, 2) transcription factors, such as Sp1, c-myc, c-myb and CREB, and 3) histone-modifying complexes, such as p300, CBP, HATs, HDACs, PRMT1, and PhoRC and INO80 [3, 4].
YY1 is involved in the transcriptional control of a large number of mammalian genes, approximately 10% of the total mammalian gene set [5]. Consequently, YY1 plays important roles in a number of biological processes, including cell cycle control, embryogenesis, viral infection, programmed cell death, oncogenesis, Polycomb Group (PcG) function and B-cell development [6]. Since it is a general transcription factor involved in so many pathways, the expression levels of YY1 must be tightly monitored for the survival of cells and organisms [4]. Accordingly, abnormal YY1 protein levels have been shown to cause defects in cell proliferation and differentiation, neural development, and the repression mediated by the PcG complex. Also, there are several types of the YY1-related diseases, such as viral infection and cancers, which are also linked to abnormal YY1 protein levels [3]. These observations clearly predict the presence of some regulatory mechanism(s) that are responsible for maintaining the appropriate transcriptional levels of YY1. Nevertheless, the mechanism of transcriptional regulation of the YY1 gene is largely unknown so far except for a few earlier observations on the promoter regions of the human and mouse YY1 gene [7, 8].
In the current study, we have identified a cluster of multiple YY1 binding sites located in the first intron of the YY1 gene. We characterized this cluster of YY1 binding sites in terms of evolutionary conservation and transcriptional activity. We also tested the possibility that YY1 may be regulated through its own binding sites. For this test, we have established and used a Tet-On inducible system, in which we can control the cellular levels of YY1 protein. Our results demonstrated that changes in the cellular levels of YY1 protein affect the transcriptional levels of its own locus, YY1, most likely through its own binding sites. This autoregulation is regarded as a homeostatic response of the YY1 locus to maintain the constant cellular levels of YY1 protein.
Identification of multiple YY1 binding sites in the 1st intron of YY1
We have identified an evolutionarily conserved region in the 1st intron of YY1 via the ECR (Evolutionary Conserved Region) browser, a tool for visualizing sequence comparison of multiple vertebrate genomes http://www.dcode.org[9]. As shown in Figure 1A, the genomic sequence of the 1st exon and intron region of human YY1 was successfully aligned with those from eight different species including tetraodon, zebrafish, frog, opossum, cow, mouse, dog, and rhesus macaque. As expected, the first exon (marked in blue) showed high levels of sequence conservation. Interestingly, however, a similar level of conservation was also detected in the beginning of the 1st intron region (marked in salmon). We further analyzed this small region, 100 bp in length, in more detail. As shown in Figure 1B, we manually aligned genomic sequences derived from 11 different vertebrates: one each from sea squirt, purple sea urchin and amphioxus, two from each of two fish, one from frog, and five from mammals. Multiple sequence alignment using ClustralW revealed 20 to 48% sequence identity between the urochordates, 71 to 82% sequence identity between the two fish, 80 to 98% sequence identity between the five mammals. Detailed inspection revealed that this small region is mainly filled with multiple YY1 binding sites. Most vertebrates contain 5 YY1 binding sites within this 100 bp region except for sea squirt (3 binding sites) and amphioxus (4 binding sites). Neither the sequence nor the length of the spacer regions between individual YY1 binding sites are conserved, which becomes more apparent in the urochordates' YY1 binding regions. The sequences of most of the YY1 binding sites are identical to the consensus sequences of the known YY1 binding motif, which display very high levels of affinity to YY1 protein [10]. However, one binding site, the 3rd one of all the vertebrates except urochordates, shows a slight sequence variation from the known consensus (marked in green), but still conserved among the vertebrates. The orientation of the individual YY1 binding sites is also conserved: the first two YY1 binding sites are localized in one direction, while the remaining three sites are in the other direction. The opposite orientation of the two outside YY1 binding sites is particularly well conserved throughout all the vertebrates. We have also examined the 1st intron of pho, a homolog of YY1 in Drosophila [11], but we did not find a similar genomic region with multiple Pho-binding sites. This indicates that this cluster of YY1 binding sites is unique to vertebrates. Chromatin immunoprecipitation (ChIP) assay was performed to confirm the in vivo binding of YY1 to its own binding sites located in the 1st intron. Homogenized mouse brain tissues were cross-linked with formaldehyde and immuno-precipitated with anti-YY1 polyclonal antibodies (Figure 1C). As expected, the multiple YY1 binding sites showed consistent enrichment levels of the immuno-precipitated DNA with this ChIP experiment, whereas the two other control regions, upstream and downstream, did not show any detectible levels of the DNA enrichment. This confirms in vivo binding of YY1 to its own multiple binding sites in the 1st intron. In sum, the 1st intron of the YY1 gene locus of all vertebrates contains a cluster of its own DNA-binding sites.
YY1 binding sites as a transcriptional activator for YY1 transcription
We performed promoter assays to determine potential roles of this cluster of YY1 binding sites in YY1 transcription. For this experiment, we first tried to clone and manipulate the 1-kb genomic region surrounding the 1st exon of the mouse YY1, but we could not amplify and stably maintain this fragment in bacterial clones mainly due to the unusually high GC composition of this region, around 80%. Therefore, we used the homologous region derived from zebrafish. According to a series of initial pretests (data not shown), the promoter region of the zebrafish YY1 has reasonably high levels of promoter activity in several mouse cell lines, confirming the feasibility of the planned experiments as well as the evolutionary conservation of the promoter activity associated with this genomic region.
We have generated a series of 12 different constructs by modifying the 1.1-kb endogenous promoter region of the zebrafish YY1. The 1.1-kb region, covering the 1st exon and the multiple YY1 binding sites, was first subcloned into a promoterless reporter system, IRES-β-Geo (Figure 2). Each of these constructs differs from the others in binding affinity, orientation and numbers of intact YY1 binding sites. This series of promoter assays excluded one particular YY1 binding site, the third, that imperfectly matches the YY1 consensus sequence (GCCATAnT instead of GCCATnTT). This difference is known to have much weaker binding affinity to YY1 protein [12]. To lower the DNA-binding affinity to the protein, the sequences of the 4 remaining YY1 binding sites were mutated from (A/C)G CCATnTT to AA CCATnTT (Construct named as Low affinity). The orientation of YY1 binding sites was also reversed in an opposite direction (Constructs named as Forward, Reverse and Both in Figure 2A). We also completely abrogated the binding potential of individual YY1 binding sites through mutating the three critical bases within the core motif of YY1 from GCC ATnTT to ATT ATnTT (Constructs named as 1 mut, 12 mut, 123 mut, All mut, 4 mut, 34 mut and 234 mut in Figure 2B). These constructs were individually transfected into two different cell lines, Neuro2A and NIH3T3, along with an internal control luciferase vector (pGL3 control) to normalize the β-gal activity.
As shown in Figure 2A and 2B, the normalized transcriptional activity of each construct was compared to the activity of the control construct containing the original unchanged 1.1-kb genomic region of the zebrafish YY1. The promoter activities of all the mutant constructs showed a dramatic decrease compared to that of the control construct. The original activity was completely abolished by any change affecting the binding affinity or orientation of the intact YY1 binding sites (Figure 2A). This is also true for the second series of mutant constructs containing the different numbers of intact YY1 binding sites. Regardless how many and which positions of YY1 binding sites remain, a single change affecting any of the YY1 binding sites completely abolished the transcriptional activity of the YY1 promoter (Figure 2B). This indicates that all these YY1 binding sites are absolutely required for the transcriptional activity of the YY1 promoter. A similar pattern was also consistently observed in both Neuro2A and NIH3T3 cells. In sum, the YY1 binding sites function as a transcriptional activator for the YY1 promoter, and this role apparently requires the absolutely intact sequence structure of all the YY1 binding sites.
Homeostatic control of the cellular levels of YY1 protein
The identification of the YY1 binding sites within the YY1 locus hinted at one intriguing possibility: YY1 may control its own transcription rate through its own binding sites. To test this possibility, we first set up an in vitro system in which we can control the cellular levels of YY1 protein. Briefly, human YY1 was added into the mouse cell line Neuro2A, and used as an exogenous gene, which allows for easy distinction between the exogenous and endogenous YY1. A Tet-On inducible system was employed to control the expression of the human YY1. We have successfully established several stable cell lines that can induce transcription of the human YY1 by the administration of an inducer, Doxycycline (Tet-On Advanced system) [13]. More detailed information is described in Methods.
