Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions

DNA polymerases amplify DNA fragments through primer extension reactions. However, polymerization behavior of short primers in the primer extension process has not been systematically explored. In this study, we examined the minimal primer length required for primer extension, and the effect of primer length, mismatches and other conditions on DNA polymerization using a nonradioactive method. Under the condition we conducted, the shortest primers polymerized by Klenow fragment (KF) and Taq DNA polymerase in our experiments were respectively heptamer and octamer. The extension efficiency was also affected by the up-stream overhanging structure of the primer -template complex. We hypothesized a simple model to interpret these observations based on the polymerase structures. Furthermore, it was found that the longer the primer, the more efficient is the primer extension. These polymerization behavior of short primers lay foundation about DNA polymerization mechanism and development of novel nucleic acid detection assays

Anthocyanins: Natural Sources and Traditional Therapeutic Uses

Anthocyanins are water-soluble naturally occurring pigments that are therapeutically beneficial and that have gained considerable interests by researchers in the field of phytopharmaceuticals and pharmacology. The evidence based scientific reports on the potential and efficacy of anthocyanins has caused an upsurge in their testing in clinical trials and formulation of herbal drug supplements since the past few decades. Their structural attributes enable them to be absorbed and react with various biomolecules in the human body, to provide beneficial physiological benefits. The anthocyanins are 2-phenylbenzopyrylium derivatives of dietary phenolics and exhibit antioxidant, anti-inflammatory and protective effects against metabolic and cardiovascular conditions. The metabolism of anthocyanins and their stability in-vivo in human body and during post-harvest storage still needs extensive investigation to fully explore their benefits. In the present chapter, we discuss the chemistry, medicinal uses in folklore/traditional medicine and the natural sources of their occurrence. The pre-clinical, clinical and pharmaceutical applications are also discussed, to emphasize the consumer demands and medicinal value of anthocyanins

1 Energy-Efficient Hybrid Beamforming for Multi-Layer RIS-Assisted Secure Integrated Terrestrial-Aerial Network

The integration of aerial platforms to provide ubiq- uitous coverage and connectivity for densely deployed terrestrial networks is expected to be a reality in emerging sixth-generation networks. Energy-effificient design and secure transmission are two crucial issues for integrated terrestrial-aerial networks. With this focus, due to the potential of RIS in substantially saving power consumption and boosting the security of private information by enabling a smart radio environment, this paper investigates the energy-efficient hybrid beamforming for multi- layer reconfigurable intelligent surface (RIS)-assisted secure in- tegrated terrestrial-aerial network for defending against simul- taneous jamming and eavesdropping attacks. Specifically, with the available of angular information based imperfect channel state information (CSI), we propose a framework for the joint optimization of user’s received precoder, terrestrial BS’s and HAP’s digital precoder, and multi-layer RIS analog precoder to maximize the system energy efficiency (EE) performance. For the design of received precoder, a heuristic beamforming scheme is proposed to convert the worst-case problem into a min-max one such that a closed-form solution is derived. For the design of digital precoder, we propose an iterative sequential convex approximation approach via capitalizing the auxiliary variables and first-order Taylor series expansion. Finally, a monotonic vertex-update algorithm with penalty convex concave procedure is proposed to obtain analog precoder with low computational complexity. Numerical results show the superiority and effective- ness of proposed optimization framework and architectur

Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells

<p>Abstract</p> <p>Background</p> <p>The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.</p> <p>Methods</p> <p>Proteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.</p> <p>Results</p> <p>At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.</p> <p>Conclusion</p> <p>GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.</p

Effects of Central Loop Length and Metal Ions on the Thermal Stability of G-Quadruplexes

The central loop of G-quadruplex molecular beacons is a key element to sense target DNA or RNA sequences. In this study, circular dichroism spectroscopy (CD), thermal difference spectrum (TDS), non-denatured non-denaturing gel electrophoresis, and thermal stability analysis were used to investigate the effect of the central loop length on G-quadruplex features. Two series of G-quadruplexes, AG3TTAG3-(TTA)n-G3TTAG3T (n = 1&#8211;8) (named TTA series) and AG3TTTG3-(TTA)n-G3TTTG3T (n = 1&#8211;8) (named TTT series) were examined in K+ and Na+ solutions, respectively. CD and TDS spectral data indicated that TTA series adopted an antiparallel G-quadruplex structure in Na+ solution and a hybrid G-quadruplex structure in K+ solution respectively. TTT series exhibited a hybrid G-quadruplex structure in both Na+ and K+ solutions. UV melting curves indicated that the stability of G-quadruplex in both series was reduced by the elongation of central loop. Thermal stability analysis concluded that the G-quadruplex destabilization with long central loop is an entropy-driven process due to more flexible and longer central loops

A Novel Design Combining Isothermal Exponential Amplification and Gold-Nanoparticles Visualization for Rapid Detection of miRNAs

