376. Characterization of DNA Integrity for Recombinant AAV Vector with Oversize Genome

Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652 adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p′-DDE), as a proxy of the total DDT exposure were determined. Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting pro-apoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas our study failed to demonstrate any relations between DDE and %TUNEL positivity and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall regions. These results suggest that CB-153 and related chemicals might alter sperm DNA integrity and Bcl-xL levels in European adult males, but not in the highly exposed Inuit men. Additional issues (genetic background, lifestyle habits and characterization of total xeno-hormonal activities) need to be investigated in order to fully assess the population variations observed.

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Characterization of AAV vector particle stability at the single-capsid level

Journal of Biological Physics ◽

10.1007/s10867-018-9488-5 ◽

2018 ◽

Vol 44 (2) ◽

pp. 181-194 ◽

Cited By ~ 10

Author(s):

Julien Bernaud ◽

Axel Rossi ◽

Anny Fis ◽

Lara Gardette ◽

Ludovic Aillot ◽

...

Keyword(s):

Vector Particle ◽

Aav Vector ◽

Particle Stability

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33. Molecular Characterization of Double-Stranded AAV Vector in Murine Liver

Molecular Therapy ◽

10.1016/s1525-0016(16)40475-2 ◽

2003 ◽

Vol 7 (5) ◽

pp. S14

Keyword(s):

Molecular Characterization ◽

Aav Vector ◽

Murine Liver

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Characterization of Transcription Termination-Associated RNAs: New Insights into their Biogenesis, Tailing, and Expression in Primary Tumors

International Journal of Genomics ◽

10.1155/2018/1243858 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Ilaria Laudadio ◽

Sara Formichetti ◽

Silvia Gioiosa ◽

Filippos Klironomos ◽

Nikolaus Rajewsky ◽

...

Keyword(s):

Cell Cycle Progression ◽

Transcription Termination ◽

Dna Integrity ◽

Transcription Start ◽

Cycle Progression ◽

Primary Tumors ◽

Transcription Start Sites ◽

Generation Sequencing ◽

Comprehensive Study

Next-generation sequencing has uncovered novel classes of small RNAs (sRNAs) in eukaryotes, in addition to the well-known miRNAs, siRNAs, and piRNAs. In particular, sRNA species arise from transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a detailed characterization of these new classes of sRNAs is still lacking. Here, we present a comprehensive study of sRNAs derived from TTSs of expressed genes (TTSa-RNAs) in human cell lines and primary tissues. Taking advantage of sRNA-sequencing, we show that TTSa-RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2, and their biogenesis does not require DICER and AGO2 endonucleolytic activity. TTSa-RNAs display a strong bias against a G residue in the first position at 5′ end, a known feature of AGO-bound sRNAs, and a peculiar oligoA tail at 3′ end. AGO-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation and DNA integrity checkpoints. Finally, we provide evidence that TTSa-RNAs can be detected by sRNA-Seq in primary human tissue, and their expression increases in tumor samples as compared to nontumor tissues, suggesting that in the future, TTSa-RNAs might be explored as biomarker for diagnosis or prognosis of human malignancies.

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Preparation and Characterization of Novel PBAE/PLGA Polymer Blend Microparticles for DNA Vaccine Delivery

The Scientific World JOURNAL ◽

10.1155/2014/385135 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Meenashi Vanathi Balashanmugam ◽

Sivagurunathan Nagarethinam ◽

Hitesh Jagani ◽

Venkata Rao Josyula ◽

Abdulmohsen Alrohaimi ◽

...

