naked DNA [keywords]
- Age-related reduction of chromatin fractal dimension in toluidine blue - stained hepatocytes. [JOURNAL ARTICLE]
- Mech Ageing Dev 2016 Jul 10.
In this study, we proposed a hypothesis that chromatin of mouse hepatocytes exhibits age-related reduction of fractal dimension. This hypothesis was based on previously published works demonstrating that complexity of biological systems such as tissues, decreases during the process of physiological aging. Liver tissue was obtained from 24 male mice divided into 3 age groups: 10-days-old (young, juvenile), 210-days-old (adult) and 390-days-old. The tissue was stained using a modification of toluidine blue (nucleic acid - specific) staining method. A total of 480 chromatin structures (20 for each animal) were analyzed. For each structure, the values of fractal dimension, lacunarity, textural angular second moment and inverse difference moment were calculated using ImageJ software and its plugins. The results indicated the age-related reduction in fractal dimension and increase in lacunarity (p<0.01). Fractal dimension is a potentially good indicator of age associated changes in chromatin structure. To our knowledge, this is the first study to show that fractal complexity of hepatocyte chromatin decreases during the process of physiological aging. Fractal analysis as a method could be useful in detection of small age-related changes in chromatin distribution not otherwise visible with naked eye on conventional tissue micrographs.
- Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases. [JOURNAL ARTICLE]
- Angew Chem Int Ed Engl 2016 Jul 8.
DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution.
- The architecture of neutrophil extracellular traps investigated by atomic force microscopy. [JOURNAL ARTICLE]
- Nanoscale 2016 Jul 8.
Neutrophils are immune cells that engage in a suicidal pathway leading to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). NETs behave as a double edged sword; they can bind to pathogens thereby ensnaring them and limiting their spread during infection; however, they may bind to host circulating materials and trigger thrombotic events, and are associated with autoimmune disorders. Despite the fundamental role of NETs as part of an immune system response, there is currently a very poor understanding of how their nanoscale properties are reflected in their macroscopic impact. In this work, using a combination of fluorescence and atomic force microscopy, we show that NETs appear as a branching filament network that results in a substantially organized porous structure with openings with 0.03 ± 0.04 μm(2) on average and thus in the size range of small pathogens. Topological profiles typically up to 3 ± 1 nm in height are compatible with a "beads on a string" model of nucleosome chromatin. Typical branch lengths of 153 ± 103 nm appearing as rigid rods and height profiles of naked DNA in NETs of 1.2 ± 0.5 nm are indicative of extensive DNA supercoiling throughout NETs. The presence of DNA duplexes could also be inferred from force spectroscopy and the occurrence of force plateaus that ranged from ∼65 pN to 300 pN. Proteolytic digestion of NETs resulted in widespread disassembly of the network structure and considerable loss of mechanical properties. Our results suggest that the underlying structure of NETs is considerably organized and that part of its protein content plays an important role in maintaining its mesh architecture. We anticipate that NETs may work as microscopic mechanical sieves with elastic properties that stem from their DNA-protein composition, which is able to segregate particles also as a result of their size. Such a behavior may explain their participation in capturing pathogens and their association with thrombosis.
- Viral and synthetic RNA vector technologies and applications. [REVIEW, JOURNAL ARTICLE]
- Mol Ther 2016 Jul 5.
Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and non-viral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of non-viral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest (GOI) as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, non-segmented negative-strand (NSNS) RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the GOI can either be incorporated directly into the viral RNA genome or as non-viral RNA. NSNS virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of GOI-encoding viral RNA. Mixed technologies combining viral and non-viral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy and gene therapy.
- Silencing NKD2 by promoter region hypermethylation promotes esophageal cancer progression by activating Wnt signaling. [JOURNAL ARTICLE]
- J Thorac Oncol 2016 Jun 30.
