Keth-seq for transcriptome-wide RNA structure mapping.

RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.

Efficient Separation of Nucleic Acids with Different Secondary Structures by Metal-Organic Frameworks.

We report the use of metal-organic frameworks (MOFs) for the selective separation of nucleic acids (DNA and RNA) with different secondary structures through size, shape, length, and capability of conformational transition. Three MOFs with precisely controlled pore environments, Co-IRMOF-74-II, -III, and -IV, composed of Co2+ and organic linkers (II, III, and IV), respectively, were used for the inclusion of nucleic acid into their pores from the solution. This was proven to be a spontaneous process from disordered free state to restricted ordered state via circular dichroism (CD) spectroscopy. Three critical factors were identified for their inclusion: (1) size selection induced by steric hindrance, (2) conformation transition energy selection induced by stability, and (3) molecular weight selection. These selection rules were used to extract nucleic acids with flexible and unstable secondary structures from complex mixtures of multiple nucleic acids, leaving those with rigid and stable secondary structures in the mother liquor. This provides the possibility to separate and enrich nucleic acids in bulk through their different structure feature, which is highly desirable in genome-wide structural measurement of nucleic acids. Unlike methods that rely on specific binding antibodies or ligand, this MOF method is capable of selecting all kinds of nucleic acids with similar secondary structure features; therefore, it is suitable for the handling of a large variety and quantity of nucleic acids at the same time. This method also has the potential to gather information about the folding stability of biomolecules with secondary structures.

A m6 A Sensing Method by Its Impact on the Stability of RNA Double Helix.

N6 -Methyladenosine (m6 A) is one of the most important RNA modifications in epigenetics. The development of detection method for m6 A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m6 A modification and target RNAs without m6 A modification. We verified the fact that the presence of m6 A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m6 A detection methods in the future.

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution.

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.

Luminescence Sensing for Qualitative and Quantitative Detection of 5-Methylcytosine.

5-Methylcytosine (5mC) is revealed as a heritable epigenetic modification in genomic DNA. It has been reported that cytosine/guanine dinucleotides (CpG) hypermethylation in the promoter regions of tumor suppressor genes is related with inappropriate gene silencing, so the determination of 5mC in the CpG islands of mammals has attracted much attention. In this paper, a luminescence sensing strategy based on bisulfite treatment, asymmetric polymerase chain reaction (PCR), and adenosine triphosphate (ATP)-releasing nucleotide is proposed. With little background, this method can provide accurate quantitative information about methylation changes at CpG sites, even at a specific site. The proposed method can be successfully employed to determine the methylation status of three hepatocellular carcinomas (HCC) related genes in clinical tissues.

Structural, Physicochemical and Functional Properties of Protein Extracted from De-Oiled Field Muskmelon (Cucumis melo L. var. agrestis Naud.) Seed Cake.

For oil plants, the oil extraction method is a crucial factor in influencing the functional characteristics of the protein. However, reports of protein functionality as affected by the oil extraction process are scarce. In this study, field muskmelon seed (FMS) protein was extracted by Soxhlet extraction method (SE), organic solvent extraction method (OSE), aqueous extraction method (AE), and pressing extraction method (PE), and its structure, amino acid profile, physicochemical properties, and functionality were determined. Molecular weight distribution was similar for all FMS proteins, whereas protein aggregates contents were most excellent for SE and OSE. FMS protein comprised predominantly glutamic acid, leucine, aspartic acid, arginine, and proline. Total amino acids content was highest for SE. Differences in functionality between four FMS proteins for different oil extraction methods were vast. PE had the highest value of solubility, and AE exhibited the lowest. AE had the greatest water and oil holding capacity. PE presented better foaming and emulsion capacities than other samples. This study demonstrated that the extraction oil method could impact the protein's physicochemical and associated functional characteristics. High-quality plant oil and protein could be simultaneously obtained by modulating the oil extraction method in future research.

The Effect of Different Extraction Methods on Extraction Yield, Physicochemical Properties, and Volatile Compounds from Field Muskmelon Seed Oil.

Field muskmelon seed oil was extracted by press extraction (PE), Soxhlet extraction (SE), organic extraction (OSE), and aqueous extraction (AE). The oils were then evaluated for their physicochemical properties, fatty acid composition, volatile compounds, and antioxidant properties. A high yield oil was found in the SE sample. The AE sample had the highest elevated acid and peroxide values, while PE and OSE had the highest oil iodine content. The oil samples did not differ significantly in their fatty acid profile depending on the extraction method. However, E-nose, HS-GC-IMS, and HS-SPME-GC-MS showed that the flavor composition of the four samples was significantly different, attributed to the changes in the composition and content of the compounds caused by the different extraction methods. Furthermore, the strongest FRAP and the free radical scavenging ability of DPPH and ABTS+ showed in the SE sample. In general, SE's seed oil has certain advantages when applied to the muskmelon seed oil industry.