With the three selected stable cell lines, we first performed time-course experiments (Figure 3A). All three cells showed increased levels, about 5 fold, of YY1 protein at 24 and 48 hours after the Dox administration as shown on western blot results. With the same set of cells, we further confirmed this induction at the transcription level by performing RT-PCR, in which we can differentiate the induced human YY1 from the endogenous mouse YY1 (Figure 3B). As expected, we observed a dramatic increase in the transcriptional level of human YY1 at 24 and 48 hours after the Dox administration. Although we observed low levels of the human YY1 prior to the Dox induction, this is believed to have been caused by genomic DNA contamination in the RNA samples. Interestingly, the transcriptional levels of the endogenous YY1 progressively decreased during the induction. Another independent measure using qRT-PCR further revealed that the transcriptional levels were reduced to 70% at 24 hours and to 50% levels at 48 hours, respectively. According to our survey on the transcription levels of other genes (Peg3, Usp29, Nespas, Nesp, Xist, and p53), the observed down-regulation was specific to the YY1 locus (data not shown). This indicates that the overall increased levels of YY1 protein, through the induction of the exogenous YY1, somehow resulted in reduction in the transcriptional levels of the endogenous YY1. This initial observation was further validated through the following scheme. We first increased the overall levels of YY1 protein through the induction of the human YY1, and later reversed the increased levels back to the normal levels of YY1 protein by withdrawing the inducer, Dox, from culture media. As predicted, this cycle of addition and withdrawal of the Dox treatment caused a concurrent rise and fall in the overall levels of YY1 protein. The levels of endogenous YY1 transcription changed in a manner inverse to the induced YY1 protein: decreasing with YY1 induction and increasing with removal of YY1 induction as shown in the results of qRT-PCR (Figure 3C). This inverse pattern is thought to be a counter-balancing response from the endogenous YY1 locus to maintain constant cellular levels of YY1 protein. In sum, these results confirm that the overall cellular levels of YY1 protein can affect the transcription levels of the YY1 locus.
YY1 is autoregulated through its own binding sites
The above results clearly demonstrated that the endogenous locus of YY1 has an unknown feedback regulatory mechanism that maintains homeostasis of the YY1 protein levels. We next tested if this observed regulation is indeed mediated through its own DNA-binding sites as part of the hypothesized autoregulation. For this test, we analyzed transcriptional responses from the YY1 promoter against a background of fluctuating cellular levels of YY1 protein. We first set up the two different cellular levels of YY1 protein with the following scheme (Figure 4). The cells to be transfected were first incubated in the absence (A) and presence (B) of Dox prior to transfection experiments. During the transfection, Dox was added (A) and removed (B) to create higher and lower levels of YY1 protein relative to the control cell, respectively. Then, the actual transfection into these cells used each of the following two reporters, Intact and All-mut constructs, containing the promoter region of the zebrafish YY1 with the intact and mutated YY1 binding sites, respectively (Figure 4). These individual transfections were also normalized by including the control luciferase vector (pGL3 control). In the first condition (Figure 4A), the promoter activity was measured while the cellular levels of YY1 protein were being increased by the Dox induction. As shown in Figure 4A, the Intact construct (containing the YY1 binding sites) showed a 0.5-fold decrease in the promoter activity when the cells started producing more YY1 protein. We also observed a similar down-regulation from the endogenous locus in response to increased levels of YY1 protein (Figure 3). This response, however, was not detected in the All-mut construct (containing the mutated YY1 binding sites), confirming that the observed response was mediated through the YY1 binding sites. In the second condition (Figure 4B), the promoter activity was measured while the cellular levels of YY1 protein were decreased by the Dox withdrawal. The Intact construct showed in a 1.7-fold increase in the promoter activity when the cells started producing less YY1 protein. This is also reminiscent of an observed up-regulation from the endogenous locus of YY1 in response to the decreased levels of YY1 protein. Collectively, the zebrafish YY1 promoter and the endogenous locus of YY1 responded very similarly to fluctuating levels of YY1 protein. These similar responses agree very well with the initial prediction that the cluster of YY1 binding sites most likely mediates the hypothesized autoregulation mechanism for the endogenous locus of YY1.
The current study reports the three unique features associated with the cluster of YY1 binding sites that is localized in the 1st intron of the vertebrate YY1 locus. First, this cluster of YY1 binding sites are well conserved throughout all the vertebrates including urochordates, suggesting that this small region has been functionally selected in the past 500 to 600 million years (Figure 1). This region is probably one of the oldest cis-regulatory regions in the vertebrate genomes. However, similar regions are not found in the pho locus, the YY1 homolog in flying insects, suggesting the formation of this region after the divergence of vertebrates and invertebrates. This sudden formation of this region in the vertebrates' YY1 locus may be linked to the different expression profiles of this gene between these two groups of animals. In insects, the homeotic genes including pho are transiently expressed by segmentation genes during early embryonic development, and their later expressions are controlled by two groups of genes, the trithorax group (trxG) and the Polycomb group (PcG) [14, 15]. Therefore, the expression pattern of pho may be restricted and focused in a developmental stage-specific manner. In contrast, YY1 is expressed ubiquitously, spatially and temporarily [16]. The ubiquitous expression may have allowed YY1 to be involved in a greater number of genes and pathways in the vertebrates than in the invertebrates. Consequently, the involvement in a large number of genes most likely has necessitated more sophisticated regulation mechanism(s) for the maintenance of appropriate cellular levels of YY1. Taken together, the cluster of YY1 binding sites most likely has coevolved with the ubiquitous expression pattern of YY1.
The second unusual feature is that this conserved cis-regulatory region has the DNA-binding sites for its own protein product, YY1. This immediately suggests a potential autoregulation mechanism for this locus. According to our cell line studies, the cellular levels of YY1 protein indeed affected the transcriptional levels of its own gene locus (Figure 3), and this control also appeared to be mediated through the cluster of YY1 binding sites (Figure 4). Besides being part of this predicted autoregulation, the cluster of YY1 binding sites is also regarded as a transcriptional activator (Figure 2) or repressor (Figure 3 and 4). Any mutations in the YY1 binding sites caused a dramatic decrease in the transcriptional activity of the YY1 promoter, indicating that YY1 is required for the transcription of its own locus. On the other hand, the same cluster of YY1 binding sites functions as a repressor in the presence of excessive cellular levels of YY1 protein. According to surveys on transcriptional regulatory networks [17], many E coli proteins, especially repressors, are also subject to similar autoregulation. Furthermore, the observed autoregulation is similarly 'negative' autoregulation: these repressors start repressing their own loci once their protein levels reach at their threshold levels. Despite these similarities, however, it is important to note that this cluster of YY1 binding sites has dual roles, repressor and activator. Interestingly, these roles have been detected at different cellular conditions: an activator role during the promoter assays with a normal level of YY1 protein (Figure 2) while a repressor role during YY1 induction with higher levels of YY1 protein (Figure 3 and 4). This suggests that the available cellular levels of YY1 protein may be a factor deciding which of the two roles to be exerted for a given condition.
Then, the next question is how this cluster of YY1 binding sites knows different cellular conditions and selects a role between an activator and repressor function. This question may be related to the third feature of this cluster of YY1 binding sites: multiplicity. One plausible scenario would be that the cluster of YY1 binding sites could function as a sensor recognizing the cellular levels of YY1 protein. In the case of higher-than-normal YY1 levels, all of the YY1 binding sites would be filled with the YY1 protein, subsequently block the traverse of this region by Pol II polymerase, and thus function as a repressor. In the case of lower-than-normal YY1 levels, on the other hand, only one or two YY1 binding sites would be filled, subsequently stimulating transcription by Pol II, and thus act as an activator. It will be very interesting to test this possibility in the near future, but the multiplicity and evolutionary conservation associated with the YY1 binding sites are very unusual, and clearly suggest some unknown functional roles in this regulatory region.
Despite a large number of genes that are controlled by YY1, there have not been a lot of studies on the transcriptional control of YY1 itself. Although the current study has mainly focused on the predicted autoregulation mechanism that might be mediated through the cluster of YY1 binding sites, we believe that this is not the only regulatory mechanism responsible for the YY1 transcription. Also, we cannot rule out the possibility that the identified cluster of YY1 binding sites might be designed for some other functions for the YY1 locus, such as a targeting site for epigenetic modification or a promoter for some unknown transcripts. In a similar line, it is important to emphasize the fact that the unusually high levels of YY1 protein are often observed in normal and cancer cells [3]. The up-regulated YY1 levels in normal cells may be an outcome of an independent regulatory program that has been preset in a given cell. On the other hand, the unusually high levels of YY1 protein observed in cancer cells might be an outcome of mis-regulated YY1 transcription. One intriguing possibility would be that some epigenetic changes, such as DNA methylation or histone modification, and subsequent malfunction of the cluster of YY1 binding sites might be responsible for this mis-regulation. In that regard, it will be interesting to test the epigenetic modification of this region in cancer cells. Overall, the cluster of YY1 binding sites described in the current study represents a very unique example of eukaryotic cis-regulatory region.
