Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer.

DNA replication timing is known to facilitate the establishment of the epigenome, however, the intimate connection between replication timing and changes to the genome and epigenome in cancer remain largely uncharacterised. Here, we perform Repli-Seq and integrated epigenome analyses and demonstrate that genomic regions that undergo long-range epigenetic deregulation in prostate cancer also show concordant differences in replication timing. A subset of altered replication timing domains are conserved across cancers from different tissue origins. Notably, late-replicating regions in cancer cells display a loss of DNA methylation, and a switch in heterochromatin features from H3K9me3-marked constitutive to H3K27me3-marked facultative heterochromatin. Finally, analysis of 214 prostate and 35 breast cancer genomes reveal that late-replicating regions are prone to cis and early-replication to trans chromosomal rearrangements. Together, our data suggests that the nature of chromosomal rearrangement in cancer is related to the spatial and temporal positioning and altered epigenetic states of early-replicating compared to late-replicating loci.

[Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells].

OBJECTIVE: To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells. METHODS: A genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone. RESULTS: The LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05). CONCLUSIONS: LINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.

Coupling coumarin to gold nanoparticles by DNA chains for sensitive detection of DNase I.

A kind of coumarin-modified gold nanoparticle by the bridge of dsDNA chains was designed and synthesized for sensitive detection of DNase I. The fluorescence of coumarin 343 at emission wavelengths of 491 nm excited at 440 nm was quenched by the gold nanoparticles due to the energy transfer process after the coumarin 343 was connected on the gold nanoparticles by DNA chains. When dsDNA chains were cut off by DNase I, the coumarin 343 molecules were released from gold nanoparticles and the fluorescence of coumarin 343 would be restored. The DNase I activity could be detected by this fluorescence assay with a high sensitivity based on the change of the energy transfer efficiency. The intensity of restored fluorescence is linearly related to the quantity of DNase I in the range from 1.0 to 40 mU/mL with a detection limit of 0.22 mU/mL. This design idea could render a useful way to develop similar molecular or enzyme sensor in analytical or biological fields.

The CpxA/CpxR Two-Component System Affects Biofilm Formation and Virulence in Actinobacillus pleuropneumoniae.

Gram-negative bacteria have evolved numerous two-component systems (TCSs) to cope with external environmental changes. The CpxA/CpxR TCS consisting of the kinase CpxA and the regulator CpxR, is known to be involved in the biofilm formation and virulence of Escherichia coli. However, the role of CpxA/CpxR remained unclear in Actinobacillus pleuropneumoniae, a bacterial pathogen that can cause porcine contagious pleuropneumonia (PCP). In this report, we show that CpxA/CpxR contributes to the biofilm formation ability of A. pleuropneumoniae. Furthermore, we demonstrate that CpxA/CpxR plays an important role in the expression of several biofilm-related genes in A. pleuropneumoniae, such as rpoE and pgaC. Furthermore, The results of electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis demonstrate that CpxR-P can regulate the expression of the pgaABCD operon through rpoE. In an experimental infection of mice, the animals infected with a cpxA/cpxR mutant exhibited delayed mortality and lower bacterial loads in the lung than those infected with the wildtype bacteria. In conclusion, these results indicate that the CpxA/CpxR TCS plays a contributing role in the biofilm formation and virulence of A. pleuropneumoniae.

Enzyme-Regulated DNA-Based Logic Device.

Herein, we report a carbazole (Cz) ligand that displays distinct turn-on fluorescence signals upon interaction with human telomeric G-quadruplex ( h-TELO) and nuclease enzymes. Interestingly, Cz selectively binds and stabilizes the mixed hybrid topology of h-TELO G-quadruplex that withstands digestion by exonucleases and nuclease S1. The distinct fluorescence signatures of Cz-stabilized h-TELO with nucleases are used to design conceptually novel DNA devices for selectively detecting the enzymatic activity of DNase I as well as performing logic operations. An INHIBIT logic gate is constructed using h-TELO and DNase I as the inputs while the inputs of h-TELO and nuclease S1 form a YES logic gate. Furthermore, a two-input two-output reusable logic device with "multireset" function is developed by using h-TELO and DNase I as inputs. On the basis of this platform, combinatorial logic systems (INHIBIT-INHIBIT and NOR-OR) have been successfully installed using different combinations of nucleases as inputs. Moreover, this new strategy of using a synthetic dual emissive probe and enzyme/DNA inputs for constructing reusable logic device may find important applications in biological computing and information processing.

