Identification of effective DNA barcodes for Triticum plants through chloroplast genome-wide analysis

The Egyptian flora is rich with a large number of Triticum plants, which are very difficult to discriminate between in the early developmental stages. This study assesses the significance of using two DNA Barcoding loci (matK and rbcL) in distinguishing between 18 different Triticum accessions in Egypt. We isolated and sequenced 15 rbcL and six matK fragments, but our analysis of the resultant sequences demonstrated a limited ability of matK and rbcL in distinguishing between Triticum accessions. Therefore, we pursued a bioinformatics approach to determine the most useful loci which may be used as DNA barcodes for the Triticum spp. We obtained the 10 available chloroplast genomes of the 10 Triticum species and sub-species from NCBI, and performed chloroplast genome-wide analysis to find the potential barcode loci. A total of 134 chloroplast genes, gene combinations, intergenic regions and intergenic region combinations were tested using a Tree-based method. We were unable to discriminate between Triticum species by using chloroplast genes, gene combinations and intergenic regions. However, a combination of the intergenic region (trnfM-trnT) with either (trnD-psbM), (petN-trnC), (matK-rps16) or (rbcL-psaI) demonstrated a very high discrimination capacity, suggesting their utilization as DNA barcodes for the Triticum plants. Furthermore, our novel DNA barcodes demonstrated high discrimination capacity for other Poaceae members.     

From NGS assembly challenges to instability of fungal mitochondrial genomes: A case study in genome complexity

The presence of repetitive or non-unique DNA persisting over sizable regions of a eukaryotic genome can hinder the genome's successful de novo assembly from short reads: ambiguities in assigning genome locations to the non-unique subsequences can result in premature termination of contigs and thus overfragmented assemblies. Fungal mitochondrial (mtDNA) genomes are compact (typically less than 100 kb), yet often contain short non-unique sequences that can be shown to impede their successful de novo assembly in silico. Such repeats can also confuse processes in the cell in vivo. A well-studied example is ectopic (out-of-register, illegitimate) recombination associated with repeat pairs, which can lead to deletion of functionally important genes that are located between the repeats. Repeats that remain conserved over micro- or macroevolutionary timescales despite such risks may indicate functionally or structurally (e.g., for replication) important regions. This principle could form the basis of a mining strategy for accelerating discovery of function in genome sequences. We present here our screening of a sample of 11 fully sequenced fungal mitochondrial genomes by observing where exact k-mer repeats occurred several times; initial analyses motivated us to focus on 17-mers occurring more than three times. Based on the diverse repeats we observe, we propose that such screening may serve as an efficient expedient for gaining a rapid but representative first insight into the repeat landscapes of sparsely characterized mitochondrial chromosomes. Our matching of the flagged repeats to previously reported regions of interest supports the idea that systems of persisting, non-trivial repeats in genomes can often highlight features meriting further attention.     

SiO2和Al2O3负载的Ni基催化剂在甲烷干重整中的催化性能差异

CH₄与CO₂干重整反应对于环境保护和天然气资源的合理利用具有重要意义。SiO₂和Al₂O₃是适用于甲烷干重整反应的两种典型的催化剂载体。为了阐明这两种载体对催化剂性能的影响,本研究采用等体积浸渍法制备了Ni/Al₂O₃和Ni/SiO₂催化剂,并利用BET、TEM、H₂-TPR、XRD、TG和Raman等技术对还原和反应后的催化剂进行了表征。结果表明,由于载体的性质不同,Ni基催化剂在甲烷干重整中的催化性能也不同。Ni/SiO₂催化剂的初始活性较高,但由于其金属-载体相互作用较弱,催化稳定性较差,在800℃下反应15h其催化活性急剧下降;较弱的金属-载体相互作用使得Ni/SiO₂催化剂上的Ni颗粒较大,有利于积炭前驱物种的生成,导致催化剂快速失活。而对于Ni/Al₂O₃催化剂,金属-载体相互作用较强,Ni颗粒较小,但由于Ni与Al₂O₃生成了NiAl_xO_y物种,有效活性位减少,其催化活性相对较低,但催化稳定性较好,干重整反应进行50h其活性保持稳定;Ni与Al₂O₃之间较强的相互作用有利于形成小且稳定的Ni粒子,能减少积炭,因而具有优异的催化稳定性。     

