Empirical insights into the benefit from implementing smart contracts

Smart contracts are highly relevant due to their support for new decentralized business models and processes. We empirically investigate the benefit of implementing smart contracts. Our approach measures the benefit by capturing the impact of implementing smart contracts on processes directly. Thus, our research supersedes previous research that uses deductive approaches for deriving beneficial effects from technical and architectural properties of smart contracts and blockchains. We conduct a systematic approach using the aspects cost, quality, time and flexibility, and their impact on the four process phases interest, agreement, fulfillment, and assessment. Our research enables decision-makers to make decisions on implementing smart contracts more precisely. Furthermore, decision-makers become able to develop more target-oriented initiatives

Geophysical Prospection of a Bronze Foundry on the Southern Slope of the Acropolis at Athens, Greece

The sanctuary of the Acropolis of Athens in Greece provided one of the first monumental bronze statues some 2500 years ago, which was dedicated to the goddess Athena. During recent decades, important understanding of the statue's manufacturing processes has been achieved by archaeological studies, and the former production site has been identified on the southern slope of the Acropolis. Two major bronze production pits have been detected and one was excavated in 2001 and 2006 and was found in an unexpected location. Therefore, in 2010 a geophysical survey of the whole production site was carried out for the first time in order to either reveal or to exclude any further sites of the bronze foundry complex. A combination of different geophysical methods was applied to survey the subsurface; magnetometry (MAG), two- and three-dimensional electrical resistivity tomography (ERT), as well as two- and three-dimensional ground-penetrating radar (GPR). Two major anomalies have been identified in the processed data, which provide evidence for additional production sites. One was a known site identified in a test trench in 2001, and our survey has outlined the extent of the former pit. The other anomaly, which was detected by ERT and GPR, was 8–10 m in length and 2–3 m in width and is oval-shaped and about 2.5 m deep. Steep vertical walls, together with two narrow points at the ends of the pit, which could reflect former entrances, were identified. Virtual ERT and GPR models generated from cross-sections of a ground-based LiDAR scan of the 2001 and 2006 excavated pit helped to interpret and understand the geophysical data of anomaly 2. This anomaly was finally interpreted as a newly detected production pit of the bronze foundry complex, and based on these findings new excavations are planned

Zinc Finger Independent Genome-Wide Binding of Sp2 Potentiates Recruitment of Histone-Fold Protein Nf-y Distinguishing It from Sp1 and Sp3

<div><p>Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The <u>S</u>pecificity <u>p</u>roteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs <i>in vitro</i>. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions <i>in vivo</i> raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters <i>in vivo</i>. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin <i>in vivo</i>.</p></div

The N-terminal domain of Sp2 can rescue target gene expression.

<p>(A) The N-terminal domain of Sp2 has the capacity to activate transcription. The Gal4 DNA-binding domain (Gal4), and Gal4-Sp1NT and Gal4-Sp2NT fusions were transfected into HEK293 cells along with a 5xUAS-luciferase reporter construct. Fold activation by Gal4-Sp1NT and Gal4-Sp2NT is expressed relative to the Gal4 DNA-binding domain set to 1. (B, C) Relative expression of the <i>Nlk</i> (B), <i>Grb2</i> and <i>Oxr1</i> (C) genes in wt MEFs, <i>Sp2ko</i> MEFs and in <i>Sp2ko</i> MEFs re-expressing full-length Sp2 (Sp2FL) or the Sp2NT and Sp2ZF mutants. (C) Schematic representation of the exon-intron structures of the <i>Grb2</i> and <i>Oxr1</i> genes and the promoters bound by either Sp1/Sp3 or by Sp2. The primer pairs P1-P2 detect specifically transcripts derived from the Sp2-bound promoters. The primer pairs P3-P4 detect all transcripts. Transcript levels in wt MEFs determined by qPCR were set to 1. <i>Gapdh</i> mRNA levels were used for normalization. Data are presented as the average of three independent experiments +/-SD.</p

The majority of the Sp2 binding sites are also bound by the heterotrimeric transcription factor Nf-y.

<p>Binding sites of the trimeric transcription factor Nf-y were determined by ChIP-seq of the Nf-ya, Nf-yb and Nf-yc subunits using wt and <i>Sp2ko</i> MEFs. (A) Venn diagrams showing the overlap of high-confidence Nf-ya, Nf-yb and Nf-yc binding sites in wild type and in <i>Sp2ko</i> MEFs. (B) Venn diagram showing the overlap of Nf-y and Sp2 binding sites. (C) The strength of Nf-y binding correlates with the strength of Sp2 binding at shared sites. Normalized ChIP-seq tag counts at individual Nf-ya and Sp2 peaks were plotted against each other. (D) Representative genome browser snapshots of promoters bound by Sp2 as well as by Nf-y (<i>Mcm3</i> and <i>Pan2</i>), only by Nf-y (<i>Wapal</i> and <i>Atxn3</i>) or only by Sp2 (<i>Fanci</i> and <i>Taf1c</i>). (E) Sequence motifs enriched at Sp2 sites also bound by Nf-y (left), at sites only bound by Nf-y (middle) or only bound by Sp2 (right). The numbers next to the logos indicate the occurrence of the motifs, and the statistical significance (<i>E</i>-value) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.ref048" target="_blank">48</a>]. (F) Central motif enrichment analysis of the motifs shown in Fig. 7E. (G) Venn diagram showing the overlap of Sp1/3, Nf-y and Sp2 binding sites. (H) Left, sequence motifs at sites that are bound by Sp1/3 but not by Sp2 and Nf-y. Right, sequence motifs at sites that are bound by Sp2 and Sp1/3 but not by Nf-y.</p

Different binding site selection of Sp1/Sp3 and Sp2 in MEFs.