Postharvest chilling diminishes melon flavor via effects on volatile acetate ester biosynthesis.

In postharvest handling systems, refrigeration can extend fruit shelf life and delay decay via slowing ripening progress; however, it selectively alters the biosynthesis of flavor-associated volatile organic compounds (VOCs), which results in reduced flavor quality. Volatile esters are major contributors to melon fruit flavor. The more esters, the more consumers enjoy the melon fruit. However, the effects of chilling on melon flavor and volatiles associated with consumer liking are yet to be fully understood. In the present study, consumer sensory evaluation showed that chilling changed the perception of melon fruit. Total ester content was lower after chilling, particularly volatile acetate esters (VAEs). Transcriptomic analysis revealed that transcript abundance of multiple flavor-associated genes in fatty acid and amino acid pathways was reduced after chilling. Additionally, expression levels of the transcription factors (TFs), such as NOR, MYB, and AP2/ERF, also were substantially downregulated, which likely altered the transcript levels of ester-associated pathway genes during cold storage. VAE content and expression of some key genes recover after transfer to room temperature. Therefore, chilling-induced changes of VAE profiles were consistent with expression patterns of some pathway genes that encode specific fatty acid- and amino acid-mobilizing enzymes as well as TFs involved in fruit ripening, metabolic regulation, and hormone signaling.

Correction: Small-molecule-based human genome G4 profiling reveals potential gene regulation activity.

Correction for 'Small-molecule-based human genome G4 profiling reveals potential gene regulation activity' by Weiwu Zeng et al., Chem. Commun., 2019, 55, 2269-2272, DOI: 10.1039/C8CC10052G.

Acrylonitrile-Mediated Nascent RNA Sequencing for Transcriptome-Wide Profiling of Cellular RNA Dynamics.

RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile-mediated uridine-to-cytidine (U-to-C) conversion (AMUC-seq) via 4-thiouridine (s4U) cyanoethylation is presented. This method converts s4U base-pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC-seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC-seq is validated by being used to detect mRNA half-lives and investigating the direct gene targets of a G-quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC-seq also confirms G-quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC-seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription-modulating drugs.

Small-molecule-based human genome G4 profiling reveals potential gene regulation activity.

Small-molecule-based G4 isolation from genomic DNA has enabled the identification of a total of 51 446 PQSs (potential G-quadruplex sites). Incubating cells with PDP for 3 h to 72 h resulted in the differential expression of a variety of genes, indicating a potential function of G4s in gene regulation.

Selective detection of N6-methyladenine in DNA via metal ion-mediated replication and rolling circle amplification.

N6-methyladenine (6mA) is reported as a potential epigenetic marker in eukaryotic genomes. However, accurate identification of the location of 6mA in DNA remains a challenging task. Here, we show that Ag+ can selectively stabilize the A-C mismatch and efficiently promote primer extension. In contrast, the complex of 6mA-Ag+-C is instable and therefore cannot be recognized by DNA polymerases, resulting in the termination of primer extension. Based on this finding, we successfully identified and quantified 6mA at the single-base level through the analysis of gel bands of extended primers and fluorescence measurements combined with rolling circle amplification. The high selectivity and sensitivity of this strategy may provide a new platform for the efficient analysis of 6mA in DNA in the future.

[A pilot study on jaw relation of edentulous patients with digital cephalometric system].

OBJECTIVE: To study the edentulous patients facio-maxillary characters, and discuss the base of recording jaw relation by swallowing action. METHODS: Forty-three edentulous patients were included in this study, age ranged from 57 to 70 yr, with new comfortable full dentures. Two lateral cephalograms for one patient were taken by SIRONA digital X-ray dental system, one of which was centric occlusion with full denture, another was the end position of swallowing action without denture. RESULTS: The vertical facial dimensions of male were a little more than that of female. The ratio of lower front facial height/upper front facial height was about 6/5, angle S-N-Po was about 78 approximately 80 degrees. These data may be useful for dentists to make diagnoses if the jaw relation is right or not. Both in vertical or anterior-posterior (horizontal) dimension, the differences between two groups (two actions in one patient) were not significant. It showed that two positions of two actions were the same. CONCLUSIONS: The SIRONA digital X-ray dental system for cephalometry was swift, and had its own characters. The way of swallowing and occluding is one of efficient methods to record the jaw relation of edentulous patients veraciously and naturally.