Blockchain-Based Distributed Marketplace

Developments in Blockchain technology have enabled the creation of smart contracts; i.e., self-executing code that is stored and executed on the Blockchain. This has led to the creation of distributed, decentralised applications, along with frameworks for developing and deploying them easily. This paper describes a proof-of-concept system that implements a distributed online marketplace using the Ethereum framework, where buyers and sellers can engage in e-commerce transactions without the need of a large central entity coordinating the process. The performance of the system was measured in terms of cost of use through the concept of ‘gas usage’. It was determined that such costs are significantly less than that of Amazon and eBay for high volume users. The findings generally support the ability to use Ethereum to create a distributed on-chain market, however, there are still areas that require further research and development

Regulation of Mouse Small Heat Shock Protein αb-Crystallin Gene by Aryl Hydrocarbon Receptor

The stress-inducible small heat shock protein (shsp)/αB-crystallin gene is expressed highly in the lens and moderately in other tissues. Here we provide evidence that it is a target gene of the aryl hydrocarbon receptor (AhR) transcription factor. A sequence (−329/−323, CATGCGA) similar to the consensus xenobiotic responsive element (XRE), called here XRE-like, is present in the αBE2 region of αB-crystallin enhancer and can bind AhR in vitro and in vivo. αB-crystallin protein levels were reduced in retina, lens, cornea, heart, skeletal muscle and cultured muscle fibroblasts of AhR−/− mice; αB-crystallin mRNA levels were reduced in the eye, heart and skeletal muscle of AhR−/− mice. Increased AhR stimulated αB-crystallin expression in transfection experiments conducted in conjunction with the aryl hydrocarbon receptor nuclear translocator (ARNT) and decreased AhR reduced αB-crystallin expression. AhR effect on aB-crystallin promoter activity was cell-dependent in transfection experiments. AhR up-regulated αB-crystallin promoter activity in transfected HeLa, NIH3T3 and COS-7 cells in the absence of exogenously added ligand (TCDD), but had no effect on the αB-crystallin promoter in C2C12, CV-1 or Hepa-1 cells with or without TCDD. TCDD enhanced AhR-stimulated αB-crystallin promoter activity in transfected αTN4 cells. AhR could bind to an XRE-like site in the αB-crystallin enhancer in vitro and in vivo. Finally, site-specific mutagenesis experiments showed that the XRE-like motif was necessary for both basal and maximal AhR-induction of αB-crystallin promoter activity. Our data strongly suggest that AhR is a regulator of αB-crystallin gene expression and provide new avenues of research for the mechanism of tissue-specific αB-crystallin gene regulation under normal and physiologically stressed conditions

Stromal Edema in Klf4 Conditional Null Mouse Cornea Is Associated with Altered Collagen Fibril Organization and Reduced Proteoglycans

PURPOSE. Klf4, one of the highly expressed transcription factors in the mouse cornea, plays an important role in maturation and maintenance of the ocular surface. In this study, the structure and proteoglycan composition of the Klf4 conditional null (Klf4CN) corneal stroma was investigated, to further characterize the previously reported Klf4CN stromal edema. METHODS. Collagen fibril spacing and diameter were calculated from scattering intensity profiles from small angle synchrotron x-ray scattering patterns obtained across the cornea along a vertical meridian at 0.5-mm intervals. Collagen fibril organization and proteoglycans were visualized by electron microscopy (EM), with or without the cationic dye cuprolinic blue. Proteoglycans and glycosaminoglycans were further analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and immunoblot analysis. Q-RT-PCR was used to measure the transcript levels. RESULTS. In the central cornea, the average collagen interfibrillar Bragg spacing increased from 44.5 nm (SD ±1.8) in wild-type to 66.5 nm (SD ±2.3) in Klf4CN, as measured by x-ray scattering and confirmed by EM. Mean collagen fibril diameter increased from 32 nm (SD ±0.4) in wild-type to 42.3 nm (SD ±4.8) in Klf4CN corneal stroma. Downregulation of proteoglycans detected by EM in the Klf4CN stroma was confirmed by FACE and immunoblot analysis. Q-RT-PCR showed that, whereas the Klf4CN corneal proteoglycan transcript levels remained unchanged, matrix metalloproteinase (MMP) transcript levels were significantly upregulated. CONCLUSIONS. The Klf4CN corneal stromal edema is characterized by increased collagen interfibrillar spacing and increased diameter of individual fibrils. The stroma also exhibits reduced interfibrillar proteoglycans throughout, which is possibly caused by increased expression of MMPs