Plasmon-Resonance-Energy-Transfer-Based Spectroscopy on Single Nanoparticles: Biomolecular Recognition and Enzyme Kinetics.

The small absorption cross sections of most molecules led to the low sensitivity of traditional optical absorption spectroscopy. This obstacle might be overcome by applying the near-field plasmon resonance energy transfer (PRET) between plasmonic nanoparticle and surrounding molecules. In this work, we utilized PRET-based spectroscopy on single gold nanostars to study the specific biomolecule recognition and enzyme kinetics choosing biotin-SA pair and DNase I as models. By analyzing the changes of absorption spectra for black hole quencher 3 (BHQ3), derived from spectra difference, we explored the kinetics of specific biomolecule recognition and enzyme digestion in different physiological environment, and we found that the viscosities of media and the sizes of molecules play vital role in biomolecular recognition and enzyme digestion. Compared with the traditional optical absorption spectroscopy techniques, PRET-based spectroscopy offers a nanoscopic resolution owing to the small size of the probe, is more sensitive and achieves detection on the order of hundreds or even dozens of molecules, and can achieve high selectivity due to the specific biomolecular recognition. This method might be used in the fields of molecular diagnostics, drug discovery, cell systems, and clinical diagnostics.

Versatile Self-Assembly and Biosensing Applications of DNA and Carbon Quantum Dots Coordinated Cerium Ions.

Self-assembly exploits noncovalent interactions to offer a facile and effective method for the construction of soft materials with multifunctionalities and diversity. In this work, fluorescence carbon quantum dots coordinated by Ce3+ ions (CQDCe) have been synthesized and exploited as building blocks to generate a series of hierarchical structures through the ionic self-assembly of CQDCe and biomolecules, namely DNA, myoglobin (Mb), and hyaluronic acid (HA). In particular, vesicles can be constructed by the simple mixing of oppositely charged CQDCe and DNA in water. The formation of unusual vesicles can be explained by the self-assembly of CQDCe with a rearranged structure and the rigid DNA biomolecular scaffolds. This facile noncovalent self-assembly method has inspired the innovative use of virgin DNA as a building block to construct vesicles rather than resorting to a sophisticated synthesis. The self-assembly of CQDCe-biopolymers was accompanied by aggregation-induced photoluminescence (PL) quenching. The biosensing platform was designed to detect polypeptides and deoxyribonuclease I through competitive binding of CQDCe and enzymatic hydrolysis of the DNA backbone, respectively. We believe that the integrative self-assembly of CQDCe and DNA will enrich the theoretical study of vesicle formation by DNA molecules and extend the application of fluorescence carbon quantum dots in the biological field.

A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells.

Human apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1) is an essential DNA repair protein. Herein, we demonstrate that avidin-oriented abasic site-containing DNA strands (AP-DNA) on the surface of silica coated magnetic nanoparticles (SiMNP) can selectively respond to APE1 while resist the digestion by other nucleases. Mechanism studies have revealed that avidin may serve as an organizer protein and recruit APE1 to the DNA substrates on the nanoparticles via strong and specific interactions. Taking advantage of this newly disclosed property, we for the first time successfully displayed the intracellular activities of APE1 in living cells by fluorescence imaging. The avidin organized AP-DNA-SiMNP assembly holds great potential for enzyme-mediated release of drugs inside tumor cells which often contain higher levels of APE1 than normal cells.