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are associated with various diseases. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis; thus, sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, and discovery of new targets for drugs. However, traditional approaches for the detection of miRNAs are usually laborious and time-consuming, with a low sensitivity. Here, we develop a simple, rapid, ultrasensitive colorimetric assay based on the combination of isothermal Exponential Amplification Reaction (EXPAR) and AuNP-labeled DNA probes for the detection of miRNAs (taking let-7a as a model analyte). In this assay, the presence of let-7a is converted to the reporter Y through EXPAR under isothermal conditions. The subsequent sandwich hybridization of the reporter Y with the AuNP-labeled DNA probes generates a red-to-purple color change. In other words, if the reporter Y is complementary to the AuNP-labeled DNA probes, the DNA-functionalized AuNPs will be aggregated, resulting in the change of solution color from red to purple/blue, while when the AuNP-labeled DNA probes are mismatched to the reporter Y, the solution remains red. This assay represents a simple, time-saving technique, and its results can be visually detected with the naked eye due to the colorimetric change. The method provides superior sensitivity, with a detection limit of 4.176 aM over a wide range from 1 nM to 1 aM under optimal conditions. The method also shows high selectivity for discriminating even single-nucleotide differences between let-7 miRNA family members. Notably, it is comparable to the most sensitive method reported to date, thus providing a promising alternative to standard approaches for the direct detection of let-7a miRNA. Importantly, through combination with specific templates, different miRNAs can be converted to the same reporter Y, which can hybridize with the same set of AuNP-labeled DNA probes to form sandwich hybrids. The color change of the solution can be observed in the presence of the target miRNA. This technique has potential as a routine method for assessing the levels of miRNAs, not only for let-7, but also for various miRNAs in the early phase of cancers. In addition, it can be a useful tool in biomedical research and clinical diagnosis, as well as diagnosis or surveillance programs in field conditions

Effects of mismatches and insertions on discrimination accuracy of nucleic acid probes

Effective discrimination of non-complementary nucleotides is an important factor to ensure the accuracy of hybridization-based nucleic acid analyses. The current study investigates the effects of the chemical nature, the positions, the numbers, and the cooperative behavior of mismatches as well as insertions on 20-mer and 30-mer duplexes. We observed the hybridization stability trend affected by mismatches: G:T ≈ G:G > G:A > A:A ≈ T:T > A:C ≈ T:C > C:C. The experimental data show that mismatches at the center of the oligonucleotide probes have a more profound destabilizing effect on the hybridization stability than those at either ends. Insertions also demonstrate a similar destabilizing effect as mismatches. These results provide useful information for designing DNA microarray nucleotide probes and for improving the discrimination accuracy of hybridization-based detections

Discrete Spectrum cover Signal Waveform Design and Generation Method

Radio Frequency-screen is one of the earliest radar active antijamming measures. It achieves antijamming by transmitting cover pulses of different frequencies before the radar pulse signal to induce enemy jammers. As the demand for antijamming measures has become increasingly urgent in recent years, Radio Frequency-screen technology has been further developed. The most representative is the use of discontinuous spectrum signals as a cover signal. However, energy utilization for sending the cover signal can be improved further. To address this problem, this paper proposes a discrete spectrum cover signal based on the discontinuous spectrum cover signal and establishes the waveform design function under the joint constraint of constant modulus and spectral amplitude. The cover signal with discrete spectrum and energy aggregation is generated using the Alternating Direction Method of Multipliers (ADMM) and spectrum shaping algorithm solution. The simulation results show that the discrete spectrum cover signal has a higher spectral amplitude of approximately 5～12 dB than the discontinuous-spectrum cover signal for the same energy and bandwidth. Moreover, the discrete spectrum cover signal can cover a larger spectral range with the same energy and close spectral amplitude, realizing a better antijamming cover effect

Effects of 2'-O-methyl nucleotide substitution on EcoRI endonuclease cleavage activities.

To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl nucleotide (2'-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2'-O-methyl nucleotide has a C3'-endo sugar pucker conformation different from the C2'-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2'-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2'-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2'-fluoro nucleic acid (2'-FNA) and locked nucleic acid (LNA) having similar C3'-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2'-endo to C3'-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2'-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine

Vibrational Analysis of Nucleic Acids. 2. Ab Initio Calculation of the Molecular Force Field and Normal Modes of Dimethyl Phosphate

The gauche-gauche (g,g) conformation of the dimethyl phosphate anion serves as a simple model for the phosphodiester group of nucleic acids. Detailed knowledge of the normal modes of vibration of dimethyl phosphate and other phosphodiester analogues is expected to provide a foundation for understanding the conformation-sensitive bands in vibrational spectra of DNA and RNA. In the previous paper of this series (Guan, Y.; et al. Biophys. J. 1994, 66, 225-235), infrared and Raman spectra of the g,g conformer of dimethyl phosphate [(CH3O)2PO2-], including deuterium [(CD3O)2PO2-] and carbon-13 [(13CH3O)2PO2-] isotopomers, were reported and assigned in conjunction with a complete normal coordinate analysis. The normal mode calculations were accomplished by use of a generalized valence force field. Here, we demonstrate that an ab initio molecular force field for the g,g conformer of dimethyl phosphate, computed using the 3-21+G* basis set, is consistent with the empirically based valence force field reported previously. We have obtained the force field scaling factors which produce satisfactory agreement between the calculated and experimental vibrational frequencies of each of the three dimethyl phosphate isotopomers in the g,g conformation. Ab initio normal mode assignments, in terms of the potential energy distribution, are in general accord with those obtained from the normal coordinate analysis. Agreement between the ab initio and normal coordinate results supports the reliability of the empirically derived force field for extension to other phosphodiester conformations of dimethyl phosphate, as well as to more complex diester derivatives of the nucleic acid orthophosphate group