<p>Binding sites of Sp1 and Sp3 in MEFs were determined by ChIP-seq. (A) Venn diagrams showing the overlap of Sp1 or Sp3 peaks obtained with two different antibodies for each factor. (B) Venn diagrams showing the overlap of Sp1 and Sp3 peaks. 3597 overlapping peaks were obtained with all four antibodies. (C) Distribution of Sp1/Sp3 binding sites in MEFs relative to annotated genes (TSS, +/- 500 bp). (D) Overlap of high-confidence Sp1/Sp3 and Sp2 binding sites. (E) Representative genome browser snapshots of promoters bound by all three Sp factors (left), by Sp1 and Sp3 but not by Sp2 (middle), or exclusively by Sp2 (right) in wild type (wt), <i>Sp2ko</i> and <i>Sp3ko</i> MEFs. (F) ChIP-qPCR validation experiments using Sp1_Ab1, Sp2_Ab1 and Sp3_Ab1. Percent of input values represent the mean of at least three independent experiments +/- SD.</p

Sequence motifs at Sp1/Sp3 and Sp2 binding sites in MEFs.

<p>(A) Sequence motifs enriched at Sp1/Sp3 and Sp2 binding sites. Logos were obtained by running MEME-ChIP [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.ref048" target="_blank">48</a>] with 300 bp summits of the top 600 Sp1/Sp3, Sp2, Sp1/Sp3-specific and Sp2-specific ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, and the statistical significance (<i>E</i>-value). (B) Central motif enrichment analysis of the motifs shown in Fig. 2A. (C) Occurrence of the canonical “CCAAT” sequence at the top 600 Sp1/Sp3, Sp2, Sp1/Sp3-specific and Sp2-specific ChIP-seq peaks. (D) Pairwise distribution of the canonical “CCAAT” sequence at the top 600 Sp1/Sp3, Sp2, Sp1/Sp3-specific and Sp2-specific ChIP-seq peaks. Each short blue line indicates a CCAAT motif. Black lines connect two CCAAT motifs in a promoter if they are located within a distance of 30 to 50 nucleotides.</p

The <i>bona fide</i> DNA-binding domain of Sp2 is dispensable for genomic binding.

<p><i>Sp2ko</i> MEFs re-expressing Flag-tagged full-length Sp2 (Flag-Sp2FL), the N-terminal domain (Flag-Sp2NT) or the C-terminal zinc finger domains (Flag-Sp2ZF) were subjected to ChIP-seq analysis. (A) Venn diagram showing the overlap of sites bound by Flag-Sp2FL, Flag-Sp2NT and Flag-Sp2ZF with sites bound by endogenous Sp2 in wild type MEFs. (B) Full-length Sp2 and the N-terminal region of Sp2 have similar chromatin binding efficiencies. ChIP-seq tag counts (normalized to 20x10⁶ reads) at individual Flag-Sp2NT and Flag-Sp2FL peaks found in both samples were plotted against each other. (C) Sites bound by Flag-Sp2NT represent high affinity binding sites of native Sp2. Individual native Sp2 peaks [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.ref018" target="_blank">18</a>] were plotted against their normalized tag counts. Those sites that were also detected by ChIP-seq in <i>Sp2ko</i> MEFs expressing the N-terminal domain of Sp2 (Flag-Sp2NT mutant) were overlaid with red dots. (D) Representative binding profiles of Sp2 in wild type MEFs (Sp2 / wt), and of Flag-Sp2FL, Flag-Sp2NT and Flag-Sp2ZF expressed in <i>Sp2ko</i> MEFs. (E) Schematic representation of the genomic binding features of Sp1/Sp3 and Sp2 based on the results shown in Figs. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.g001" target="_blank">1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.g004" target="_blank">4</a>.</p

Nf-y is necessary for recruitment of Sp2 to shared sites.

<p>(A) Sp2 and Nf-y are bound simultaneously to their target sites. Sp2 and Nf-y occupancy at selected target promoters was analyzed by sequential ChIP (re-ChIP) using anti-Sp2 and anti-Nf-yb antibodies as indicated. The <i>Sp2</i>, <i>Osbp</i>, <i>Sp1</i>, <i>Amd1</i>, <i>Nxt1</i> and <i>Nipal3</i> promoters are co-occupied by Nf-y and Sp2; the <i>Fanci</i> promoter is only bound by Sp2 but not by Nf-y; the <i>Atxn3</i> promoter is only bound by Nf-y but not by Sp2, and the <i>Raf1</i> promoter is neither bound by Sp2 nor by Nf-y. The percent of input values are mean +/- SD (n = 3). (B, C) MEFs were treated with a control siRNA (sicontrol) or siRNAs targeting <i>Nf-ya</i>, <i>Nf-yb</i> or <i>Nf-yc</i>. (B) Top panel, immunoblot analysis of Nf-ya and Nf-yb showing the knockdown efficiency. Bottom panel, knockdown of Nf-yc was controlled by RT-qPCR due to the poor performance of the Nf-yc antibody in immunoblots. (C) Binding of Nf-y subunits and Sp2 to selected promoters after siRNA treatment was analyzed by ChIP-qPCR. The <i>Sp1</i>, <i>Osbp</i>, <i>Amd1</i> and <i>Dctn4</i> promoters are co-occupied by Nf-y and all three Sp factors; the <i>Oxr1</i> and <i>Plcl1</i> promoters are bound by Nf-y and Sp2 but not by Sp1 and Sp3; the <i>Fanci</i> and <i>Taf1c</i> promoters are bound by all three Sp factors but not by Nf-y; and the <i>Raf1</i> promoter is bound by Sp1 and Sp3 but neither by Sp2 nor by Nf-y. The percent of input values are mean +/- SD (n = 3).</p