Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease

Identification of a Candidate Proteomic Signature to Discriminate Multipotent and Non-Multipotent Stromal Cells

<div><p>Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105−) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC <em>in vitro</em>.</p> </div

Implementation of quantum key distribution network simulation module in the network simulator NS-3

As the research in quantum key distribution (QKD) technology grows larger and becomes more complex, the need for highly accurate and scalable simulation technologies becomes important to assess the practical feasibility and foresee difficulties in the practical implementation of theoretical achievements. Due to the specificity of the QKD link which requires optical and Internet connection between the network nodes, to deploy a complete testbed containing multiple network hosts and links to validate and verify a certain network algorithm or protocol would be very costly. Network simulators in these circumstances save vast amounts of money and time in accomplishing such a task. The simulation environment offers the creation of complex network topologies, a high degree of control and repeatable experiments, which in turn allows researchers to conduct experiments and confirm their results. In this paper, we described the design of the QKD network simulation module which was developed in the network simulator of version 3 (NS-3). The module supports simulation of the QKD network in an overlay mode or in a single TCP/IP mode. Therefore, it can be used to simulate other network technologies regardless of QKD.Web of Science1610art. no. UNSP 25

Purification of murine stroma based on CD105 expression allows the identification of proteins that are unique to multipotent and non-multipotent stroma.

<p>(A) CD105 expression demarcates MSC and non-multipotent stromal cells in C57BL/6 bone marrow derived cultures. CD105− (R1) and CD105+ (R2) stromal cells were purified by FACS and cultured under conditions that support adipocyte, osteocyte and chondrocyte differentiation. (n = 3) (B) Schematic of method used to compare the proteome of multipotent and non-multipotent stroma. CD105+ and CD105− stroma were isolated from 3 separate cultures. Proteins were isolated from the nuclear, cytoplasmic and membrane fractions and analyzed by LC MS/MS. Proteins identified in each of the 3 experiments were pooled to generate data sets representative of MSC (blue) or non-multipotent stroma (yellow). Data from the two populations was generated from both the NLM and Swiss Prot data bases and compared using Microsoft Access™ to generate lists of both unique and common proteins.</p

Standard and serum free human stromal cultures containing MSC, harbour cells that express candidate markers of murine MSC at varying quantities.

<p>Human bone marrow derived stromal cultures were initiated from 4 different donors in either serum containing (SC) or serum free (SF) conditions. (A) Human stromal cultures containing MSC can be derived in the either SC or SF media. Cultures were analyzed after first passage by both flow cytometry and in multipotent differentiation assays according to ISCT established criteria for MSC. Gates established based on unstained controls were used to compare the frequency of CD105, CD73, CD90 and CD45 expressing cells. Evidence of adipocyte, osteocyte and chondrocyte differentiation from each culture was verified by Oil Red O, Alizarin Red and Alcian Blue staining respectively (n = 4). (B) Extracts from human MSC containing stromal cultures express murine MSC specific proteins at frequencies that vary according to both donor and culture condition. An equal quantity of whole cell protein extracts was analyzed by Western Blotting for the presence of murine MSC associated proteins. The molecular weight of each protein is shown at the right of each blot. Nrp1 antibody recognized both modified (130 kDa) and unmodified (120 kDa) forms. Individual blots were analyzed for either actin or vinculin as a control for protein loading.</p

Real-time PCR and Western Blot analysis validate potential markers of MSC that are associated with distinct functional networks.

<p>The expression levels of 16 proteins that were identified as either common to all stroma, or unique to CD105+ cells, were chosen for verification based on the availability of reliable antibodies. (A) Comparison of gene expression levels in CD105+ and CD105− cells. An aliquot of the CD105+ and CD105− cells used for LC MS/MS was utilized for mRNA isolation and converted to cDNA for real-time RT PCR comparison of gene expression (n = 3). Results were normalized to GAPDH and fold change in gene expression was determined in comparison to levels detected in CD105− cells. (B) Western Blot analysis of protein expression levels in CD105+ and CD105− cells. In three separate experiments, whole proteins extracts were obtained from CD105+ and CD105− cells grown to the identical passage as used for LC-MS/MS experiments. Equivalent amounts of each protein extract were probed by Western Blot for the presence of common or unique proteins. Analysis of either actin or vinculin expression was completed on each blot to act as a loading control.</p

List of proteins identified by LC MS/MS in CD105+ cells that participate in specific canonical pathways as determined by IPA analysis.

<p>Proteins detected only in CD105+ cell extracts were analyzed by IPA software to determine their potential role in canonical signalling pathways. Proteins identified to participate in the top four canonical pathways are listed in alphabetical order.</p

List of 25 CD105+ cell unique proteins with the highest Scaffold ranking.

<p>Proteins identified by LC MS/MS analysis of the nuclear, cytoplasmic and membrane subfractions of CD105+ cells were compared to those identified from CD105− cells in each of 3 replicate experiments. Unique proteins were sorted according to their Scaffold rank scores. The 25 highest ranking proteins are listed along with the number of unique peptides identified and percentage of protein coverage from a representative experiment (n = 3). Also listed are the subcellular fractions from which each protein was consistently isolated in each of the three experiments.</p