The current study has identified a very unusual cluster of YY1 binding sites within its own gene locus, prompting a potential autoregulation mechanism for YY1 transcription. Consistently, the overall cellular levels of YY1 protein were shown to affect (or control) the transcription levels of the endogenous YY1 locus. Also, this control appears to be mediated through its own YY1 binding sites. This suggests that YY1 is likely self-regulated through its own binding sites.
Sequence alignment
The cluster of YY1 binding sites in the first intron of YY1 was discovered through the ECR (Evolutionary Conserved Region) browser http://www.dcode.org[9]. This conserved region was manually aligned from genomic sequences of each genome ranging from urochordate (sea squirt) to placental mammals. A series of database searches were conducted using the BLAST program http://www.ncbi.nim.gov/BLAST to obtain the genomic sequences of the YY1 locus from several species. The aligned sequences are as follows: sea urchin (Scaffold81718: 20284–20353), sea squirt (ENSCSAVG00000003120: 624148–624214), amphioxus (JGI scaffold_8: 4922034–4922089), zebrafish_1 (NC_007128: 40211168–40211267), zebrafish_2 (NW_001877837: 59200–59296), fugu_1 (chrUn: 161085228–161085087), fugu_2 (chrUn: 51268538–51268643), human (NC_000014: 99776064–99776201), mouse (AC_000034: 110030364–110030467), cow (NC_007319: 58875242–58875872), dog (NW_876327: 66420933–66421058), opossum (ENSMODG00000013124: 318625672–318625815), and frog (JGI4.1:scaffold_222: 1017795–1034375).
ChIP assay
ChIPs were performed according to the protocol provided by Upstate Biotechnology with some modification as described previously [18]. Briefly, the mouse brain tissues (0.1 g per tissue) were homogenized in 10 ml PBS for ChIP assay. The samples were treated with formaldehyde to a final concentration of 1% and incubated at 37°C for 10 min. Treated samples were sheared by sonication to derive DNA fragments averaging 500 bp in length. Sheared chromatin was immunoprecipitated with anti-YY1 antibody (sc-1703; Santa Cruz Biotechnology). Precipitated DNA and protein complexes were reverse crosslinked and purified through phenol/chloroform extraction. Purified DNA was used as templates for PCR amplification. Three primer sets were used: ChmYYup1(+), 5'-GGCACTTTTGTCACTGTTGCACCGCG-3' and ChmYYup2(-), 5'-CAACTCCTCAACCCCGAGCCCAGATCTC-3'; ChmYYb3(+), 5'-GGGAGCAGAAGCAGGTGCAGATCAAG-3' and ChmYYb4(-), 5'-CTCAACCGGCCCCGCCGCACGTCCGTTG-3'; ChmYYdw5(+), 5'-CTGCACGGTAGGTTATCAGGAGCTGTATG-3' and ChmYYdw6(-), 5'-CGATTCATCAACACCACACTTGACGAAG-3'. PCR reactions with an inclusion of 1% DMSO were carried out for 35 cycles (94°C, 30 s; 64°C, 30 s; 72°C, 30 s) using the Maxime PCR premix kit (Intron Biotech). The resulting PCR products were analyzed by running on 1.5% agarose gel containing ethidium bromide. The animal experiment was performed in conditions approved by the Louisiana State University Institutional Animal Care and Use Committee (IACUC), protocol #07–051.
Promoter assay
We used one β-Geo vector that has been modified from pGT1.8iresβgeo [19]. We have transferred a Bam HI partial digested fragment containing the IRES-LacZ-Neo cassette into the Bam HI site of pBluescript II SK (+). All constructs derived from zebrafish YY1 (BX546455; 5043–6110) were cloned into the Not I site of this IRES-β-Geo vector. Several mutations on 4 YY1 binding sites were performed by PCR-based mutagenesis with modified oligonucleotides. Neuro2A and NIH3T3 cells were maintained in MEM and DMEM medium with 10% fetal bovine serum and 1% antibiotic-antimycotic (GibcoBRL), respectively. 2×105 cells were plated in each well of a six-well plate. On the next day, cells were co-transfected with GeneJuice transfection reagent according to the manufacturer's protocol (Novagen). Two days after transfection, the cells were harvested, washed with PBS, and treated with 100 μl of lysis buffer (0.25 M Tris-HCl, pH 7.8 + 0.1% NP40) for 30 min at 4°C and cellular debris was removed by centrifugation for 10 min. For the β-galactosidase assay, 30 μl of cell lysate was mixed with the same volume of 2× β-galactosidase assay buffer (Promega) in a 96-well flat bottom clear plate. The plate was incubated at 37°C, monitored visually and terminated with 90 μl of 1 M sodium carbonate. The absorbance was measured at 405 nm with Wallac 1420 multilabel counter VICTOR3 (PerkinElmer). For the luciferase assay, 20 μl of cell lysate was combined with 100 μl of Luciferase Assay Reagent (Promega) in a 96-well flat bottom white plate (Corning). Luminescence was measured with Wallac 1420 multilabel counter VICTOR3. To control for transfection efficiency in each well, β-galactosidase activity was normalized to luciferase activity.
YY1 inducible cell line
pTet-On Advanced, pTRE-tight vectors and HeLa Tet-On cell line were purchased from Clontech. The cDNA fragments encoding human and mouse YY1 (GenBank accession No. NM_003403 and NM_009537) were inserted into BamHI-NheI sites and BamHI-NotI sites of the pTRE-tight vector, respectively. Neuro2A Tet-On cell line was established by stable transfection of the pTet-On Advanced vector under the presence of 500 ug/ml G418. The human YY1 and mouse YY1 cloned pTRE-tight vectors were cotransfected into Neuro2A and HeLa Tet-On cell lines with the linearized Hygr gene at a 10:1 molar ratio, respectively. Eight single colonies in the Neuro2A Tet-On cell line only were isolated in the presence of 250 ug/ml G418 and 500 ug/ml hygromycin B. The clones were analyzed to determine the cellular levels of YY1 protein by western blot analysis after doxycycline treatment.
Western blot analysis
For our western blot analysis, the cells were lysed after different incubation times following doxycycline treatment for 30 min at 4°C using lysis buffer (0.25 M Tris-HCl, pH 7.8, plus 0.1% NP-40). Cellular debris was removed by centrifugation for 10 min. Protein concentrations were determined by the Bradford assay kit (Pierce). Thirty micrograms of lysate was separated on 10% SDS-PAGE gels and transferred to PVDF membrane (Hybond-P; Amersham) using a Mini Trans-Blot transfer cell (Bio-Rad). Membranes were blocked for 1 h in Tris-buffered saline containing 5% skim milk and 0.05% Tween 100 and incubated at 4°C overnight with anti-YY1 (sc-1703; Santa Cruz Biotechnology) or anti-GAPDH (MAB374; Chemicon) antibodies. These blots were incubated for an additional 1 h with the secondary antibody linked to horseradish peroxidase (Sigma). The blots were developed using a Western blot detection system according to the manufacturer's protocol (Intron Biotech).
RT-PCR and qRT-PCR
Total RNAs were first purified from transfected cells using Trizol as described by the manufacturer (Invitrogen); second, first-strand cDNA was reverse transcribed using the SuperScript First-Strand Synthesis System (Invitrogen); and finally PCR amplifications were performed using the following primer sets: human YY1 (hYY1F-2, 5'-GACCTCTCAGATCCCAAA-3' and hYY1R-1, 5'-TTGTTTTTGGCCTTAGCA-3'), mouse YY1 (mYY1F-2, 5'-GACCTCTCAGACCCTAAG-3' and mYY1R-1, 5'-TTGTTTTTGGCTTTAGCG-3'), and mouse GAPDH (GAPDH-RT-F, 5'-ATGACATCAAGAAGGTGGTG-3' and GAPDH-RT-R, 5'-CATACCAGGAAATGAGCTTG-3'). Also, quantitative real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad) using the icycler iQ multicolor real-time detection system (Bio-Rad). All qPCRs were carried out for 40 cycles under standard PCR conditions. We analyzed the results of quantitative real-time PCR based on the ΔΔCt method [20].
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Simon J, Chiang A, Bender W: Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products. Development. 1992, 114 (2): 493-505.
Luo C, Lu X, Stubbs L, Kim J: Rapid evolution of a recently retroposed transcription factor YY2 in mammalian genomes. Genomics. 2006, 87 (3): 348-355. 10.1016/j.ygeno.2005.11.001
Alon U: Network motifs: theory and experimental approaches. Nat Rev Genet. 2007, 8 (6): 450-461. 10.1038/nrg2102
Kim J, Kollhoff A, Bergmann A, Stubbs L: Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3. Hum Mol Genet. 2003, 12 (3): 233-245. 10.1093/hmg/ddg028
Mountford P, Zevnik B, Duwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A: Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci USA. 1994, 91 (10): 4303-4307. 10.1073/pnas.91.10.4303
Winer J, Jung CK, Shackel I, Williams PM: Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem. 1999, 270 (1): 41-49. 10.1006/abio.1999.4085
We would like to thank Dr. Kurt Svoboda for providing zebrafish; Jennifer M. Huang for critical reading of the manuscript. This research was supported by National Institutes of Health R01 GM66225 (J.K).
Additional information
Authors' contributions
JDK analyzed sequences and constructed all the vectors for the promoter assays, and also performed the ChIP experiment and wrote the paper. SY established the YY1 inducible cell lines and performed RT-PCR, western blot and the promoter assays. JK provided the original concept of the study, supervised the study, and contributed to writing the paper. All authors read and approved the final manuscript.
Jeong Do Kim, Sungryul Yu contributed equally to this work.
Authors' original submitted files for images
Below are the links to the authors' original submitted files for images.
Rights and permissions
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.
About this article
Cite this article
Kim, J.D., Yu, S. & Kim, J. YY1 is autoregulated through its own DNA-binding sites. BMC Molecular Biol 10, 85 (2009). https://doi.org/10.1186/1471-2199-10-85
DOI: https://doi.org/10.1186/1471-2199-10-85
- Promoter Assay
- Endogenous Locus
- Multilabel Counter VICTOR3
- Inducible Cell Line
- Control Luciferase Vector

