Solvent extraction and determination of scandium(III) with Cyanex272 as an extractant

2658-2660A novel method for the solvent extraction separation of scandium (III) at pH 5.0 using 5×10-4  M Cyanex272 has been developed. Scandium(III) has been stripped from the organic phase with 1.0 M HNO3 and determined spectrophotometrically as its complex with Arsenazo(III) a 675 nm . The method developed has been applied for selective separation of scandium(III) from some closely associated elements. viz ., La (III), Y(III), Ce(IV), Zr(IV), Dy(III), Nd (III), Pr(III), Yb(III). etc. in their binary mixtures. The method permits mutual separation of scandium(III)yttrium(III)-lanthanum(III) and scandium(III)-yttrium(III)cerium (IV) and for recovery of scandium(III) from USGS standard soil sample GXR-2, Japanese stream sediment sample Jsd-3 and ashes from Hutti gold mines in Karnataka, The method presented is simple, rapid and selective for isolation of scandium(III) from complex mixtures. The method developed is reproducible with a relative standard deviation of 0.4%.</span

Differential sequences and single nucleotide polymorphism of exosomal SOX2 DNA in cancer.

Glioblastoma multiforme (GBM) is the most common form of brain cancer, with an average life expectancy of fewer than two years post-diagnosis. We have previously reported that cancer cell originated exosomes, including GBM, have NANOG and NANOGP8 DNA associated with them. The exosomal NANOG DNA has certain differences as compared to its normal counterpart that are of immense importance as a potential cancer biomarker. NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. Similarly, SOX2 is another stemness gene highly expressed in cancer stem cells with an intimate involvement in GBM progression and metastasis as well as promotion of tumorigenicity in Neuroblastoma (NB). Since exosomes are critical in intercellular communication with a role in dissipating hallmark biomolecules responsible for cancer, we conducted a detailed analysis of the association of the SOX2 gene with exosomes whose sequence modulations with further research and appropriate sample size can help to identify diagnostic markers for cancer. We have detected SOX2 DNA associated with exosomes and have identified some of the SNPs and nucleotide variations in the sequences from a GBM and SH-SY5Y sample. Although a further systematic investigation of exosomal DNA from GBM and NB patient's blood is needed, finding of SOX2 DNA in exosomes in the current study may have value in clinical research. SOX2 is known to be misregulated in cancer cells by changes in miRNA function, such as SNPs in the binding sites. Our finding of cancer-specific SNPs in exosomal SOX2 DNA sequence may reflect those changes in the cancer stem cells as well as cancer cells. A series of our study on embryonic stem cell gene analysis in exosomal DNA may lead to a minimally invasive exosome-based diagnosis, and give us a key in understanding the mechanisms of cancer formation, progression, and metastasis

DNA Associated with Circulating Exosomes as a Biomarker for Glioma

Cancerous and non-cancerous cells secrete exosomes, a type of nanovesicle known to carry the molecular signature of the parent for intercellular communications. Exosomes secreted by tumor cells carry abnormal DNA, RNA, and protein molecules that reflect the cancerous status. DNA is the master molecule that ultimately affects the function of RNA and proteins. Aberrations in DNA can potentially lead a cell to malignancy. Deviant quantities and the differential sequences of exosomal DNA are useful characteristics as cancer biomarkers. Since these alterations are either associated with specific stages of cancer or caused due to a clinical treatment, exosomal DNA is valuable as a diagnostic, prognostic, predictive, and therapeutic-intervention response biomarker. Notably, the exosomes can cross an intact blood&ndash;brain barrier and anatomical compartments by transcytosis. As such, the cancer-specific trademark molecules can be detected in systemic blood circulation and other body fluids, including cerebrospinal fluid, with non-invasive or minimally invasive procedures. This comprehensive review highlights the cancer-specific modulations of DNA associated with circulating exosomes that are beneficial as glioma biomarkers

VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule. While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis. The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression

Differential Sequences Of Exosomal Nanog Dna As A Potential Diagnostic Cancer Marker

NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis

Analysis of regulatory sequences in exosomal DNA of NANOGP8.

Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined

Site-Specific Integration of Agrobacterium tumefaciens T-DNA via Double-Stranded Intermediates

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome

Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker

<div><p>NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis.</p></div

22 BP insertion occurrence.

<p>Percentage of clones within a cell source containing the 22 bp insert into NANOGP8 3’UTR of exosomal DNA. Specific values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197782#pone.0197782.t001" target="_blank">Table 1</a>, primer set IV.</p

Confocal microscopy of HEK 293 exosome clusters.

<p>Confocal microscopy images of HEK 293 exosome clusters, demonstrating the preserved integrity of exosome structure of the samples used within this study. (A) Green fluorescence from lipophilic stain DiO. (B) Red fluorescence from packed RFP (red fluorescent protein). (C) Co-localization of both DiO staining and RFP.</p