Controlling DNA-nanoparticle serum interactions.

Understanding the interaction of molecularly assembled nanoparticles with physiological fluids is critical to their use for in vivo delivery of drugs and contrast agents. Here, we systematically investigated the factors and mechanisms that govern the degradation of DNA on the nanoparticle surface in serum. We discovered that a higher DNA density, shorter oligonucleotides, and thicker PEG layer increased protection of DNA against serum degradation. Oligonucleotides on the surface of nanoparticles were highly resistant to DNase I endonucleases, and degradation was carried out exclusively by protein-mediated exonuclease cleavage and full-strand desorption. These results enabled the programming of the degradation rates of the DNA-assembled nanoparticle system from 0.1 to 0.7 h-1 and the engineering of superstructures that can release two different preloaded dye molecules with distinct kinetics and half-lives ranging from 3.3 to 9.8 h. This study provides a general framework for investigating the serum stability of DNA-containing nanostructures. The results advance our understanding of engineering principles for designing nanoparticle assemblies with controlled in vivo behavior and present a strategy for storage and multistage release of drugs and contrast agents that can facilitate the diagnosis and treatment of cancer and other diseases.

One-pot Synthesis of Quencher Labeled Hairpin DNA-CdTe QDs Conjugate for Target DNA and Deoxyribonuclease I Detection.

A black-hole quencher (BHQ-2) labeled DNA (Q-DNA) with a phosphorothioate backbone was covalently conjugated to the CdTe QDs during the QDs synthesis procedure. The hairpin structure of Q-DNA shortened the distance of the CdTe QDs and the BHQ-2 group, which resulted in fluorescence quenching of the QDs. The addition of target DNA or deoxyribonuclease I (DNase I) could move the BHQ-2 group away from the QDs. As a result, the fluorescence of the CdTe QDs recovered. This work provides a new way for target DNA and DNase I detection.

Homogeneous Immunochemical Assay on the Lateral Flow Strip for Measurement of DNase I Activity.

Deoxyribonuclease I (DNase I) is an important enzyme that cleaves both double-stranded and single-stranded DNA at their phosphate backbone. DNase I is a useful biomarker. Previous studies have shown that patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity, and patients with myocardial infarction exhibit increased DNase I activity. Current methods of measuring DNase I relies either on an immunochemical assay, which requires multiple washing steps, or on a single radial enzyme diffusion assay, which requires a long digestion time and an expensive fluorescence detection system. We have developed a lateral flow immunochemical assay for the measurement of DNase I activity on the test strip. The assay utilized a dually labeled double-stranded DNA as the reporter probe. The biotin-labeled terminal of the probe bound to the streptavidin immobilized on the lateral flow test strip, and the fluorescein-labeled terminal bound to the antibody-conjugated gold nanoparticles, resulting in a visible test line. The presence of DNase I would cleave the reporter probe and lead to reduced test line intensity. Using the DNase I test strip, we have successfully measured the DNase I activity and determined the factors that influence the sensitivity and linear dynamic range of the assay. We have also investigated the conditions that inhibited the DNase I activity. The combined advantage of a wash-free assay format and colorimetric readout would make the lateral flow DNase I test strip a suitable platform for point-of-care diagnostics.

Installing logic gates in permeability controllable polyelectrolyte-carbon nitride films for detecting proteases and nucleases.

Proteases and nucleases are enzymes heavily involved in many important biological processes, such as cancer initiation, progression, and metastasis; hence, they are indicative of potential diagnostic biomarkers. Here, we demonstrate a new label free and sensitive electrochemiluminescent (ECL) sensing strategy for protease and nuclease assays that utilize target-triggered desorption of programmable polyelectrolyte films assembled on graphite-like carbon nitride (g-C3N4) film to regulate the diffusion flux of a coreactant. Furthermore, we have built Boolean logic gates OR and AND into the polyelectrolyte films, capable of simultaneously sensing proteases and nucleases in a complicated system by breaking it into simple functions. The developed intelligent permeability controlled enzyme sensor may prove valuable in future medical diagnostics.