subset: commoncrawl


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AI game theory tool to strengthen military cyber defenseNews
August 28, 2019
CAMBRIDGE, Mass. Charles River Analytics Inc., developer of intelligent systems solutions, has received additional funding from the U.S. Air Force to build a Game-Theoretic Reasoning and Analysis of Vulnerability (GRAVITY) tool.
GRAVITY analyzes vulnerabilities and executes rich models of behaviors to reason about systems in a game-theoretic manner.
Company officials claim that with GRAVITY, tools are being developed to analyze and respond to threats that exist in the gray area between peace and conflict within the military's embedded systems and networked technologies.
Charles River Analytics says that the GRAVITY tool includes a game-theoretic engine that takes stakeholder and system models as inputs and computes possible vulnerabilities, adversary strategies, and defensive counterstrategies. This modeling enables analysts to identify, visualize, and prioritize vulnerabilities.


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The whole party comes to life.
You can bring this experience to your next event by hiring Brass Animals!
Brass Animals is a cover band that is very familiar with jazz classics. Our skilled musicians can flawlessly imitate the sounds of jazz greats like Duke Ellington, John Coltrane, Miles Davis… and the list goes on.
Our jazz bands will provide your event with a unique, classy atmosphere that brings you back to the days of women's suffrage and bathtub gin. Our infectious enthusiasm will ensure that all of your guests are out of their seats and grooving!
So, for your next event, book Brass Animals for something unique, classy and fun that your guests will remember for years to come!
We play all kinds of events
Watch us perform some of our favorite songs
We've performed for some of the coolest companies around..
"We had the pleasure of having the Brass Animals grace our stage at a little brewery in south Austin. Nobody knew what to expect but they brought the Fun/ funk/ heat/ love/ grooves. They covered familiar songs but in new and provocative ways. I highly recommend Brass Animals for any of your events!"
Libby B., St. Elmos Brewing Company
Austin, TX


text: AAFL Memorandum: The DACM Office requested that each Army Acquisition Functional Leader (AAFL) provide an updated Recommended Continuous Learning Point (CLP) Memorandum for the currently cycle that runs from October 1, 2020 through September 30, 2022. These documents provide specific guidance to the Army Acquisition Workforce (AAW) professionals within the AAFL's designated Acquisition Career Field (ACF) on current and emerging critical technical areas. They also contain recommendations for courses/activities that can be counted as part of an AAW professional's Continuous Learning requirement, in order to further enhance knowledge and skills in the ACF. There is a signed AAFL Recommended CLP Memorandum for the following ACFs: Business-Cost Estimating (BUS-CE), Business-Financial Management (BUS-FM), Facilities Engineering (FE), Life Cycle Logistics (LCL), Production Quality and Manufacturing (PQM), Science and Technology Manager (STM), and Test and Evaluation (T&E). The Army DACM Office is still awaiting memorandums for Contracting (CON), Engineering (ENG), Industrial Contract Property Management (ICPM), Information Technology (IT), Program Management (PM), and Purchasing (PUR).


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Virtual reality technology is the next big thing in marketing and is expected to have a major impact on how the B2C and B2B brands plan their marketing campaigns. Soon, virtual reality platforms will no longer be the domain of only video gamers and early adopters.
The concept of augmented reality or virtual reality is already a hit. A survey by Greenlight Insights revealed that 71% of the consumers believe that brands using virtual reality are progressive, and 62% said they will feel more connected with a brand that uses VR technology.
The Future of VR
The future has big plans for VR, and walking around a virtual room is going to be a reality in no time. You will be able to shop in a virtual reality store, walk around, and add things to your cart by using hand controllers. You will also be able to browse through virtual clothing shelves, and pick the ones you like.
- The projected VR Active-User growth is on the rise, and in 2017, the VR product revenue is also expected reach $4.6 billion.
- In 2017, the consumer-facing brands under Forbes Global 2000 will be experimenting with virtual reality and augmented reality.
- VR marketing can influence the three parts of the brain which are responsible for forming perceptions and reactions in individuals in respect to their surroundings.
A Few Suggestions for VR Marketing Campaigns
VR marketing, like any other form of marketing, focuses on strategy and implementation. Here are a few tactics that can be used in VR Marketing:
- Panoramic Video – Enable customers to get a 360 degree view, so that they are able to experience it from all angles.
- Design a Cardboard VR Device – A branded cardboard device allows consumers to experience a VR campaign in the most cost-effective way. And if you design a branded cardboard VR device yourself, you can link yourself to both the experience and the device.
- Develop a VR App – Your brand can keep in touch with your customers continuously, and provide updates on new VR experiences with a VR app.
There is a lot more to virtual reality marketing. Find out other interesting facts about it in the following gifographic created and shared by Marketo and Column five


text: There is just no getting around the fact that almond paste is indispensable for these meringue-like cookies; it transforms their flavor and helps them brown evenly. Some brands sell in a tube; others will be in a can in the baking aisle. Using the tube is ideal since the canned might be looser, but either works for this recipe.


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Throughout the three-night excursion, tourists are becoming vaccinated with a Russian personal clinic in a resort and unite it with sightseeing of lockdown-free Moscow. "Plenty of Germans wish to visit Moscow to find the vaccinations and also to join the trip with vacation or with cultural schedule, therefore it functions," stated one of those tour organizers Hans Blank.
For 46-year-old software programmer from Stuttgart, Uwe Keim, vaccination in Russia is the only alternative available at the present time after Serbia halted vaccination for thieves. "In Germany found that acquiring a normal vaccine was roughly five to ten months later on, therefore it had been too long for me personally," said Keim.
Though Sputnik V hasn't been accepted for use in the EU, German vacationers anticipate the research published in the British medical journal The Lancet claims that the vaccine is 91% successful and seems to stop inoculated people from getting seriously sick with COVID-19.
"It is essentially like AstraZeneca, only very little differences so that I said okay, it is great enough for me," explained Enno Lenze who traveled to Russia for the first time. The Treaty tour is coordinated by means of a Norwegian travel operator Globe Buyers that have helped Russians residing in Germany to go to Moscow for vaccination.
Co-owner of all World Visitor and mind of this vaccination tours endeavor Albert Sigl asserts that ONF has little to do with the next excursion with German tourists. In accordance with Sigl, the company has just provided a service letter that should facilitate communicating with state bodies.
"We as the tour operator don't have any intention, political intent," states Sigl. "For all of us, it's a really easy business model" As Italian tourists like the simplicity of being vaccinated at the hotel, some Russian regions are short of vaccine supplies. Russian President Vladimir Putin's envoy to the Far East whined a week of shortage of vaccines at the Far East areas.
At precisely the exact same time, Deputy Prime Minister Tatyana Golikova said more than 8 million Russians have been vaccinated which is approximately 5 percent of the entire population, while based on Putin's statements of the month Russia has generated 20.1 million jabs.


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First Things First
If you see a 1, 2 or PET on the bottom of the bottle or jar, drop it in your local recycling bin. All of our cartons and glass bottles are locally recyclable, too!
To take care of everything else (like pumps, jars, caps & droppers), we have proudly partnered with TerraCycle®
This free, easy and innovative recycling program recycles all Bliss packaging into new, useful items -- resulting in zero waste.
How It Works:
Register for our free recycling program at terracycle.com
Rinse out your empties, box them up and ship to TerraCycle® using their pre-paid label.
That's it! They take it from there and find innovative ways to recycle all of it.
Thank you for joining us in our continued commitment towards sustainability!


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It's 2AM and your phone has just buzzed you awake, filling the room in white-blue LED light. You have a message. It's a photo. No words, no explanation. Just a photo. Tell us all about it. And what happens next. Creative Writing Challenge: 2AM Photo
Bling, bling! Bling, bling! Whenever I receive a text, my phone makes this awesome noise that my daughters call "Barbie Sparkles". I like the sound because it's loud and annoying enough to wake me up. But they are right, that's what it sounds like.
Groggily, I roll over to pick up the glowing, vibrating phone from the nightstand. Even though I'm still mostly asleep I notice the time is 2AM. My brain knows it's either the bank telling me that a deposit has cleared my account (I hate that sometimes I get those texts at 2AM!) or it's my sexy Loverman sending me a sweet dream (I really enjoy those texts at 2AM).
I flip the phone open and open up the awaiting Multimedia Message. There are no words, just a picture message from Loverman of the driver's side of the inside of my truck. The steering wheel looks like something smashed it into the dashboard, the airbag is deployed and deflated. Below I can see two legs sprawled out, mangled, jeans torn and bloody. I know exactly what happened. Loverman was in a car accident!
The grog immediately clears and I start to panic: Oh! My! God! Do I call the police? I don't know where he is! Who am I going to call for help? He is the one I always call for help! I have to go out and find him! SSSSHHHHIIIITTTT!!!!
I frantically grab the clothes laying on the top of the dresser and spend 5 minutes falling all over myself because, seemingly, I cannot remember how to get dressed. It's taking too much time and I am becoming more and more tangled with myself. "ARGH! This isn't helping!!!" I think. But, I can't think straight — what should I do? keeps screaming through my head over and over like a manic mantra. I am blinding myself with my fear, emotionally paralyzing myself in a state of utter panic.
It's not until I get to my left shoe and I start putting it on when I start to realize how frantic I'm being. I need to slow down and be careful. I think about Loverman again and how disappointed he will be with me if I re-break that darn ankle so close to being deemed "healed". Methodically, I concentrate on putting my left shoe on my healing foot and then I walk over to check myself out in the mirror because I don't want to look like a crazy-meth-head-on-the-loose (even though I kind of felt like one).
I look acceptable. Freaked out! But, acceptable. I shake my body all over, take one more deep breath (inhale… exhale…) and I am on my way down the stairs to retrieve my jacket and my keys. Kitty was sitting at the top of the staircase watching me and he decides that he wants to go downstairs at the exact time that I do. And in the exact same footfalls, too. I avoid his elusive furriness for the first couple of stairs, but his fuzzy body lands on the third stair at the same time as my left foot.
"Fuck this!", I hear myself say. I feel my body rolling down the last several steps and see my head hit the wall at the bottom. It's strange how it all seems like an out-of-body experience. Am I unconscious? Am I dead? Did anyone hear and are they coming to help me? What have I done?
… … …
My eyes open with a start! I am laying back in my bed. There's no pain. That's strange. I move my head back and forth slowly. My neck's not sore. Hmmmmm… I move my left leg around underneath the covers. Hmmmmm, again. The ankle is fine. It doesn't even feel sore…
Remembering what woke me up so abruptly, I look over at my phone and see that there is a new message. Hoping that it's Loverman with a sweet and sexy bedtime message for me, I open it. I could use something to take my mind off that crazy dream! I notice, as I open up my phone, that it's 2AM…
5 responses to "2AM Photo Scare"
Wow! Great job! I have never really done creative writing! Only journaling. My thoughts and feelings and blah,blah,blah….
It helps, but really sometimes I look back and see what I have written again and again and it just gets old. My marriage sucks and I have issues with accepting the limitations between MM and I. Okay. And what has changed? Nothing. I guess it's just that in those moments, spilling all of my thoughts onto paper (or rather, into my keyboard) just keeps me sane. So it's fine of course. But definitely NOT creative.
I may need to try one of these. That would truly be an exercise of my mind and possibly teach me a new style of writing and of expressing myself.
Great read. The ending leaves you hanging, yet….it doesn't. Great job!
[…] 2AM Photo Scare | smittenwithhim […]
[…] 2AM Photo Scare (smittenwithhim.wordpress.com) […]
[…] the text noise is really loud and annoying and would wake anyone up (my daughters call it "Barbie Sparkles" because it sounds like if Barbie was 6 and had a […]