Basonuclin-Null Mutation Impairs Homeostasis and Wound Repair in Mouse Corneal Epithelium

At least two cellular processes are required for corneal epithelium homeostasis and wound repair: cell proliferation and cell-cell adhesion. These processes are delicately balanced to ensure the maintenance of normal epithelial function. During wound healing, these processes must be reprogrammed in coordination to achieve a rapid re-epithelialization. Basonuclin (Bnc1) is a cell-type-specific transcription factor expressed mainly in the proliferative keratinocytes of stratified epithelium (e.g., corneal epithelium, epidermis and esophageal epithelium) and the gametogenic cells in testis and ovary. Our previous work suggested that basonuclin could regulate transcription of ribosomal RNA genes (rDNA) and genes involved in chromatin structure, transcription regulation, cell-cell junction/communication, ion-channels and intracelllular transportation. However, basonuclin's role in keratinocytes has not been demonstrated in vivo. Here we show that basonuclin-null mutation disrupts corneal epithelium homeostasis and delays wound healing by impairing cell proliferation. In basonuclin-null cornea epithelium, RNA polymerase I (Pol I) transcription is perturbed. This perturbation is unique because it affects transcripts from a subset of rDNA. Basonuclin-null mutation also perturbs RNA polymerase II (Pol II) transcripts from genes encoding chromatin structure proteins histone 3 and HMG2, transcription factor Gli2, gap-junction protein connexin 43 and adheren E-cadherin. In most cases, a concerted change in mRNA and protein level is observed. However, for E-cadherin, despite a notable increase in its mRNA level, its protein level was reduced. In conclusion, our study establishes basonuclin as a regulator of corneal epithelium homeostasis and maintenance. Basonuclin likely coordinates functions of a subset of ribosomal RNA genes (rDNA) and a group of protein coding genes in cellular processes critical for the regulation of cell proliferation

Conformation Effects of CpG Methylation on Single-Stranded DNA Oligonucleotides: Analysis of the Opioid Peptide Dynorphin-Coding Sequences

Single-stranded DNA (ssDNA) is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide PDYN (prodynorphin) fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4°C, and some also at 37°C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA

Comparative genomics reveals functional transcriptional control sequences in the Prop1 gene

Mutations in PROP1 are a common genetic cause of multiple pituitary hormone deficiency (MPHD). We used a comparative genomics approach to predict the transcriptional regulatory domains of Prop1 and tested them in cell culture and mice. A BAC transgene containing Prop1 completely rescues the Prop1 mutant phenotype, demonstrating that the regulatory elements necessary for proper PROP1 transcription are contained within the BAC. We generated DNA sequences from the PROP1 genes in lemur, pig, and five different primate species. Comparison of these with available human and mouse PROP1 sequences identified three putative regulatory sequences that are highly conserved. These are located in the PROP1 promoter proximal region, within the first intron of PROP1, and downstream of PROP1. Each of the conserved elements elicited orientation-specific enhancer activity in the context of the Drosophila alcohol dehydrogenase minimal promoter in both heterologous and pituitary-derived cells lines. The intronic element is sufficient to confer dorsal expansion of the pituitary expression domain of a transgene, suggesting that this element is important for the normal spatial expression of endogenous Prop1 during pituitary development. This study illustrates the usefulness of a comparative genomics approach in the identification of regulatory elements that may be the site of mutations responsible for some cases of MPHD

Identification of Y-Box Binding Protein 1 As a Core Regulator of MEK/ERK Pathway-Dependent Gene Signatures in Colorectal Cancer Cells

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties