Biochemical and biophysical properties of positively supercoiled DNA

In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (-) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (-) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (-) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (-) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (-) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (-) sc DNA

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes ( and DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes ( DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, , , and . We found that for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5\u27-triphosphate binding to DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

A refined spectral element model for wave propagation in multiscale hybrid epoxy/carbon fiber/graphene platelet composite shells

The propagation of elastic waves in heterogeneous media is of interest for impact dynamics and non-destructive detection. This work presents a refined spectral element model (RSEM) to study the wave propagation in multiscale hybrid composite (MHC) shells subjected to impulsive loadings. The doubly-curved MHC shell consists of epoxy, carbon fibers, and graphene platelets (GPLs). The GPLs are functionally distributed along the thickness of the shell. For the three-phase MHC, the Halpin-Tsai micromechanical model in conjunction with the Mori-Tanaka approach is exploited to determine the effective material properties. In the framework of four-variable shear deformation theory, the governing equations along with the natural boundary conditions are derived using Hamilton's principle. A two-node spectral shell element is developed according to the closed-from solutions. The accuracy of the RSEM is verified by comparison with published results in aspects of the natural frequency and transient responses. The wave dispersion characteristics, including the wave number, phase velocity, and group velocity are examined. In the context of high frequency and short wavelength, the proposed RSEM achieves high computational efficiency benefiting from its independence of mesh structure. The time domain responses clearly indicate the wave-boundary interactions, e.g., wave reflection, dispersion, and interference. It is revealed that the present model can well capture the fundamental wave modes of the MHC shell. Moreover, the inclusion of GPLs plays a significant role in improving transverse moduli and mitigating the discontinuities of inter-laminar shear stress

Large scale preparation of the mammalian high mobility group protein A2 for biophysical studies

Due to asymmetrical charge distribution of the mammalian high mobility group protein A2 (HMGA2), which makes HMGA2 bind to both cation- and anion-exchange columns, we developed a rapid procedure for purifying HMGA2 in the milligram range. This purification procedure greatly facilitated biophysical studies, which require large amounts of the protein

Buckling Analysis on Resin Base Laminated Plate Reinforced with Uniform and Functional Gradient Distribution of Carbon Fiber in Thermal Environment

The present paper aims to investigate the buckling load of functionally graded carbon-fiber-reinforced polymer (FG-CFRP) composite laminated plates under in-plane loads in a thermal environment. The effective material properties of the CFRP composite are calculated by the Mori–Tanaka homogenization method. The theoretical formulations are based on classical laminate plate theory (CLPT) and the von Kármán equations for large deflections. The governing equations are derived based on the principle of virtual work and then solved through the Navier solution. Results are obtained for the critical buckling load and temperature effect of a simply supported plate subjected to in-plane loading. A detailed numerical study is conducted to provide important insights into the effects of the functionally graded carbon fiber (CF) distribution pattern and volume fraction, total number of layers, temperature, geometrical dimension and lamination angle on the buckling load of functionally carbon-fiber-reinforced composite plates. Finally, the validation is compared with the Reddy and finite element analyses, which show consistency with each other