Keyword(s):

Dna Vaccine ◽

Polymer Blend ◽

Delivery System ◽

Transfection Efficiency ◽

Vaccine Delivery ◽

Double Emulsion ◽

Dna Integrity ◽

Effective Combination ◽

Cargo Capacity

Context. Poly(beta-amino ester) (PBAE) with its pH sensitiveness and Poly(lactic-co-glycolic acid) (PLGA) with huge DNA cargo capacity in combination prove to be highly efficient as DNA delivery system.Objective. To study the effectiveness of novel synthesized PBAE polymer with PLGA blend at different ratios in DNA vaccine delivery.Methods. In the present study, multifunctional polymer blend microparticles using a combination of PLGA and novel PBAE polymers A1 (bis(3-(propionyloxy)propyl)3,3′-(propane-1,3-diyl-bis(methylazanediyl))dipropanoate) and A2 (bis(4-(propionyloxy)butyl)3,3′-(ethane-1,2-diyl-bis(isopropylazanediyl))dipropanoate) at different ratios (85 : 15, 75 : 25, and 50 : 50) were prepared by double emulsion solvent removal method. The microparticles were characterized for cytotoxicity, transfection efficiency, and DNA encapsulation efficiency.Result. It was evident from results that among the microparticles prepared with PLGA/PBAE blend the PLGA : PBAE at 85 : 15 ratio was found to be more effective combination than the microparticles prepared with PLGA alone in terms of transfection efficiency and better DNA integrity. Microparticles made of PLGA and PBAE A1 at 85 : 15 ratio, respectively, were found to be less toxic when compared with microparticles prepared with A2 polymer.Conclusion. The results encourage the use of the synthesized PBAE polymer in combination with PLGA as an effective gene delivery system.

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Nep1, a Schizosaccharomyces pombe deneddylating enzyme

Biochemical Journal ◽

10.1042/bj20041991 ◽

2005 ◽

Vol 389 (2) ◽

pp. 307-314 ◽

Cited By ~ 16

Author(s):

Lihong Zhou ◽

Felicity Z. Watts

Keyword(s):

Schizosaccharomyces Pombe ◽

Cell Cycle Progression ◽

Dna Integrity ◽

Main Function ◽

Cycle Progression ◽

D Cells ◽

Modified Forms

Nedd8 is a ubiquitin-like modifier that is attached to the cullin components of E3 ubiquitin ligases. More recently, p53 has also been shown to be Nedd8-modified. Nedd8 attachment occurs in a manner similar to that observed for other ubiquitin-like modifiers. In the present study, we report on the characterization of Nep1, a deneddylating enzyme in fission yeast (Schizosaccharomyces pombe). Unlike loss of ned8, deletion of the nep1 gene is not lethal, although nep1.d cells are heterogeneous in length, suggesting a defect in cell-cycle progression. Viability of nep1.d cells is dependent on a functional spindle checkpoint but not on the DNA integrity checkpoint. Deletion of a related gene (nep2), either alone or in combination with nep1.d, also has little effect on cell viability. We show that Nep1 can deneddylate the Pcu1, Pcu3 and Pcu4 cullins in vitro and that its activity is sensitive to N-ethylmaleimide, consistent with the idea that it is a member of the cysteine protease family. nep1.d cells accumulate Nedd8-modified proteins, although these do not correspond to modified forms of the cullins, suggesting that, although Nep1 can deneddylate cullins in vitro, this is not its main function in vivo. Nep1 can be co-precipitated with the signalosome subunit Csn5. Nep1 itself is present in a high-molecular-mass complex, but the presence of this complex is not dependent on the production of intact signalosomes. Our results suggest that, in vivo, Nep1 may be responsible for deneddylating proteins other than cullins.

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Nucleophosmin Is Required for DNA Integrity and p19Arf Protein Stability

Molecular and Cellular Biology ◽

10.1128/mcb.25.20.8874-8886.2005 ◽

2005 ◽

Vol 25 (20) ◽

pp. 8874-8886 ◽

Cited By ~ 143

Author(s):

Emanuela Colombo ◽

Paola Bonetti ◽

Eros Lazzerini Denchi ◽

Paola Martinelli ◽

Raffaella Zamponi ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Genetic Alterations ◽

Dna Integrity ◽

Senescent Phenotype ◽

Tumor Suppressive Function ◽

Proliferation In Vitro

ABSTRACT Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19Arf and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.

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