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Map of Winter Haven Municipal Airport
Here is the location map of Winter Haven Municipal Airport.
Other maps of Winter Haven Municipal Airport: satellite map, google map, contour map, topographic map.
Where is located Winter Haven Municipal Airport?
Airport is located in Winter Haven, Polk County, Florida, United States. Winter Haven Municipal Airport is located 8 mi from Florida Polytechnic University and 24 mi from Sherwood Forest RV Resort. The nearest lake Lake Jesse is 1 mi away. The nearest beach Melt-Away Bay is 23 mi away. The nearest museum Florida Southern College Planetarium is 12 mi away. The nearest park Reedy Creek Mitigation Bank is 17 mi away. The nearest castle Cinderella Castle is 27 mi away. The nearest city Auburndale is 2 mi away.
- Orlampa Inc. Airport is 8 mi away
- Cherokee Ranch Airport is 12 mi away
- Chalet Suzanne Air Strip is 12 mi away
- Lakeland Linder Regional Airport is 17 mi away
- Plant City Municipal Airport is 25 mi away
- Zephyrhills Municipal Airport is 27 mi away
- Wauchula Municipal Airport is 39 mi away
- Orlando International Airport is 37 mi away
- Arbuckle Airfield is 38 mi away
- Tampa Executive Airport is 36 mi away
- Wimauma Air Park is 40 mi away
- Tampa North Aero Park is 39 mi away
- Orlando Executive Airport is 42 mi away
- Orlando Apopka Airport is 45 mi away
- Winter Haven Hospital is 3 mi away
- Heart of Florida Regional Medical Center is 11 mi away
- Bartow Regional Medical Center is 11 mi away
- Lakeland Regional Health Medical Center is 12 mi away
- Lake Wales Medical Center is 16 mi away
- Florida Hospital Celebration Health is 22 mi away
- South Florida Baptist Hospital is 24 mi away
- Osceola Regional Medical Center is 27 mi away
- Florida Hospital Kissimmee is 27 mi away
- Central Florida Behavioral Hospital is 30 mi away
- Dr. P. Phillips Hospital is 30 mi away
- Orlando Health Medical Pavilion - Spring Lake is 32 mi away
- Florida Hospital Zephyrhills is 30 mi away
- Saint Cloud Regional Medical Center is 30 mi away
Map of Monroe County Map of Marion County Map of Broward County Map of Lee County Map of Bay County Map of Glades County Map of Okeechobee County Map of Putnam County Map of Wakulla County Map of Leon County Map of Hardee County Map of Baker County Map of Lafayette County Map of Seminole County Map of Lakeland Map of Fellsmere Map of The Villages Map of Sanibel Map of Eustis Map of Marco Island Map of Plantation Map of Miami Gardens Map of Longboat Key Map of Naples Map of Loxahatchee Groves Map of West Melbourne Map of Deerfield Beach Map of Cooper City Map of Green Cove Springs Map of Pinecrest


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Showing 1 to 11 of 11 (1 Pages)


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Editors' Note: Apartment613 is crowdsourcing a directory of Ottawa restaurants offering takeout and delivery throughout COVID-19. Click here for the growing list.
Love Local Delivery is a delivery service that launched mid-March to connect local eateries with the patrons who miss them dearly. With drivers from Responsible Choice (a designated driving service), local restaurants are able to deliver ready-to-serve and heat-and-serve meals right to your door. In their own words, extraordinary circumstances bring out extraordinary responses, and our caring community is no exception.
View this post on Instagram
Welcome to @lovelocaldelivery, a new locally run food delivery service in support of your favourite local restaurants 😋 ⠀ ⠀ Next time you're taking a break from cooking, share some local love and order from one of our current participating restaurants:⠀ ⠀ •⠀ @olacocinataqueria •⠀ @daslokalrestaurant •⠀ @eatatbibis •⠀ @heartbreakerspizza •⠀ @northandnavy •⠀ @grayjay613 •⠀ @gruntottawa ⠀ Contact each restaurant directly to order and schedule your delivery. A charge of $5 applies for deliveries within a 5km radius of restaurants. Additional charges apply for further distances. #lovelocaldelivery #ottawa #613 #yow #ottawaeats #ottawafood #ottawarestaurants #ottawarestaurant #yowfood #yoweats #myottawa #igersottawa #ottawafoodie #ottawafoodies #ottawastrong #support613 #supportlocal #locavore
The list of participating restaurants grows every time I take a look. Hours and days vary by restaurant, and you order directly through the participating restaurant. Payment is accepted by credit card over the phone. Delivery is a set $5 fee and open to anyone within a 5km radius of the restaurant. Curbside pickup is also an option.
While the service started out in three neighbourhoods, the list has grown to over 30 participating restaurants in many of the downtown neighbourhoods. Are you a local restaurant who wants to get involved? Get in touch with Love Local Delivery online.
Participating restaurants (updated as of April 2, 2020)
ByWard Market/New Edinburgh/Vanier
- Ola Cocina Taqueria
- Das Lokal
- Bibi's Middle Eastern Kitchen
- Baccanalle
- Bistro Ristoro
- Fraser Café
- Khao Thai
- Ahora
- Coconut Lagoon
- Sushi Me
- Zak's Diner
- Heartbreakers Pizza
- Grunt
- Churchills
- Absinthe
- Thyme & Again
- Wellington Butchery
Glebe/Old Ottawa South
- Eldon's
- Oat Couture
- Pelican Seafood
- Patty's Pub
- Sweet Basil
- Thanjai
- North & Navy
- Gray Jay
- Orto
- Pirho
- Pascale's ice cream
- The Buchipop Burrow Shop
- La Fiesta Latina
- Kuidaore
- Cumin Indian Grill
- The Prescott
For the latest additions, follow Love Local Delivery on Instagram @lovelocaldelivery or visit their website.


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Last week I put a good amount of miles behind me driving a Hybrid Toyota Camry across the Mid & Northwest which for all intents and purposes could have also doubled as an oasis of silence, shielding me from the constant flow of information that we are all bombarded with on a constant basis through various media outlets. There is a lot to be said for tuning out, but I opted not to as I jumped from destination to destination. On my long drives, some as long as 850 miles in a single day, I passed the time enjoying extended stretches of silence to brainstorm creative ideas while intermitently listening to NPR radio and other local stations. The funny thing about having a little more time to think while traveling as I did is that subtle events and not so subtle events have a way of adding up to more clearly take form as a larger thought or issue.
Just before I left on my trip I received a comment on Flickr from Patrick Smith, a photographer here in Northern California, commenting on the merit of my watermark and how image theft is overblown. I shared my experience and moved on. After all even I would rather talk about other things than taking safeguards against image theft. Then while on my trip news broke of a family photo sent out as a Christmas card that somehow was taken off a family blog and used in a large commercial advertisement in Europe
- NPR report – Text and Audio
- Dear Social Media – the photographs take on the situation
- A Mom Goes Viral, Attracts Haters – more photos of the store front
Then days later I heard DJ's on a pop radio station in Utah discussing this, one DJ noting that "if it's on the Internet it's free." Lastly to come full circle I read a blog entry titled Creativity! by Patrick Smith upon my return about how one of his photos was being used in a "Photoshop This" thread on Fark.com.
So my question to you, "Did you get the memo that your online content is free?"
Clearly I'm a believer that my content is not free if it is placed online and I'm consistently appalled that people continue to claim others content online as being free. For some reason I find that people who do not create content whether an article, a photograph, etc. have a tough time wrapping their head around the concept of "Intellectual Property". It's much easier to just assume the world is their oyster… it's all there for the taking. One other observation I've made is that those who are broadcasting types (TV and Radio) do think of online content as being free far more than say writers or photographers.
I've written plenty on this subject and I think all of you know very well how I fall on this subject. For those of you that are new to my blog you can find my past thoughts on the following topics here: copyright infringement and photo watermarking.
What is your take on this mythical culture of free that people seem to errantly propogate in relation to your online photographs or writing?
I almost forgot… this came out on Sunday:
Use Their Work Free? Some Artists Say No to Google – NY Times
[tags]Photography, Copyright, Photo, Watermark[/tags]


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Gordon Hayward scored a career-high 44 points as the visiting Charlotte Hornets snapped a three-game losing streak with a 102-94 win over the Atlanta Hawks on Wednesday.
Hayward was 15-for-25 from the field and 4-for-9 on 3-pointers. Hayward broke his previous career high of 39 he last accomplished against Cleveland on Nov. 5, 2019.
Hayward also helped thwart an Atlanta comeback.
Atlanta overcame a 24-point first-half deficit and took an 86-85 lead on a three-point play from John Collins with 5:22 left. But Hayward answered with a basket and followed with a three-point play to restore Charlotte's lead to four points, at 90-86, with 3:55 left.
LaMelo Ball came off the bench to add 16 points, eight rebounds and five assists. Terry Rozier scored 14.
The Atlanta offense never got in gear. Trae Young, who entered the game averaging 28.6 points, scored only two points in the first half and finished with a season-low seven points. Young made only 2 of 9 shots, was 0-for-3 on 3-pointers and had seven turnovers.
Collins led the Hawks with 23 points and 11 rebounds. Kevin Huerter came off the bench to make a season-high five 3-pointers to score 19 and De'Andre Hunter added 17. Clint Capela had a season-high 19 rebounds.
Atlanta has lost three straight and four of its last five. The Hawks were 0-3 on the homestand.
The Hornets took control from the first quarter. Hayward scored 12 in the opening quarter and Charlotte led 27-11. It was the fewest points scored by Atlanta in a quarter this season.
The Hornets took advantage of Atlanta's poor perimeter shooting — 4-for-18 (22.2 percent) from the 3-point line in the first half — and led by as many as 24 points with a minute remaining. Charlotte led 59-40 at intermission.
Atlanta's second unit helped trim the lead and a Bogdan Bogdanovic basket with 2:25 cut the lead to 10 points. But the Hornets, with a driving layup from Ball with 1.6 seconds left, took an 81-67 lead after three quarters.
The two teams play again on Saturday in Charlotte.
–Field Level Media


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Smart Meetings is reporting on all the excitement at the IMEX America 2017 trade show, taking place Oct. 10–12. The first day of the seventh annual show was filled with incredible booth displays, great networking and business opportunities—not to mention lots of fun.
IMEX America, held in Las Vegas at Sands Expo and Convention Center, is the meetings and events industry's premier trade show. If you missed it, check out our footage of Day One highlights, and stay tuned! There's still much more to come. And if you're at IMEX America, come say hello at booth #A3213—we'll make you a Smart Meetings cover star!