Naked cuticle homolog 2 (NKD2) was found to be frequently methylated in human breast and gastric cancers. However, the epigenetic changes and mechanisms of NKD2 in human esophageal cancer remain unclear.Nine esophageal cancer cell lines and 154 cases of primary esophageal cancer samples were analyzed using methylation specific PCR, immunohistochemistry, western blot and xenograft mouse models.Loss of NKD2 expression and complete methylation were found in KYSE150 and TE1 cells. Reduced NKD2 expression and partial promoter region methylation were observed in KYSE30, KYSE70, KYSE410, KYSE140 and COLO680 cells. High levels of NKD2 expression and unmethylation were detected in KYSE450 and TE8 cells. Re-expression of NKD2 was induced by 5-aza-2'-deoxycytidine in NKD2 unexpressed cells or cells in which NKD2 expression was reduced. NKD2 was methylated in 53.2% (82/154) of human primary esophageal cancer samples, and promoter region hypermethylation was significantly associated with reduced expression of NKD2 (p<0.01). NKD2 methylation was associated with TNM stage and lymph node metastasis (p<0.01). Our results suggest that NKD2 is regulated by promoter region methylation, and methylation of NKD2 may serve as a prognostic marker in esophageal cancer. Our further studies demonstrate that NKD2 suppresses cell proliferation, colony formation, cell invasion and migration, as well as induces G1/S check point arrest in esophageal cancer cells. NKD2 suppressed xenograft tumor growth and inhibited Wnt signaling in human esophageal cancer cells.NKD2 is frequently methylated in human esophageal cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses esophageal cancer progression by inhibiting Wnt signaling both in vitro and in vivo.
- Unraveling the message: insights into comparative genomics of the naked mole-rat. [Journal Article, Review]
- Mamm Genome 2016 Aug; 27(7-8):259-78.
Animals have evolved to survive, and even thrive, in different environments. Genetic adaptations may have indirectly created phenotypes that also resulted in a longer lifespan. One example of this phenomenon is the preternaturally long-lived naked mole-rat. This strictly subterranean rodent tolerates hypoxia, hypercapnia, and soil-based toxins. Naked mole-rats also exhibit pronounced resistance to cancer and an attenuated decline of many physiological characteristics that often decline as mammals age. Elucidating mechanisms that give rise to their unique phenotypes will lead to better understanding of subterranean ecophysiology and biology of aging. Comparative genomics could be a useful tool in this regard. Since the publication of a naked mole-rat genome assembly in 2011, analyses of genomic and transcriptomic data have enabled a clearer understanding of mole-rat evolutionary history and suggested molecular pathways (e.g., NRF2-signaling activation and DNA damage repair mechanisms) that may explain the extraordinarily longevity and unique health traits of this species. However, careful scrutiny and re-analysis suggest that some identified features result from incorrect or imprecise annotation and assembly of the naked mole-rat genome: in addition, some of these conclusions (e.g., genes involved in cancer resistance and hairlessness) are rejected when the analysis includes additional, more closely related species. We describe how the combination of better study design, improved genomic sequencing techniques, and new bioinformatic and data analytical tools will improve comparative genomics and ultimately bridge the gap between traditional model and nonmodel organisms.
- Advanced design of dumbbell-shaped genetic minimal vectors improves non-coding and coding RNA expression. [JOURNAL ARTICLE]
- Mol Ther 2016 Jun 30.
Dumbbell-shaped DNA minimal vectors lacking non-therapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, non-viral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of non-coding or coding RNA. We minimised shRNA or miRNA expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimised shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimised miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.
- In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles. [Journal Article]
- Int J Nanomedicine 2016.:2675-83.
A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies.
- Neovahlkampfia nana n. sp. Reinforcing an Underrepresented Subclade of Tetramitia, Heterolobosea. [JOURNAL ARTICLE]
- J Eukaryot Microbiol 2016 Jun 21.
The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.
- Multidimensional colorimetric sensor array for discrimination of proteins. [JOURNAL ARTICLE]
- Biosens Bioelectron 2016 Jun 14.:56-61.
An extensible multidimensional colorimetric sensor array for the detection of protein is developed based on DNA functionalized gold nanoparticles (DNA-AuNPs) as receptors. In the presence of different proteins, the aggregation behavior of DNA-AuNPs was regulated by the high concentrations of salt and caused different color change; while DNA-AuNPs grew induced by the reduction of HAuCl4 and NH2OH as a reductant on the surface of nanoparticles exhibited different morphologies and color appearance for different proteins. The transducers based on AuNPs modified by specific and nonspecific DNA enables naked-eye discrimination of the target analytes. This extensible sensing platform with only two receptors could simultaneously discriminate ten native proteins and their thermally denatured conformations using hierarchical cluster analysis (HCA) at the concentration of 50nM with 100% accuracy. This opens up the possibility of the sensor array to investigate the different conformational changes of biomacromolecules, and it gives a new direction of developing multidimensional transduction principles based on plasmonic nanoparticle conjugates. Furthermore, the sensing system could discriminate proteins at the concentration of 500nM in the presence of 50% human urine, which indicated this sensor array has great potential ability in analyzing real biological fluids. In addition, the multidimensional colorimetric sensor array is suitable for analysis of target analytes in the resource-restricted regions because of rapid, simple, low cost, and in-field detection with the naked eye.