Epigenotoxicity of environmental pollutants evaluated by a combination of DNA methylation inhibition and capillary electrophoresis–laser-induced fluorescence immunoassay

A variety of environmental pollutants may cause abnormal DNA methylation, which further disturb gene expression. In this work, we developed a rapid and sensitive method for the characterization and identification of the epigenotoxicity of environmental pollutants in terms of DNA methylation. The method combines in vitro inhibition reactions of a model DNA methyltransferase (DNMT) with rapid and sensitive capillary electrophoresis–laser-induced fluorescence (CE-LIF) immunoassays. This method was first assessed using two known DNMT inhibitors, (–)-epigallocatechin-3-gallate and RG108, and then applied to epigenotoxic evaluation of four aldehydes and six benzo-1,4-quinones. It was found that all these electrophilic chemicals could inhibit DNMT activity, probably due to their interactions with the active sites of DNMT. Interestingly, benzo-1,4-quinones displayed more inhibitory effects on DNMT activity than aldehydes. Among the tested six benzo-1,4-quinones, halogenated benzo-1,4-quinone showed higher inhibitory activity than non-halogenated p-benzo-1,4-quinone. Owing to its speed and sensitivity, our method will be potentially applicable for fast epigenotoxic screening of environmental pollutants and mechanistic study of environmental epigenetics.

Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of ??-actin gene. A total of 377 and 720 clones with insert size >250?bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59?%) or hypothetical functions (23 and 18?%) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.     

Deciphering nucleic acid modifications by chemical derivatization-mass spectrometry analysis

Chemical derivatization in combination with mass spectrometry (MS) analysis is a promising strategy for the sensitive and effective analysis of nucleic acid modifications. In this review, we summarize the recent advances for deciphering modifications in DNA and RNA by chemical derivatization-MS analysis.     

Reconstituted biosynthesis of fungal meroterpenoid andrastin A

Andrastins (andrastin A-D), produced by several Penicillium species, exhibit inhibitory activity against ras farnesyltransferase, suggesting that these compounds could be promising leads for antitumor agents. Although the genome sequence of Penicillium chrysogenum, an andrastin-producing species, is available, the genetic and molecular bases for the biosynthesis of andrastins have not been elucidated. Here we report the identification and characterization of the gene cluster for andrastin biosynthesis. We reconstituted the biosynthetic pathway in Aspergillus oryzae, a fungal expression host, by the co-expression of five genes, including that of a terpene cyclase, and of four genes encoding the tailoring enzymes, required for the generation of andrastins. Remarkably, we successfully obtained andrastin A, the most complex andrastin molecule, as the metabolite of nine gene products, thus confirming the potential of the fungal expression system to synthesize useful natural products.     

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5′-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.     

Copper(II) complexes of methylated glycine derivatives: Effect of methyl substituent on their DNA binding and nucleolytic property

A set of copper(II) complexes of glycine and methylated glycine derivatives, Cu(aa)₂, consisting of C-dimethylglycine, l-alanine, N-dimethylglycine and sarcosine, was investigated for their DNA binding and nucleolytic properties by means of EPR and visible spectroscopy, and electrophoresis. They bind weakly to DNA with apparent binding constants in the range 1.8–2.9 × 10³ M⁻¹ with very similar orientation. No DNA cleavage is observed in the absence of exogenous agents. Copper(II) complexes of N-methylated derivatives bind to DNA more stereo-specifically and less strongly, and their oxidative DNA cleavage is less efficient than those of the corresponding C-methylated derivatives in the presence of hydrogen peroxide (H₂O₂) alone, or sodium ascorbate (NaHA) alone or tandem H₂O₂–NaHA. The oxidative DNA cleavage mechanism in the three systems involves a common copper(I) species. Neocuproine can inhibit DNA cleavage by these complexes.     

