Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al³⁺ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. “Sebastian”, showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in “Sebastian”. The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.     

Influences of magnetic fields on current–voltage characteristics of gold-DNA-gold structure with variable gaps

Achieving highly sensitive magnetic sensors by means of Metal-DNA-Metal (MDM) structure is a key issue. DNA, being a genetic information carrier in living cells reveals tunable semiconducting response in the presence of external electric and magnetic fields, which is promising for molecular electronics. The influence of magnetic fields up to 1200 mT on the current–voltage (I–V) behavior of Gold-DNA-Gold (GDG) structure having variable gap sizes from 20–50 μm are reported in this work. These structures were fabricated using UV lithography, DC magnetron sputtering and thermal evaporation techniques. DNA strands were extracted from Boesenbergia rotunda plant via standard protocol. The acquired I–V characteristics display the semiconducting diode nature of DNA in GDG structures. The potential barrier for all the structures exhibit an increasing trend with the increase of externally imposed magnetic field irrespective of variable gap sizes. Furthermore, the potential barrier in GDG junction at higher magnetic field strengths (>1000 mT) is found to be considerably enhanced. This enhancement in the junction barrier height at elevated magnetic fields is attributed to the reduction of carrier mobility and augmentation of resistance. The achieved admirable features of magnetic sensitivity suggest the viability of using these GDG sandwiches as a prospective magnetic sensor.     

Membrane disruption and DNA binding of Staphylococcus aureus cell induced by a novel antimicrobial peptide produced by Lactobacillus paracasei subsp. tolerans FX-6

Previously, we have isolated a novel bacteriocin, peptide F1 from Tibetan Kefir, and demonstrated its superior antimicrobial activity. However, its antimicrobial mechanism is still undefined. This study was aimed to elucidate the antimicrobial mechanism of peptide F1 against Staphylococcus aureus. The antimicrobial effects of peptide F1 were characterized by the following methods: chemical assay to quantify cytoplasmic β-galactosidase leakage, atomic absorption spectrometry to measure the released potassium ions, transmission electron microscopy to visualize the cellular morphological changes, and electrophoresis analysis and atomic force microscopy together to exam the DNA binding activity. Our results revealed that peptide F1 exerted its bactericidal effects by damaging bacterial cell membranes and by binding to the genomic DNA in the cytoplasm, which both led to rapid cell death.     

Efficiency of ITS Sequences for DNA Barcoding in Passiflora (Passifloraceae)

DNA barcoding is a technique for discriminating and identifying species using short, variable, and standardized DNA regions. Here, we tested for the first time the performance of plastid and nuclear regions as DNA barcodes in Passiflora. This genus is a largely variable, with more than 900 species of high ecological, commercial, and ornamental importance. We analyzed 1034 accessions of 222 species representing the four subgenera of Passiflora and evaluated the effectiveness of five plastid regions and three nuclear datasets currently employed as DNA barcodes in plants using barcoding gap, applied similarity-, and tree-based methods. The plastid regions were able to identify less than 45% of species, whereas the nuclear datasets were efficient for more than 50% using “best match” and “best close match” methods of TaxonDNA software. All subgenera presented higher interspecific pairwise distances and did not fully overlap with the intraspecific distance, and similarity-based methods showed better results than tree-based methods. The nuclear ribosomal internal transcribed spacer 1 (ITS1) region presented a higher discrimination power than the other datasets and also showed other desirable characteristics as a DNA barcode for this genus. Therefore, we suggest that this region should be used as a starting point to identify Passiflora species.     

当归红芪超滤物对重离子12C6+辐射肝癌细胞DNA损伤修复的影响

将人肝癌H22细胞分成4组,分别为对照组、药物组(100 mg/L)、辐射组(2 Gy)及联合组(100 mg/L药物 + 2 Gy照射),采用CCK-8法、单细胞凝胶电泳、γ-H2AX免疫荧光原位杂交技术以及Western Blotting印迹法,研究当归红芪超滤物(Radix Angelicae Sinensis and Radix Hedysari, RAS-RH)对重离子¹²C⁶⁺辐射引起人肝癌H22细胞DNA损伤修复的影响和其可能的机制。结果表明,在0～72 h和给药剂量为5～200 mg/L范围内,RAS-RH对人肝癌H22细胞的增殖抑制作用具有时间和剂量依赖性,其20%抑制浓度IC₂₀为(117.6±2.15)mg/L;单细胞凝胶电泳显示联合组头部DNA含量低于辐射组,而尾部DNA含量、尾距TM、Olive尾距OTM均高于辐射组;γ-H2AX免疫荧光原位杂交技术发现RAS-RH不增加重离子¹²C⁶⁺辐射引起的DNA损伤,但在2—12 h,DNA双链断裂的γ-H2AX foci修复作用被RAS-RH抑制,DNA损伤持续存在;Western Blotting显示RAS-RH通过下调Ku70/80及Rad51的蛋白表达,抑制γ-H2AX的聚集。以上结果说明RAS-RH对人肝癌H22细胞的辐射增敏作用可能是下调DNA损伤修复相关因子Ku70/80及Rad51的表达。     

Histone Tail Sequences Balance Their Role in Genetic Regulation and the Need To Protect DNA against Destruction in Nucleosome Core Particles Containing Abasic Sites

Abasic sites (AP) are produced 10 000 times per day in a single cell. Strand cleavage at AP is accelerated ≈100-fold within a nucleosome core particle (NCP) compared to free DNA. The lysine-rich N-terminal tails of histone proteins catalyze single-strand breaks through a mechanism used by base-excision-repair enzymes, despite the general dearth of glutamic acid, aspartic acid, and histidine—the amino acids that are typically responsible for deprotonation of Schiff base intermediates. Incorporating glutamic acid, aspartic acid, or histidine proximal to lysine residues in histone N-terminal tails increases AP reactivity as much as sixfold. The rate acceleration is due to more facile DNA cleavage of Schiff-base intermediates. These observations raise the possibility that histone proteins could have evolved to minimize the presence of histidine, glutamic acid, and aspartic acid in their lysine-rich N-terminal tails to guard against enhancing the toxic effects of DNA damage.     

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 10⁴ copies per library member are used as the input.     

Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sites—that is, AATT double strand—in the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.