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Two Countries is a Malayalam movie 2015 directed by Shafi. The movie starring Dileep, Mamta Mohandas, Mukesh, Aju Varghese, Jagadeesh, Ashokan, Suraj Venjarumood in lead roles. Music is composed by Gopi Sundar and Nadhirshah. Lyrics is written by B K Harinarayanan and Nadhirshah.
Posted by Web Desk on November 30, 2019
Posted by Web Desk on December 11, 2019


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To facilitate smoother fund management and easy financial reporting of the four commodities (maize, rice, wheat, and cassava) under the SARD-SC project, a 3-day session on financial management was held for accountants, some commodity specialists, monitoring and evaluation specialists, procurement specialists, and other staff from AfricaRice, ICARDA, and IITA. The training took place 23-25 July at the Africa Rice Center, Cotonou, Benin Republic, and was organized by the SARD-SC project. Oluseyi Fashokun, IITA/SARD-SC Project Accountant, facilitated the meeting.
The training aimed to, among other things, familiarize project staff the required processes and format of financial reporting and the fund management guidelines of the African Development Bank, the sponsor of the SARD-SC project. The use of proper reporting procedures and templates will aid in the timely submission of fund requests, accountability, and hence timely execution of the project. The discussion centered on finance and issues relating to processes and procedures inhibiting speedy financial reporting.
Some of the issues discussed and resolved include the following: Acceptable format for project financial statements, modality for timely project financial reporting acceptable to AfDB, process involved on reimbursement request, process and procedure for special accounts replenishment, process and procedure for direct payment request, external audit requirements, next external auditor's institution visit and work schedule, and plans and brainstorming for the next replenishment
Dr Chrys Akem, Project Coordinator, chaired the last day of the meeting and discussed issues as well as upcoming events in the year such as the following: preparation for the project's mid-term review, relevance and validity of the Project Implementation Manual, content and volume of all reports required by AfDB for the project, SARD- SC Project web site, AFDB's review and last External Auditor's Report.


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Words and Photos by Aaron Richter
I'd like to preface self-titled's South By Southwest wrap-up with a disclaimer. I made a simple rule this year: to follow my own tastes–strictly want-to-sees, no supposed-to-sees. Some of these happened to coincide with mass quantities of beer, but that's besides the point. Here are 10 artists I'm actually looking forward to hearing more from this year…
Only after a certain number of Lone Star tallboys can I really feel comfortable hurtling myself into a rowdy pit with three grand in camera gear stowed rather unsecurely over my shoulder. But JEFF is one of those bands that just makes you want to be in it all, as close as possible and holding up singer Jake Orrall as he crowd-surfs with his three-stringed guitar. Despite a few mic problems, their Saturday show at Mohawk probably had the best, fullest sound of anything I caught in Austin. Note: I came to town with relatively new, embarrassingly clean Chucks; this set dirtied 'em up proper.
I never managed to catch a full Yuck set, yet probably heard all of their songs scattered throughout three shows I straggled to late. My music-fanatic teeth were cut on '90s alt radio, and Yuck adheres so perfectly to my nostalgia receptors for the sort of post-DIY modern-rock crunch an In Utero–shirted, Columbia House–subscribing grade-school Aaron loved discovering.
I'll admit to a budding man-crush on Dirty Beaches' Alex Zhang Hungtai, who can hold his own on a stage all by his lonesome, playing guitar and singing like Roy Orbison over dreamy, Suicide-style loops. I'm a strong booster of the idea that music should be cool, and Dirty Beaches–all slicked-back hair with a comb in his pocket and rockabilly lip curl–is certainly that. But he backs up his image with moody execution, giving songs that might be more apt for late nights at home an edge of captivating intimacy. The set's highlight was a haunting cover of the Stooges' "No Fun." We'll start a campaign now to make it the soundtrack to the next Gaspar Noé flick.
Let's just say I'm somewhat of a dissenter when it comes to our issue No. 9 cover star Zola Jesus. Canadian singer Katie Stelmanis, on the other hand, does gothy, Fever Ray-ish dance music in her newish act Austra without alienating those who'd prefer their high drama tempered with pop cues, Ã la Bat for Lashes. And the best thing about it: Austra has singles–huge, massive ones that you can't help but wonder when you'll hear them graduating to big-room venues like Webster Hall. So much potential here. Can't wait to see where it takes them.
Nothing quite makes me want to boot a blogger in the throat quite like the "I had no idea what to expect†lede when "reviewing†live shows. But I'm gonna do exactly that right here. (Fuck off:I'm tired.) I really had no idea what to expect with King Tuff, his full-length being one of my absolute favorites in recent years. This project by Kyle Thomas–who also sings in Happy Birthday and Witch–always seemed much more like a sorely missed myth than an act I'd one day see live. The full band was rowdy, the bassist rocked a Carbonas tee, and Thomas bubbled with goofy charm. Gotta love a group that smiles onstage as much as these guys–no pose, no untouchable pretension, just a whole lot of dumb fun.
Caught these guys (and girl) twice in Austin after falling for them hard at a show in Atlanta this past year. Canada's White Wires are for anyone who shed a tear at the demise of Exploding Hearts–tight, energetic power pop with infectious bubblegum choruses. The best test of a fest like SXSW is which band you're listening to the week after you get back, and I've had the band's White Wires II and Girly Girly Girly albums on repeat since touching back down in Brooklyn.
At one point during Yelawolf's blistering Fader Fort set, I paused to contemplate licking him square across his tatted torso. Nothing much more needs to be said, though during self-titled's official SXSW panel discussion (alongside Pitchfork, The Fader, XLR8R and Kill Screen), Fader publisher Andy Cohn made a point about how eye-numbingly boring it is to see magazine covers that purvey in chiseled abs:at which point we both took a moment to acknowledge that Yelawolf is absolutely welcome to take his shirt off on a magazine cover any day without our judgment.
I remember my first time seeing Bloc Party–some Silent Alarm–era Coachella past–and being blown away at how fast they played their songs live–the guys just locked in and took off and you really had to hang on to keep up. Any band that can rocket-charge the tempo of their songs onstage earns an instant edge live, and Twin Shadow transfers what is essentially an intimate one-man bedroom-pop record into an exhilarating full-group stage show.
Michael Cera, playing bass live with this Sub Pop group, gets the most cheers from the crowd. But Mister Heavenly will earn its share from the undeniable chemistry between its core members, Man Man's Ryan Kattner and Islands' Nick Thorburn. Hearing them play together at Emo's, it's clear that each have a yet-unindulged musical itch that needs a serious scratching.
If anyone deserves to carry the Jay Reatard torch, it's this Texas group, who've gained remarkable stage presence as a trio since I last caught them about a year ago. Or maybe that extra confidence arrived via the all-too-familiar liquid variety in the case of guitarist/singer Orville Neeley, who seemed pretty obliterated onstage at Shangri-La on Saturday. Not that it hindered the music at all; in fact, his already-ferocious guitar playing sparked with added vitality, and for Bad Sports' sputtered brand of shredded punk, a sloppy, blurry-eyed show should be worn like a badge of honor.


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The Central Bank will conduct a "deep dive" into the role of property funds in the domestic commercial real estate sector and assess whether a macroprudential response is needed to any potential risks, Governor Gabriel Makhlouf said today.
Research from the Central Bank showed that Irish investment funds now account for more than 35% of the rapidly growing commercial real estate market, having invested a total of €18 billion in property and land here
"My prime motivation is to understand much, much more and shine a light on exactly what's going on because one thing I want to absolutely avoid is getting on the back foot if something happens and it has an impact on the financial system as a whole," Mr Makhlouf told a news conference today.
The Central Bank also announced that it would not force lenders here to hold more capital under two buffers it levies on them - the countercyclical capital buffer (CCyB) and Other Systemically Important Institutions (O-SIIs).
Under the O-SIIs, banks identified as systemically important to the domestic economy because of their size and market share must hold additional capital.
Barclays and Bank of America were designated as O-SIIs for the first time today after they moved significant operations to Ireland as a result of Brexit.
The bank's Irish subsidiaries will have to set aside 0.75% of risk-weighted assets.
Meanwhile, UniCredit and Depfa Bank's Irish units are no longer designated as O-SIIs, the Central Bank said.
Central Bank Governor Gabriel Makhlouf, one of the newest members of the European Central Bank's Governing Council, also said today that negative interest rates were an effective measure.
But he added that that they would be more effective if governments backed them up with fiscal measures.
"Are negative rates working? I think they are working, which is why the governning council took the decisions it did back in September," Mr Makhlouf told today's news conference in Dublin.
"The point that Mr Draghi made at the time is that monetary policy would work much more effectively if it was supported by other tools, in particularly fiscal tools," he added.


