DNA sequence and methylation prescribe the inside-out conformational dynamics and bending energetics of DNA minicircles

Eukaryotic genome and methylome encode DNA fragments’ propensity to form nucleosome particles. Although the mechanical properties of DNA possibly orchestrate such encoding, the definite link between ‘omics’ and DNA energetics has remained elusive. Here, we bridge the divide by examining the sequence-dependent energetics of highly bent DNA. Molecular dynamics simulations of 42 intact DNA minicircles reveal that each DNA minicircle undergoes inside-out conformational transitions with the most likely configuration uniquely prescribed by the nucleotide sequence and methylation of DNA. The minicircles’ local geometry consists of straight segments connected by sharp bends compressing the DNA’s inward-facing major groove. Such an uneven distribution of the bending stress favors minimum free energy configurations that avoid stiff base pair sequences at inward-facing major grooves. Analysis of the minicircles’ inside-out free energy landscapes yields a discrete worm-like chain model of bent DNA energetics that accurately account for its nucleotide sequence and methylation. Experimentally measuring the dependence of the DNA looping time on the DNA sequence validates the model. When applied to a nucleosome-like DNA configuration, the model quantitatively reproduces yeast and human genomes’ nucleosome occupancy. Further analyses of the genome-wide chromatin structure data suggest that DNA bending energetics is a fundamental determinant of genome architecture.     

Thin‐Film Solar Cells with InP Absorber Layers Directly Grown on Nonepitaxial Metal Substrates

The design and performance of solar cells based on InP grown by the nonepitaxial thin‐film vapor–liquid–solid (TF‐VLS) growth technique is investigated. The cell structure consists of a Mo back contact, p‐InP absorber layer, n‐TiO₂ electron selective contact, and indium tin oxide transparent top electrode. An ex situ p‐doping process for TF‐VLS grown InP is introduced. Properties of the cells such as optoelectronic uniformity and electrical behavior of grain boundaries are examined. The power conversion efficiency of first generation cells reaches 12.1% under simulated 1 sun illumination with open‐circuit voltage (V_OC) of 692 mV, short‐circuit current (J_SC) of 26.9 mA cm^?2, and fill factor (FF) of 65%. The FF of the cell is limited by the series resistances in the device, including the top contact, which can be mitigated in the future through device optimization. The highest measured V_OC under 1 sun is 692 mV, which approaches the optically implied V_OC of ≈795 mV extracted from the luminescence yield of p‐InP.     

Trace analysis of N‐acetyl‐L‐cysteine using luminol–H2O2 chemiluminescence system catalyzed by silver nanoparticles

N‐Acetyl‐L‐cysteine (NAC) can inhibit the luminol–H₂O₂, reaction, which is catalyzed by silver nanoparticles. Based on this phenomenon a new method was developed for NAC determination. Under optimum conditions, a linear relationship between chemiluminescence intensity and NAC concentration was found in the range 0.034–0.98 µg/mL. The detection limit was 0.010 µg/mL (S/N =3), and the relative standard deviation (RSD) was <5% for 0.480 µg/mL NAC (n =5). This simple, sensitive and inexpensive method has been applied to measure the concentration of NAC in pharmaceutical tablets. Copyright © 2014 John Wiley & Sons, Ltd.     

Study on the interaction of a cyanine dye with human serum transferrin

Complexation between the primary carrier of ligands in blood plasma, human serum transferrin (Tf), and a cyanine dye, 3,3′‐di(3‐sulfopropyl)‐4,5,4′,5′‐dibenzo‐9‐phenyl‐thiacarbocyanine‐triethylam monium salt (PTC) was investigated using fluorescence spectra, UV/Vis absorption spectra, synchronous fluorescence spectra, circular dichroism (CD) and molecular dynamic docking. The experimental results demonstrate that the formation of PTC–Tf complex is stabilized by van der Waal's interactions and hydrogen bonds, and the binding constants were found to be 8.55 × 10⁶, 8.19 × 10⁶ and 1.75 × 10⁴ M^?1. Moreover, fluorescence experiments prove that the operational mechanism for the fluorescence quenching is static quenching and non‐radiative energy transfer. Structural investigation of the PTC–Tf complexes via synchronous fluorescence spectra and CD showed that the structure of Tf became more stable with a major increase in the α‐helix content and increased polarity around the tryptophan residues after PTC binding. In addition, molecular modeling highlights the residues located in the N‐lobe, which retain high affinity for PTC. The mode of action of the PTC–Tf complex is illustrated by these results, and may provide an effective pathway for the transport and targeted delivery of antitumor agents. Copyright © 2015 John Wiley & Sons, Ltd.     

Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzyme

Bacillus thermocatenulatus lipase 2 (BTL2) is a promising industrial enzyme used in biodiesel production. Although BTL2 has high thermostability and good resistance to organic solvents, the activity of BTL2 is suboptimal for industrial processes. To improve BTL2 activity, we engineered BTL2 lipase by modulating hydrophobicity of its lid domain. Through site‐directed mutagenesis, we constructed three mutants, namely Y225F+S232A, S232A+T236V and Q185L, to cover all uncharged hydrophilic amino acids within the lid domain. Activities of these mutants were characterized. Our findings suggest that one mutant (Y225F+S232A) showed ～35% activity increase in catalyzing heterogeneous hydrolytic reactions relevant for industrial applications. A mathematical framework was established to account for different molecular events that contribute to the observed apparent catalytic activities. Increases in hydrophobicity of lid domains were associated with increased interfacial adsorption of lipases and lower molecular enzymatic activities. The measured apparent activities of lipases include contributions from both events. Lid hydrophobicity can thus result in different changes in lipase activities depending on the mutation site. Our work demonstrates the feasibility of increasing BTL2 activity by modulating the hydrophobicity of lid domains and provides some guidelines for further improving BTL2 activity.     

Bis‐ and Tetrakis(carboxylato)platinum(IV) Complexes with Mixed Axial Ligands – Synthesis,Characterization, and Cytotoxicity

A series of twelve novel diamminetetrakis(carboxylato)platinum(IV) and 18 novel bis(carboxylato)dichlorido(ethane‐1,2‐diamine)platinum(IV) complexes with mixed axial carboxylato ligands was synthesized and characterized by multinuclear ¹H‐, ¹³C‐, ¹⁵N‐, and ¹⁹⁵Pt‐NMR spectroscopy. Their cytotoxic potential was evaluated (by MTT assay) against three human cancer cell lines derived from ovarian teratocarcinoma (CH1/PA‐1), lung (A549), and colon carcinoma (SW480). In the cisplatin‐sensitive CH1/PA‐1 cancer cell line, diamminetetrakis(carboxylato)platinum(IV) complexes showed IC₅₀ values in the low micromolar range, whereas, for the most lipophilic compounds of the bis(carboxylato)dichlorido(ethane‐1,2‐diamine)platinum(IV) series, IC₅₀ values in the nanomolar range were found.     

Cofactor‐induced reversible folding of Flavodoxin‐4 from Lactobacillus acidophilus

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN‐binding mechanism to flavodoxins‐4 were obtained from the NMR structures of the apo‐protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO ) and exhibits the characteristic flavodoxin fold, with a central five‐stranded parallel β–sheet and five α‐helices forming an α/β‐sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β‐sheet and covers the FMN‐binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.     

First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR‐Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase‐Thymidylate Synthase (DHFR‐TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod‐2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off‐target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR‐Cas9‐mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.     

Relationships between taxonomic and functional components of diversity: implications for conservation of tropical freshwater fishes

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