Monitoring the multitask mechanism of DNase I activity using graphene nanoassemblies.

Here we have demonstrated that graphene serves as a remarkable platform for monitoring the multitask activity of an enzyme with fluorescence spectroscopy. Our studies showed that four different simultaneous enzymatic tasks of DNase I can be observed and measured in a high throughput fashion using graphene oxide and oligonucleotide nanoassemblies. We have used phosphorothioate modified oligonucleotides to pinpoint the individual and highly specific functions of DNase I with single stranded DNA, RNA, and DNA/DNA and DNA/RNA duplexes. DNase I resulted in fluorescence recovery in the nanoassemblies and enhanced the intensity tremendously in the presence of sequence specific DNA or RNA molecules with different degrees of amplification. Our study enabled us to discover the sources of this remarkable signal enhancement, which has been used for biomedical applications of graphene for sensitive detection of specific oncogenes. The significant difference in the signal amplification observed for the detection of DNA and RNA molecules is a result of the positive and/or reductive signal generating events with the enzyme. In the presence of DNA there are four possible ways that the fluorescence reading is influenced, with two of them resulting in a gain in signal while the other two result in a loss. Since the observed signal is a summation of all the events together, the absence of the two fluorescence reduction events with RNA gives a greater degree of fluorescence signal enhancement when compared to target DNA molecules. Overall, our study demonstrates that graphene has powerful features for determining the enzymatic functions of a protein and reveals some of the unknowns observed in the graphene and oligonucleotide assemblies with DNase I.

Apoptosis resistance-related ABCB5 and DNaseX (Apo10) expression in oral carcinogenesis.

BACKGROUND: Apoptosis resistance is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). METHODS: Expression of apoptosis resistance-related ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] and DNaseX (Apo10) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. RESULTS: Expression of ABCB5 and Apo10 were significantly increased in the carcinogenesis of OSCC compared with normal tissue. Compared with SIN I-III, ABCB5 expression was significantly decreased in OSCC. Apo10 expression did not significantly differ from OSCC compared with SIN I-III. CONCLUSIONS: This study provides the first evidence of the expression of ABCB5 and Apo10 in the multi-step carcinogenesis of OSCC. Overcoming drug resistance of ABCB5+ and Apo10+ cells in precursor lesions and tumors by natural compounds may act as sensitizers for apoptosis or could be useful for chemoprevention.

A barium based coordination polymer for the activity assay of deoxyribonuclease I.

A new coordination polymer which shows an unusual 2D inorganic connectivity was constructed. This compound exhibits distinct fluorescence quenching ability to the dye-labeled single-stranded DNA probes with different lengths, based on which an analytical method was developed for the activity assay of deoxyribonuclease I.

Novel high-sensitive fluorescent detection of deoxyribonuclease I based on DNA-templated gold/silver nanoclusters.

Herein, fluorescent DNA-templated gold/silver nanoclusters (DNA-Au/Ag NCs) are presented as a novel probe for sensitive detection of deoxyribonuclease I (DNase I). The procedure is based on quenching fluorescence of DNA-Au/Ag NCs by DNase I digestion of the DNA (5'-CCCTTAATCCCC-3') template. This decrease in fluorescence intensity permitted sensitive detection of DNase I in a linear range of 0.013-60 µg mL(-1), with a detection limit of 3 ng mL(-1) at a signal-to-noise ratio of 3. Furthermore, the practicality of this probe for detection of DNase I in human serum and saliva samples was validated, demonstrating its advantages of simplicity, selectivity, sensitivity and low cost. Importantly, satisfactory agreement between results obtained by the fluorescent method described here and high performance liquid chromatography (HPLC) further confirmed the reliability and accuracy of this approach.

A graphene-based real-time fluorescent assay of deoxyribonuclease I activity and inhibition.

