Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer‐adjacent tissues collected from 30 CRC patients and then bisulfite‐converted. The exon regions of 5 targeted genes (CNRIP1 , HIC1 , RUNX3 , p15 , and SFRP2 ) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC‐MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer‐adjacent individuals, while that of HIC1 , p15 , and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC‐MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.     

Application of reversed-phase high-performance liquid chromatography with fluorimetric detection for simultaneous assessment of global DNA and total RNA methylation in Lepidium sativum: effect of plant exposure to Cd(II) and Se(IV)

In the present work, application of the previously established reversed-phase liquid chromatography procedure based on fluorescent labeling of cytosine and methylcytosine moieties with 2-bromoacetophenone (HPLC-FLD) is presented for simultaneous evaluation of global DNA and total RNA methylation at cytosine carbon 5. The need for such analysis was comprehended from the recent advances in the field of epigenetics that highlight the importance of non-coding RNAs in DNA methylation and suggest that RNA methylation might play a similar role in the modulation of genetic information, as previously demonstrated for DNA. In order to adopt HPLC-FLD procedure for DNA and RNA methylation analysis in a single biomass extract, two extraction procedures with different selectivity toward nucleic acids were examined, and a simplified calibration was designed allowing for evaluation of methylation percentage based on the ratio of chromatographic peak areas: cytidine/5-methylcytidine for RNA and 2´-deoxycytidine/5-methyl-2′-deoxycytidine for DNA. As a proof of concept, global DNA and total RNA methylation were determined in Lepidium sativum hydroponically grown in the presence of different Cd(II) or Se(IV) concentrations, expecting that plant exposure to abiotic stress might affect not only global DNA but also total RNA methylation. The results obtained showed the increase of DNA methylation in the treated plants up to concentration levels 2 mg?L^?1 Cd and 1 mg?L^?1 Se in the growth medium. For higher stressors’ concentration, global DNA methylation tended to decrease. Most importantly, an inverse correlation was found between DNA and RNA methylation levels (r?=??0.6788, p?=?0.031), calling for further studies of this particular modification of nucleic acids in epigenetic context.     

DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity

In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 unit mL⁻¹. Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M.SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism.     

Development of CE-C4D Method for Determination Tropane Alkaloids

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C⁴D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) β-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters—precision (CV): 1.07–2.08%, accuracy of the assay (recovery, RE): 101.0–102.7% and matrix effect (ME): 92.99–94.23%. Moreover, the optimized CE-C⁴D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.     

Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

In this article, we show that methylation‐specific multiplex PCR (MS‐multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS‐multiplex PCR to simultaneously amplify three sequences: the 3′ ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation‐sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader–Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS‐multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS‐multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in‐house‐designed MS‐multiplex PCR protocol is a relatively simple, cost‐effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.     

Determination of Dopamine Using 2-(4-Boronophenyl)quinoline-4-carboxylic Acids as Fluorescent Probes

The selective identification of dopamine is a significant issue because this compound is an important neurotransmitter closely related to Parkinson’s disease and other mental disorders. 2-(4-Boronophenyl)quinoline-4-carboxylic acid (PBAQA) has been previously reported as a water-soluble fluorescent probe for catechol. However, there are no significant differences in the binding constants between catechol and catecholamines, such as dopamine or levodopa. Here a series of bis-boronic acid compounds based on PBAQA were synthesized and the binding activities were characterized. As a representative compound, the binding constant of 4-(4-((3-(3-borono-4-chlorobenzamido)propyl)carbamoyl)quinolin-2-yl)boronic acid to dopamine is up to 10⁴?L?mol^?1 and much higher than previously reported boronic acid probes. Dopamine selectivity may be achieved by the variation of the substituents in the probe molecules. 4-(4-((3-(3-Borono-4-methoxybenzamido)propyl)carbamoyl)quinolin-2-yl)boronic acid has a stronger binding affinity to dopamine (K_a=5204?±?106?L?mol^?1) than catechol (K_a=2588?±?273?L?mol^?1) or levodopa (K_a=2383?±?273?L?mol^?1). This fluorescence response was used for determining dopamine in a range from 5?×?10^?5?mol?L^?1 to 5?×?10^?4?mol?L^?1 with a detection limit of 7.7?×?10^?6?mol?L^?1. This compound has been successfully used for the assay of dopamine in rabbit plasma, exhibiting excellent specificity. It is believed that synthesized compounds hold great promise as practical platforms to monitor dopamine levels.     