Essential Oil Composition and DNA Barcode and Identification of Aniba species (Lauraceae) Growing in the Amazon Region

Lauraceae species are widely represented in the Amazon, presenting a significant essential oil yield, large chemical variability, various biological applications, and high economic potential. Its taxonomic classification is difficult due to the accentuated morphological uniformity, even among taxa from a different genus. For this reason, the present work aimed to find chemical and molecular markers to discriminate Aniba species collected in the Pará State (Brazil). The chemical composition of the essential oils from Aniba canelilla, A. parviflora, A. rosaeodora, and A. terminalis were grouped by multivariate statistical analysis. The major compounds were rich in benzenoids and terpenoids such as 1-nitro-2-phenylethane (88.34–70.85%), linalool (15.2–75.3%), α-phellandrene (36.0–51.8%), and β-phellandrene (11.6–25.6%). DNA barcodes were developed using the internal transcribed spacer (ITS) nuclear region, and the matK, psbA-trnH, rbcL, and ycf1 plastid regions. The markers psbA-trnH and ITS showed the best discrimination for the species, and the phylogenic analysis in the three- (rbcL + matK + trnH − psbA and rbcL + matK + ITS) and four-locus (rbcL + matK + trnH − psbA + ITS) combination formed clades with groups strongly supported by the Bayesian inference (BI) (PP:1.00) and maximum likelihood (ML) (BS ≥ 97%). Therefore, based on statistical multivariate and phylogenetic analysis, the results showed a significant correlation between volatile chemical classes and genetic characteristics of Aniba species.     

5-Formyluracil targeted biochemical reactions with proteins inhibit DNA replication,induce mutations and interference gene expression in living cells

Covalent DNA–protein cross-links are toxic DNA lesions that interfere with essential biological processes, which can cause serious biological consequences, such as genomic instability and protein misexpression. 5-Formyluracil (5fU) as an important modification in DNA, which is mainly from oxidative damage, exists in a variety of cells and tissues. We have reported that 5fU mediated DNA–protein conjugates could exist in human cells [Zhou et al. CCS Chem. 2 (2020) 54–63]. We now aimed to explore its potential biological effects in vitro and in vivo. In this paper, we firstly reported that 5fU intermediated DNA–peptide or DNA–protein conjugates (both were called DPCs) could inhibit different polymerases bypass or cause mutations. Then we further investigated the functional impacts caused by 5fU-mediated DPCs, which appeared in different gene expression components [in the promoter sequence or 5′-untranslated regions (UTR)]. These results together may contribute to a broader understanding of DNA–protein interactions as well as the biological functions associated with 5fU.     

DNA hybridization-enhanced porous silicon corrosion: mechanistic investigations and prospect for optical interferometric biosensing

Hybridization of DNA oligonucleotides in neutral aqueous solutions with complementary sequences immobilized on highly doped p-type porous silicon matrix is shown to result in an unexpectedly large shift in the Fabry-Perot interference pattern to lower wavelengths implying a decrease in effective optical thickness of the porous matrix. We have determined that the observed optical effects are due to enhanced corrosion (oxidation-hydrolysis) of the porous silicon layer triggered by the formation of complementary DNA duplexes. Scanning force microscopy and reflectance spectroscopy were employed at various stages of the signal evolution process to monitor and establish the material changes induced by the DNA binding events. We postulate that the slow background corrosion process initiated at the exposed Si-H_x groups is dramatically enhanced as a result of the change in carrier charge density of the porous silicon layer in response to the local increase in the electrostatic field generated by the nucleic acid hybridization. The proposed mechanism is consistent with the experimental observations that the characteristics of the porous silicon matrix and the charge density of the hybridized DNA complexes can both influence the corrosion process. Functionalized porous silicon matrices prepared from highly doped silicon wafers (resistivity 1 mΩ·cm) produce large corrosion rates and improved signal to noise ratios. Moreover, the enhanced decrease in the effective optical thickness could be prevented by either shielding the negative charges of the DNA duplex in the presence of Mg²⁺ ions, or by using backbone charge neutral peptide nucleic acids (PNA) in the DNA hybridization experiments. The observed phenomenon is thus an example of an active sensor matrix in which the molecular recognition signal is transduced and amplified by a profound change in the chemical reactivity and physical property of the solid support itself. With the signal amplification mechanism described, binding of unlabeled complementary DNA oligonucleotides of approximately 0.1 amol/mm² has been detected suggesting the potential utility of this new approach in DNA sensing.