Using the remarkable difference in the affinity of graphene oxide (GO) with double strand DNA (dsDNA) and short DNA fragments, we report for the first time a GO-based nonrestriction nuclease responsive system. Our system was composed of GO and a fluorescent dye fluorescein amidite (FAM)-labeled dsDNA substrate (F-dsDNA). At first, the fluorescence of this F-dsDNA substrate was quenched upon addition of GO. When nuclease was added to the mixture of dsDNA and GO, hydrolysis of dsDNA was initiated and small DNA fragments were produced. As a result, the short FAM-linked DNA fragments were released from GO due to the weak affinity of GO with short DNA fragments, and the fluorescence got a restoration. At present, many sensing systems are based on the fact that GO prefers to bind long single strand DNA (ssDNA) over dsDNA or short ssDNA. As for our system, GO has a prior binding with dsDNA over short DNA fragments. Compared with previous methods, this assay platform has some advantages. First, since GO can be prepared in large quantities from graphite available at very low cost, this method shows advantages of simplicity and cost efficiency. Besides, the proposed GO-based nuclease assay provides high sensitivity due to the super quenching capacity of GO. Using deoxyribonuclease I (DNase I) as a model system, DNase I activity can be quantitatively analyzed by the velocity of the enzymatic reaction, and 1.75 U mL(-1) DNase I can be significantly detected. Moreover, the fluorescent intensity with various concentrations of nuclease becomes highly discriminating after 3-8 min. Thus, it is possible to detect nuclease activity within 3-8 min, which demonstrates another advantage of quick response of the present system. Finally, use of dsDNA as substrate, our method can achieve real-time nuclease activity/inhibition assay, which is time-saving and effortless.

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles.

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.

Comprehensive comparison of two new biodegradable gene carriers.

Safety and high transfection efficiency are the prerequisites for an ideal gene vector. Polyethylenimine (PEI), especially PEI 25k (25 kDa), is a well-known cationic gene carrier with high transfection efficiency. However, the high toxicity, depended on its molecular weight, has limited its use as a potential gene carrier. In our research, for the purpose of reducing the toxicity and increasing the transfection efficiency and further to inspect where the degradation of these biodegradable polymers take place would be more beneficial, in cytoplasm or in endocytic vesicles, two kinds of degradable polymers were synthesized. One is an acid-liable PEI derivate (PEI-GA) which was cross-linked by PEI 2k with glutadialdehyde (GA) through imine linkages and the other one (PEI-TEG) was cross-linked PEI 2k with modified triethyleneglycol (TEG) through biscarbamate linkages and can be degraded at neutral environment. By the use of a series of assay methods both in vitro and in vivo, the results showed that PEI-TEG was found to be biodegradable at neutral environment and exhibit high transfection ability with low toxicity, which indicated its potential as a candidate carrier for gene therapy.

A technical feasibility study of dornase alfa delivery with eFlow® vibrating membrane nebulizers: aerosol characteristics and physicochemical stability.

Dornase alfa (Pulmozyme®) is an inhaled mucus-active drug that decreases viscoelasticity of sputum in vitro, improves lung function and reduces respiratory exacerbations in cystic fibrosis (CF) patients of 5 years age and older. The regulatory approval of dornase alfa 15 years ago stipulated that only certain jet nebulizer-compressor combinations should be used to deliver the drug. Since that time there have been significant advances in aerosol delivery technology, including development of electronic perforated vibrating membrane devices. Three independent laboratories studied aerosol characteristics, nebulization time, dose delivery, and stability of dornase alfa after nebulization to determine the feasibility of using perforated vibrating membrane devices to deliver the drug. These studies determined that the eFlow® vibrating membrane technology delivers dornase alfa more rapidly and efficiently than jet nebulizers, and does not affect the physicochemical properties of the drug. These in vitro results demonstrate only the technical feasibility of using vibrating membrane devices to deliver dornase alfa. Clinical studies will be required before any conclusions can be made regarding clinical safety and efficacy of these drug-device combinations for cystic fibrosis.