(3R,4S)-isostreptenol III from Penicillium purpurogenum MM

Abstract

In continuation of the search for new compounds from the terrestrial fungus Penicillium purpurogenum MM, (3R,4S)-isostreptenol III (1), a further new natural compound is reported. The chemical structure of 1 was confirmed by extensive 1D and 2D NMR and ESI HR mass measurements, and by comparison with literature data. The absolute configuration was determined by ab initio calculations of ECD, ORD, and NMR data. The antimicrobial and cytotoxic activities of the crude extract and 1 were reported using a set of microorganisms and by the brine shrimp assay, respectively.     

Conformational Analysis of Germacrane Sesquiterpene Lactones. 1(10)Z,4E- and 1(10)E,4Z-Germacranolides

Molecular mechanics was used to study the structures of 1(10)Z,4E- and 1(10)E,4Z-germacranolides. Possible conformers, their probabilities, and barriers to conformational transitions were determined     

Syntheses and structural characterizations of Fe4S4 cubane-like cluster compounds containing cycloalkylthiolate ligands

In a reaction system consisting of FeCl₂, tetrathiometallate and cycloalkylthiolate, two Fe₄S₄ cubanelike cluster compounds were obtained with the following crystallographic data: (PhCH₂NMe₃)₂ [ Fe₄S₄ (SC₅H₉)₄] (I), monoclinic space groupP2₁/c,a = 1.632 7(4),b=1.122 9(3),c = 2. 802 5(10) nm, β= 94.63(2)°, Z=4, andR= 0.074; (Et₄N)₂[F₄S4(SC₆H_lI)₄] (II), tetragod space group ,a = l.167 05(9),b = 1.167 06(2),c = 2.063 26(5) nm,Z = 2, D_obs = 1. 28 g/cm³, andR = 0. 078. The participation of cycloalkylthiolate ligand does not obviously arouse the change of the Fe₄S₄ core structure. Meanwhile, the influence of the cation on the structural symmetry of the Fe₄S₄ cluster dianion is also discussed. Project supported by the National Natural Science Foundation of China and the Climbing Program Foundation of China.     

Anti-inflammatory bibenzyls from the stems of Dendrobium huoshanense via bioassay guided isolation

Abstract

The stems of Dendrobium huoshanense have long been used to prevent various diseases, including inflammatory diseases. This study was aimed to explain the anti-inflammatory effect of D. huoshanense stems in LPS-induced RAW 264.7 macrophages and to discover potential anti-inflammatory compounds. Results exhibited that D. huoshanense stems ethanol extract could significantly inhibit LPS-induced production of NO, TNF-α and IL-1β. Based on bioassay guided strategy, four bibenzyls (1–4) were isolated from D. huoshanense stems for the first time. Anti-inflammatory assay showed 1–4 could remarkably inhibit the production of NO in LPS-induced macrophages. Moreover, quantitative RT-PCR analysis displayed that the mRNA levels of iNOs, TNF-α and IL-1β could also be significantly reduced by 1–4. These results suggested that D. huoshanense stems ethanol extract and bibenzyls 1–4 might be well developed as therapeutic agent to prevent inflammatory diseases.     

Global DNA Methylation Level Monitoring by methyl-CpG Binding Domain-Fused Luciferase

DNA methylation is an epigenetic modification that represses gene expression. In cancer cells, alterations of the DNA methylation state in promoter regions and repetitive DNA sequences are observed; therefore, DNA methyltransferase inhibitors have been the focus of interest as potential anticancer drugs. We previously reported a simple global DNA methylation level-sensing assay using methyl-CpG binding domain (MBD) fused to luciferase (MBD-luciferase). In the assay, the MBD-luciferase binds to methyl-CpG sites on genomic DNA. Subsequently, bioluminescence resonance energy transfer (BRET) between the luciferase and a fluorescent DNA intercalating dye generates a signal that is dependent on DNA methylation level. In this study, we investigated whether global DNA hypomethylation induced by a DNA methyltransferase inhibitor or nutrient can be monitored by the BRET assay. 5-Aza-2′-deoxycytidine and folic acid were utilized as the DNA-methyltransferase inhibitor and nutrient that affect DNA methylation in cells. The HeLa cells were cultured with the inhibitor or in folic acid-deficient medium and their global DNA methylation levels measured. Both time- and concentration-dependent hypomethylation were detected by the BRET assay. These results demonstrate that global DNA hypomethylation can be monitored by the BRET assay, indicating that the assay is applicable to cell-based screening of DNA-methyltransferase inhibitors.     