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Bollywood fashionista's always look their best during movie promotions. SC rounded up last month's best dressed celebrities and we'll tell you how their styles can fit to suit your own body shapes perfectly!
Sonam Kapoor always makes it a point to dress to impress and her fashion choices for Khoobsurat promotions are no exception. While making the promotional rounds, Sonam opted for a midi skirt from Asos, teamed with an Alexander Wang top and a Rajesh Pratap Singh jacket. Dolce & Gabbana heels completed her look. This look is a great option for women with column body types. The loose, flaring silhouette cleverly hides the lack of curves.
At another promotional event, the actor looked pretty in a Stefanel maxi dress layered with a chic Pankaj and Nidhi bomber. This outfit will work well on hourglass body types (the cinched waist will emphasise the narrow waist perfectly). The bomber jacket is a great way to cover up plump arms. This look works as both, as a glamorous day look and for a casual dinner out – or if you're meeting your girls for drinks!
Deepika Padukone looks effortlessly chic in the casual outfits that she has been wearing for her Finding Fanny promotions. While in Hyderabad, she wore a blush pink sleeveless blazer from Frou Frou by Archana Rao, fresh off the LFW runway. She teamed this with a white tank top and a pair of white jeans, keeping the outfit simple. This look will work well with almost all the body types and sizes.
Esha Gupta has been making some waves with her elegant fashion choices that have us impressed to no end. She chose to wear a belted tie and dye maxi dress from Ikai with nude oxfords to the screening of Finding Fanny. Though Ms. Gupta is quite tall, this look will work well for short and petite girls too, as the single dark color creates an unbroken line thus providing an illusion of height.
Priyanka Chopra promoted Mary Kom in Jaipur wearing a yellow top by QuirkBox, from their newest Mumbai-inspired collection for Lakme Fashion Week. She teamed it with a pair of draped dhoti pants by Pankaj & Nidhi which made for a smart, balanced outfit. This kind of look will work to great advantage for women with goblet or lollipop (top-heavy) body types as it draws the eye to the lower part of the figure. The cowls in the pants create a broader silhouette for the legs, making the figure look balanced.
Check back with us for more celebrity style updates and sign up now to chat with our stylists!


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A look at sports and sports memorabilia popularity south of the border in Mexico
Fernando Valenzuela pitched in the major leagues for six teams in his 17-year career from 1980 to 1997, highlighted by his first 11 seasons with the Los Angeles Dodgers.
The lefthanded pitcher compiled a 173-153 lifetime record, with six All-Star seasons. His unorthodox windup, his screwball pitch brought Fernandomania to worldwide fame.
In 1981, Valenzuela skyrocketed to stardom, winning his first eight starts (five of them shutouts). Valenzuela finished the strike-shortened '81 campaign with a 13-7 record and a 2.48 ERA. He became the first, and still the only, player to win both Cy Young and Rookie of the Year awards in the same season.
Valenzuela is still the man in this tourist-filled beach city – and throughout Mexico.
"He was a big name (from Mexico), maybe the biggest from the big four (male team sports)," said Isaac Galvez, the director of restaurants and bars at the Marriott Puerto Vallarta Resort & Spa, which features a memorabilia-filled Champions Sports Bar on property – and where his office is located.
"Whenever I hear the name Fernando Valenzuela, I always see the LA logo, for the Los Angeles Dodgers. He was a huge name of Mexican sports."
Valenzuela – who hails from Navojoa, Sonora, Mexico – was a National League (NL) All-Star in each season from 1981-1986, and won a major league-leading 21 games in 1986, though Mike Scott of the Houston Astros beat him out in the Cy Young Award voting that year.
Valenzuela also was a heralded hitter, smacking 10 career home runs. In fact, former Dodgers manager Tommy Lasorda even used Valenzuela as a pinch-hitter at times.
Valenzuela threw a no-hitter on June 29, 1990, against the visiting St. Louis Cardinals.
Ironically, Dave Stewart of the Oakland Athletics threw a no-hitter earlier that day, too.
After leaving the Dodgers, Valenzuela spent time with the California Angels, Baltimore Orioles, Philadelphia Phillies, San Diego Padres, and St. Louis Cardinals.
Valenzuela threw the ceremonial first-pitch at game two of the 2017 World Series, drawing cheers from the Los Angeles faithful – and memory smiles from a generation that always will remember, Fernandomania.
Valenzuela first appeared on cards in 1981, with his Topps Traded card (No. 850) selling for about $7. He also appeared on the 1981 Topps card No. 302 of three Dodgers Future Stars, along with Mike Scioscia and Jack Perconte. Plus, he is on card No. 140 in the 1981 Fleer set.
Despite Fernandomania, baseball is not that big in Puerto Vallarta.
Soccer – or futbol, as it's known – is the sport-of-choice in this city, Galvez said.
"Soccer is the No. 1 sport in Puerto Vallarta, and the entire country," he said. "For most Mexicans, it's soccer and pretty much that's it."
Locals are clearly divided on their allegiances.
Club Deportivo Guadalajara is the official team name, though it's often just known as Guadalajara and most commonly, Chivas. This team is based in Guadalajara, Jalisco – and is one of the 10 founding members of the Mexican First Division – and is, arguably, the most popular.
Many locals also side with Club de Fútbol América S.A. de C.V., commonly known as Club América, or simply as América. This team is based in Mexico City.
Hugo Sanchez, a soccer star from Mexico in the 1980s and 1990s, is often called the best Mexican soccer player ever and one of the greatest players of his generation.
Many locals also recall the goaltending heroics, and flashy uniforms, of Jorge Campos.
Other noteworthy sporting icons among locals in Puerto Vallarta include Tony Romo, Eduardo Nájera and Horacio Llamas.
"The city (of Puerto Vallarta) is mostly dedicated to tourism, so it doesn't seem that people want to pursue a career in sports," said Galvez, noting that none from PV have made it into the sporting spotlight of baseball, basketball, football or hockey.
Basketball is the second-biggest sport among locals, particularly NBA, though NCAA March Madness annually brings out a strong group of fans. At Champions, Galvez said, the restaurant-bar is filled with fans of numerous teams. Golden State is, not surprisingly, the most popular NBA team among locals.
Football follows basketball in popularity among locals, mostly because of the heat making it difficult to play much tackle football. That said, Galvez noted a version of flag football, played locally by women, "is pretty popular." He said Pittsburgh, New England and Carolina are the three most popular NFL teams in PV.
When asked about hockey and the NHL, Galvez said there is no interest, mostly because there are no rinks in Puerto Vallarta. That said, Champions and other area sports bars are filled with fans for the Stanley Cup and the NHL playoffs, though it is mostly Americans or Canadians on vacation.
Golf and tennis are popular, particularly on TV, in Puerto Vallarta, and there are numerous amateur volleyball tournaments held there.
Galvez said the five most popular WWE stars among locals are, no surprise: The Undertaker, John Cena, The Rock, Hulk Hogan and "Stone Cold" Steve Austin. Mexico also has produced its own pro wrestling – lucha libre, as its none in Mexico – stars, such as El Santo, Blue Demon, Rey Mysterio Jr., Tinieblas and Atlantis.
Galvez, who still plays soccer locally, was a baseball card-collecting kid. But he doesn't have any of his cards anymore.
There are no card shops in Puerto Vallarta, but, "I think you will find a shop or two that will sell trading cards," he said."But (the cards) will be pricey."
Champions at the Marriott Puerto Vallarta Resort & Spa is filled with sports memorabilia, including signed jerseys, bats, photos and more.
"Memorabilia is not too big (or popular) in Mexico; there are very few people here who collect memorabilia," said Galvez.
Champions has memorabilia on display of Fran Tarkenton, Cal Ripken, Brett Favre, Bob Griese, Wayne Gretzky, Michael Jordan, Larry Bird, Dan Marino, Joe Montana and others.
They do not change the memorabilia at Champions, "but I want to," Galvez said.
Ross Forman is a freelance contributor to Sports Collectors Digest. He can be reached at email@example.com.


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LADbible x
PlayStation 5
Art Direction, Social Media, Mobile & Web
for the LADbible Group launch campaign
of PlayStation 5.
Creating a social media style campaign that references having the day off.
I was commissioned by the LADbible Group to create their launch campaign of PlayStation 5.
The campaign was mainly focused around their Social Media platforms to give the impression that all their staff have the day off to play the new PlayStation 5 on the worldwide launch date and also run a competition for their audience for a chance to win a PS5 console.
Each channel needs to have an in-feed Instagram Post announcing the chance to win a PS5.
The channels which were included in the campaign were: LADbible, SPORTbible, GAMINGbible, UNILAD and UNILAD Tech.
Launch Date: 19 Nov 2020
Over 500k engagements
Over 100k new followers
Banners (Static & Motion)
A variety of digital banners were developed in static and motion form to be showcased across the websites of the company.
The banners will drive the audience to the competition landing page where they can enter their best excuse and their details.
Social Media Posts (Static & Motion)
Banners (Static & Motion)
Streaming Assets
As part of this campaign, additional images were created to hype up the idea that LADbible Group has been given the day off to the staff
to play the new PS5 all day.
These images had to feel like they are really coming from the staff, so need to look like screenshots from peoples WhatsApp groups/ workplace groups and out of office emails and Messenger.


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24/7 Telehealth Services
We're making sure you get quality care, whenever and wherever you need it.
In this difficult time, we're introducing free telehealth services so you can talk with licensed providers from home, either over the phone or via videochat.
We're offering several options to meet every need:
- Talk with someone about everyday stress, anxiety, or other problems you're facing. Get free counseling by calling the Laborers' Support Network at 800-522-6763 and requesting a telehealth referral. Arcadian Telepsychiatry will then reach out to you to schedule an appointment. Learn more here.
- Consult a physician for minor medical or emotional health concerns. Access free care by calling or downloading the WellConnection app. Learn more here.
- Access existing in-network care. The Fund will cover all telehealth visits if your provider is in the Blue Cross Blue Shield of Massachusetts EPO network.
- Get tips online on how to manage stress and anxiety. The Laborers' Support Network's online resources are available 24/7 at hmc.personaladvantage.com, access code: MALAB.
If you have questions about accessing these services, please call our Health Coach Kristin Woodruff at 781-496-5715.