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine‐O⁶‐DNA‐Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non‐methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non‐linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.     

N4‐Methylation of Cytosine Drastically Favors the Formation of (6‐4) Photoproducts in a TCG Context

Methylation of cytosine is a common biological process both in prokaryotic and eukaryotic cells. In addition to 5‐methylcytosine (5mC), some bacterial species contain in their genome N⁴‐methylcytosine (N4mC). Methylation at C5 has been shown to enhance the formation of pyrimidine dimeric photoproducts but nothing is known of the effect of N4 methylation on UV‐induced DNA damage. In the present work, we compared the yield and the nature of bipyrimidine photoproducts induced in a series of trinucleotides exhibiting a TXG sequence where X is either T, C, 5mC or N4mC. HPLC associated to tandem mass spectrometry was used to quantify cyclobutane pyrimidine dimers (CPD), (6‐4) photoproducts (64PP) and their Dewar valence isomer. Methylation at position N4 was found to drastically increase the reactivity of C upon exposure to both UVC and UVB and to favor the formation of 64PP. In contrast methylation at C5 increased the yield of CPD at the expense of 64PP. In addition, enhancement of photoreactivity by C5 methylation was much higher in the UVB than in the UVC range. These results show the drastic effect of the methylation site on the photochemistry of cytosine.     

Electrocatalytical Oxidation of Nitrite and Its Determination Based on Au@Fe3O4 Nanoparticles

A promising electrochemical nitrite sensor was fabricated by immobilizing Au@Fe₃O₄ nanoparticles on the surface of L ‐cysteine modified glassy carbon electrode, which was characterized by scanning electron microscopy, X‐ray photoelectron spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The proposed sensor exhibited excellent electrocatalytic activity toward nitrite oxidation. The kinetic parameters of the electrode reaction process were calculated, (1–α)n_α was 0.38 and the heterogeneous electron transfer coefficient (k) was 0.13 cm s^?1. The detection conditions such as supporting electrolyte and pH value were optimized. Under the optimized conditions, the linear range for the determination of nitrite was 3.6×10^?6 to 1.0×10^?2 M with a detection limit of 8.2×10^?7 M (S/N=3). Moreover, the as‐prepared electrode displayed good stability, repeatability and selectivity for promising practical applications.     

Efficient Route to Highly Functionalized Chalcone-Based Pyranocoumarins via Iodine-Promoted Michael Addition Followed by Cyclization of 4-Hydroxycoumarins

Molecular iodine is used as an efficient promoter in the regioselective synthesis of highly functionalized chalcone-based pyranocoumarin derivatives using 4-hydroxycoumarin in acetic acid solvent at 100 °C. Under optimized reaction conditions, our protocol (Michael addition followed by intermolecular cyclization) has tolerance for many functional groups and gave products in good to excellent yields (75–98%) within 1–2 h.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource: Full experimental and spectral details.]     

The Henicosaphosphide Iodides of Potassium and Rubidium,K4P21I and Rb4P21I

The novel ternary polyphosphides M₄P₂₁I (M = K, Rb) have been synthesized from the elements in single crystalline form, representing further examples for the formation of mixed crystals between simple salts and binary phosphides. They form as ruby‐red platelets and dark‐red prisms, respectively, and are only slightly sensitive to moisture and oxygen. The compounds are isotypic (Ccmm (no 63); Z = 4; oP104; K₄P₂₁I: a = 12.853Å; b = 21.795Å; c = 9.748Å; 1168 hkl, R = 0.033; Rb₄P₂₁I: a = 13.281Å; b = 21.868Å; c = 9.771Å; 777 hkl, R = 0.053) and feature corrugated 2D networks formed from two different types of polymerized P₇ units. The networks form large cavities filled by M⁺ and I^‐ ions. Zigzag chains of condensed trigonal M₆ prisms, centered by the I^‐ anions, separate the polyphosphide nets. The mean homoatomic P‐P bond length (d = 2.216Å) corresponds to a P‐P single bond. However, the individual P‐P distances vary with position and function (2.126 ‐ 2.247Å) and these are compared with those of the isolated P₂₁^‐3 anion.     

Label‐Free Quantification of 5‐Azacytidines Directly in the Genome

Azacytidines (AzaC and AzadC) are clinically relevant pharmaceuticals that operate at the epigenetic level. They are integrated into the genome as antimetabolites to block DNA methylation events. This leads to a reduction of the 5‐methyl‐2′‐deoxycytidine (m⁵dC) level in the genome, which can activate epigenetically silenced genes. Because of the inherent chemical instability of Aza(d)Cs, their incorporation levels in DNA and RNA are difficult to determine, which hinders correlation of therapeutic effects with incorporation and removal processes. Existing methods involve radioactive labeling and are therefore unsuitable to monitor levels from patients. We report here a new direct chemical method that allows absolute quantification of the levels of incorporated AzaC and AzadC in both RNA and DNA. Furthermore, it clarifies that Aza(d)C accumulates to high levels (up to 12.9 million bases per genome). Although RNA‐based antimetabolites are often 2′‐deoxygenated in vivo and incorporated into DNA, for AzaC we see only limited incorporation into DNA. It accumulates predominantly in RNA where it, however, only leads to insignificant demethylation.     

Identification of methylated regions with peak search based on Poisson model from massively parallel methylated DNA immunoprecipitation-sequencing data

DNA methylation is one of the most important epigenetic modification types, which plays a critical role in gene expression. High efficient surveying of whole genome DNA methylation has been aims of many researchers for long. Recently, the rapidly developed massively parallel DNA‐sequencing technologies open the floodgates to vast volumes of sequence data, enabling a paradigm shift in profiling the whole genome methylation. Here, we describe a strategy, combining methylated DNA immunoprecipitation sequencing with peak search to identify methylated regions on a whole‐genome scale. Massively parallel methylated DNA immunoprecipitation sequencing combined with methylation DNA immunoprecipitation was adopted to obtain methylated DNA sequence data from human leukemia cell line K562, and the methylated regions were identified by peak search based on Poison model. From our result, 140 958 non‐overlapping methylated regions have been identified in the whole genome. Also, the credibility of result has been proved by its strong correlation with bisulfite‐sequencing data (Pearson R²=0.92). It suggests that this method provides a reliable and high‐throughput strategy for whole genome methylation identification.     

Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity

The present work demonstrates a novel signal-off electrochemical method for the determination of DNA methylation and the assay of methyltransferase activity using the electroactive complex [Ru(NH₃)₆]³⁺ (RuHex) as a signal transducer. The assay exploits the electrostatic interactions between RuHex and DNA strands. Thiolated single strand DNA1 was firstly self-assembled on a gold electrode via Au–S bonding, followed by hybridization with single strand DNA2 to form double strand DNA containing specific recognition sequence of DNA adenine methylation MTase and methylation-responsive restriction endonuclease Dpn I. The double strand DNA may adsorb lots of electrochemical species ([Ru(NH₃)₆]³⁺) via the electrostatic interaction, thus resulting in a high electrochemical signal. In the presence of DNA adenine methylation methyltransferase and S-adenosyl-l-methionine, the formed double strand DNA was methylated by DNA adenine methylation methyltransferase, then the double strand DNA can be cleaved by methylation-responsive restriction endonuclease Dpn I, leading to the dissociation of a large amount of signaling probes from the electrode. As a result, the adsorption amount of RuHex reduced, resulting in a decrease in electrochemical signal. Thus, a sensitive electrochemical method for detection of DNA methylation is proposed. The proposed method yielded a linear response to concentration of Dam MTase ranging from 0.25 to 10 U mL⁻¹ with a detection limit of 0.18 U mL⁻¹ (S/N = 3), which might promise this method as a good candidate for monitoring DNA methylation in the future.