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HDA's 2020 Traceability Seminar will now take place online! While we are not able to gather in Washington, D.C., seminar attendees can expect the same high-quality educational programming delivered annually by DSCSA and traceability experts. This event is designed for pharmaceutical distributors, pharmacies, manufacturers and other supply chain stakeholders responsible for the implementation of traceability strategy, systems and processes.Registration will open in the coming weeks. For questions, contact Lisa Anderson at (703) 885-0220.
Companies in this article


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February 19, 2017
Most Philadelphians know that living in the big city means you need to constantly be on the lookout for unexpected happenings.
Especially in the City of Brotherly Love, a place that is home to a rich history, a vibrant culture and a vast array of characters. Adventure lurks around every corner.
In just the past two months, the city has featured subway riders dropping their drawers, a scratch-off loser phoning in violent threats against lottery officials, a television meteorologist denying rumors of being on house arrest, and the Mummers Parade. Just to name a few things.
The city delivered another odd story when SB Nation writer Josh Priemski walked into a "random store in Old Philly that just sells old stuff to hipsters."
While inside, he saw a 1991 Sports Illustrated magazine. Nothing particularly special, but a cool find. The issue features NBA legend Michael Jordan on the front, but it's famous for its holographic cover.
Almost bought this a moment ago pic.twitter.com/EX8xGiZ3Ge— josh (@HoopPlusTheHarm) February 18, 2017
Priemski left the store, but something was bothering him about the magazine. Five minutes later, it dawned on him:
WAIT A SECOND DOES THIS MEAN THAT pic.twitter.com/lWJekUKV34— josh (@HoopPlusTheHarm) February 18, 2017
Could this really be former Philadelphia 76ers great Charles Barkley's copy?
The address corresponds to a Bala Cynwyd house inside a gated community on the Main Line and Barkley was playing his final season with the Sixers at the time.
The evidence checked out for several Twitter users and Priemski, who rushed back to buy it.
$5. I am now the proud owner of an old copy of SI with a holographic image of Jordan on the cover once owned by THE Charles Barkley.— josh (@HoopPlusTheHarm) February 18, 2017
"The things you find in Philly, man," Priemski wrote.


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Nails aren't just for shredding furniture!
Scratching is a normal feline behavior. Many people think that scratching is a destructive behavior when it is actually a necessary part of your cat's physical and emotional well-being. Even cats who have been declawed continue to exhibit scratching-like behavior. Scratching serves many vital functions, as we shall learn.
One important part of nail health is the removal of the dead outer nail sheath so that the new nail growth underneath is exposed. When a cat digs its claws into an object it is helping to pull off that outer sheath (that's part of the reason many cats love leather couches!). The ability to scratch effectively on an object is also a great way for a cat to fully stretch its body.
Scratching also serves as a marking behavior. Cats leave a visual mark by raking their claws down an object and also leave a scent mark through scent glands in their paw pads. Visual scratch marks allow approaching cats to see the mark at a distance and may help prevent potential conflicts. Cats who come up close will be able to interpret the scent communication.
Scratching is also used as a displacement behavior. An anxious or excited cat will often use scratching to displace some of the pent-up energy. A good example of this is the cat that runs to scratch on something in anticipation of dinner being served or after a tense encounter with a companion cat.
So, how do we minimize the damage to our living space? The best way is having one–or several–dedicated scratching areas (i.e., scratching posts). Scratching areas can come in all shapes and sizes and various materials, depending on your cat's preferences. Some cats prefer a horizontal scratching area (cats that scratch on the carpeting) and will do well with cardboard or carpet scratchers. Other cats prefer vertical spaces (door frames, backs of couches). These cats do well with scratching posts covered in thicker material, such as rope or leather. Homemade rope scratching posts are easy to make by gluing thick rope around a cardboard tube. Another easy way to help limit damage is to routinely trim your cat's nails every 4 – 6 weeks. If done properly, most cats can be trained to easily accept nail trimming. If you have never trimmed your cat's nails and would like to learn how, we can show you how at the clinic. The final way to help deter unwanted scratching is the placement of Soft Paws. These are rubber caps that glue over the nails. They are painless and will naturally fall off after 4 – 8 weeks. We do NOT recommend declawing as a way to stop unwanted scratching behavior. Declawing is not simply removal of the nail: it is amputation of the toe and the closest joint to the nail. It can possibly cause chronic pain, worsen or bring on behavioral issues, or have post-surgical complications.
Again, if you have any concerns about your kitty's scratching behavior, give us a call at (406)728-0022. We're happy to help! Hope you're all having a wonderful Summer!


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Table of Contents
The bitcoin ledger is the software that underlies virtual monies and aids in the appropriate behavior and recording of all activities. However, the bitcoin blockchain isn't only a financial value; bitcoin traders may use it for several purposes. Bitcoins Blockchain technologies make online recordings nearly everlasting and unchangeable. A significant quantity of computational resources and collaboration is required to undo a transfer and mask a person's traces. Which is the reason the bitcoins distributed ledgers is known as the "long chain of certainty." Additional Bitcoin blockchain apps, websites like Bitcoin, and the money created to take advantage of Bitcoin's distributed ledger technology. They stated that there are four bitcoins distributed ledger ideas that got developed currently.
1. Smart Contracts
Bitcoins distributed ledger applications' potential usefulness is already being uncovered. Smart contracts seem to be adaptable electronic agreements that seem to be enforceable by law and stored on the bitcoins blockchain. Rather than relying on a particular centralized power, programmers design law agreements as parameters and assertions to transfer money utilizing the bitcoin blockchain as a "third company administrator." For example, suppose two persons wish to trade five hundred dollars once at a specified moment in the long term. In that case, if a series of prerequisites are fulfilled, it will create a smart contract with those circumstances, participants' information, and rewards. When the innovative contract blockchain's standards get fulfilled, this would collect the money and transmit it to the correct entity according to the agreements. Bitcoins distributed ledger specialists would concentrate their attention on testing and adapting the bitcoin ledger to all community sections in the upcoming decades. Irrespective of whether the product is adopted again worldwide, the bitcoins ledger remains to change how our civilization operates.
2. Supply Chain Management
Manufacturing connectivity is a mechanism that connects a company with its suppliers. The Bitcoin Blockchain assures us to develop an option for stockpile connection problems through the computerization of advantages. It considers goods to be tagged and assigned with one-of-a-kind letters that are subsequently transferred over onto the bitcoin database, which is unchangeable, transparent, and safe. The Bitcoin Database assists in tracking important product information such as the product's condition, use period, timestamp, and location. Bitcoin Database could successfully transfer the manufacturing system onto a shop system using bitcoins blockchain-powered asset digitalization. A bitcoins blockchain-enabled distribution network allows customers to confirm the legitimacy of things bought. Goods could be traced precisely throughout multiple sites and phases in the distribution chain. As a result, parties would be able to identify and address any possible issues.
3. Paying Employees
It only stands to reason that perhaps the bitcoins database, which owes its origins in economics, might be utilized to recompense workers. If your firm constantly provides payments to immigrant workers, introducing Bitcoins into the payment process might get you a lot of money. Management and workers could conserve the cash and effort by using a Bitcoin-based employment solution to avoid the hefty prices of foreign currency transfers and the period required for those monies to transit from institution to institution. You could know precisely wherever the cash is everywhere in the process by utilizing a shared blockchain that records all activities in sequential sequence. Now there is the issue of compensating subcontractors and distant workers. This kind of payment is a massive part of the private company, and it's anything that a lot of big businesses (and institutions) rely on each year.
4. Electronic Voting
DPOS seems to be the quickest, most economical, most decentralized, and most adaptable agreement methodology accessible, according to a globally dispersed network. DPOS uses shareholder endorsement polling to address problems of agreement fairly and transparently. Appointed representatives may adjust any system parameters, including fee structures, block durations, and payment sizes. Payments are processed in a minimum of one second, thanks to predictable block generator choice. The agreement mechanism, maybe most critically, is meant to safeguard all parties from unwelcome governmental meddling. The bitcoins blockchain's prospects may brighten in the following years. I've submitted around six inventions in this field in the last six months. Surprisingly, there were around twelve hundred inventions filed that included the bitcoins ledger. It'll only get bigger.


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You can tour this property via live video chat. We perform video walkthroughs while you are on the line via your favourite mobile chat app (Whatsapp or Viber). Request a viewing now.
A good size bungalow built on an exceptionally massive 90% building density plot of 1307sq. This property needs to be viewed to fully appreciate its massive plot size. This property offers further scope for expansion both above and around the big plot as its 90% building density allows it. It is however in need of full renovation. The side entrance to the property (currently used as the main entrance) leads onto the open plan kitchen/diner and living area. Off the kitchen area is a hallway leading to the 3 spacious bedrooms, all with built in wardrobes. There is a large bathroom with vanity unit and a WC. The garden is of exceptional size with ample room to add a swimming pool. Separate Annex of around 74sq at the corner off the plot is also available. House is situated not far from the commercial centre of town.
Stamp duties is tax paid by property purchasers to the land registry when the property sales agreement is registered with the land registry. Transfer fees paid by property purchasers to the land registry when the title deed of the property is transferred to their name.
Disclaimer - Property reference 227941. The information displayed about this property comprises a property advertisement. BuySellCyprus.com makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and BuySellCyprus.com has no control over the content. This property advertisement does not constitute property particulars.