Qualitative and Quantitative Analytical Techniques of Nucleic Acid Modification Based on Mass Spectrometry for Biomarker Discovery.

Nucleic acid modifications play important roles in biological activities and disease occurrences, and have been considered as cancer biomarkers. Due to the relatively low amount of nucleic acid modifications in biological samples, it is necessary to develop sensitive and reliable qualitative and quantitative methods to reveal the content of any modifications. In this review, the key processes affecting the qualitative and quantitative analyses are discussed, such as sample digestion, nucleoside extraction, chemical labeling, chromatographic separation, mass spectrometry detection, and data processing. The improvement of the detection sensitivity and specificity of analytical methods based on mass spectrometry makes it possible to study low-abundance modifications and their biological functions. Some typical nucleic acid modifications and their potential as biomarkers are displayed, and efforts to improve diagnostic accuracy are discussed. Future perspectives are raised for this research field.

Evaluation of the effectiveness of amendments derived from vermicompost combined with modified shell powder on Cd immobilization in Cd-contaminated soil by multiscale experiments.

The widespread heavy metal contamination of agricultural soils poses an enormous challenge to food safety. To evaluate the Cd immobilization potential of vermicompost combined with modified shell powder (VMSP) on Cd-contaminated soil, batch adsorption tests and field experiments were conducted. First, the Cd2+ removal characteristics and adsorption mechanisms of vermicompost (V), vermicompost combined with shell powder (VSP), and VMSP in an aqueous solution were investigated by batch tests. Then, 3 kg·m2 V, VSP, and VMSP doses were applied to Cd-contaminated farmland soils as soil amendments to plant green garlic (Allium sativum L.) and investigate their Cd immobilization effects in Cd-contaminated soils. Batch adsorption tests showed that VMSP was most effective for Cd2+ removal, with adsorption rates as high as 85.7-99.79% and desorption rates of approximately 1.25-1.34%. Combining further characterization analysis of VMSP, it was demonstrated that the adsorption mechanism of Cd2+ was monolayer chemisorption, mainly involving the complexation reaction of Cd2+ with organic functional groups and the precipitation reaction of Cd2+ with mineral elements. The field experiment showed that adding V, VSP, and VMSP effectively inhibited the enrichment of Cd in green garlic, and the Cd content was reduced by 42.18%, 46.88%, and 68.75%, respectively. However, only the Cd content of green garlic treated with VMSP was lower than the national standard for food safety in China (Cd≤ 0.2 mg·kg-1). V, VSP, and VMSP additions improved soil fertility and reduced Cd bioavailability in the soil by 15.5%, 18.9%, and 36.3%, respectively. In addition, V, VSP, and VMSP addition increased bacterial diversity and improved bacterial communities and functions in the soil by improving basic soil properties and reducing Cd-related toxicity. The results indicated that VMSP is a promising amendment for Cd immobilization in Cd-contaminated farmland soils.

In-source fragmentation of nucleosides in electrospray ionization towards more sensitive and accurate nucleoside analysis.

Nucleosides have been found to suffer in-source fragmentation (ISF) in electrospray ionization mass spectrometry, which leads to reduced sensitivity and ambiguous identification. In this work, a combination of theoretical calculations and nuclear magnetic resonance analysis revealed the key role of protonation at N3 near the glycosidic bond during ISF. Therefore, an ultrasensitive liquid chromatography-tandem mass spectrometry system for 5-formylcytosine detection was developed with 300 fold signal enhancement. Also, we established a MS1-only platform for nucleoside profiling and successfully identified sixteen nucleosides in the total RNA of MCF-7 cells. Taking ISF into account, we can realize analysis with higher sensitivity and less ambiguity, not only for nucleosides, but for other molecules with similar protonation and fragmentation behaviors.

Ultrasensitive HPLC-MS Quantification of S-(2-Succino) Cysteine Based on Ethanol/Acetyl Chloride Derivatization in Fumarate Accumulation Cells.

Succination is a nonenzymatic and irreversible post-translational modification (PTM) with important biological significance, yielding S-(2-succino) cysteine (2SC) residue. This PTM is low in abundance and often requires a large amount of protein samples for 2SC quantification. In this work, an efficient quantification method based on ethanol/acetyl chloride chemical derivatization was developed. The three carboxyl groups of 2SC were all esterified to increase hydrophobicity, greatly improving its ionization efficiency. The sensitivity was increased by 112 times; the limit of detection was reduced to 0.885 fmol, and the protein usage was reduced by at least 10 times. The established method was used to detect the overall concentration of 2SC in fumarate accumulation cells quantitatively.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

Heavy metal and δ13C value variations and characterization of dissolved organic matter (DOM) during vermicomposting of pig manure amended with 13C-labeled rice straw.

In this work, a mixture of pig manure and 13C-labeled rice straw was vermicomposted with Eisenia fetida for 40 days. The results showed that after they acclimated to the vermicomposting environment, the earthworms helped to degrade the substrate residues. After 40 days, the vermicomposting led to much higher pH, EC, C/N, available K, available P, available Zn, and CEC values but much lower available N and available Cu values in the substrate residues compared to the initial values. The earthworms accumulated 13C, Cu, and Zn with a heavy metal enrichment capacity in the order of Cu > Zn. Furthermore, the correlation analysis showed that the Cu and Zn content in the DOM was mainly controlled by the Cu and Zn content in the pig manure. The earthworms accumulated Cu and Zn by feeding on the substrate residues, but the DOM from the 13C-labeled rice straw helped to extract Zn from the substrate residues and promoted the migration of Zn into the earthworm tissues and/or DOM. Moreover, the characterization of the DOM extracted from the substrate residues revealed a decrease in the content of the aliphatic alcohols or polysaccharide-like substances and an increase in the aromatic compounds and fulvic or humic acid-like substances in the DOM as the vermicomposting time increased. This indicated that the higher humification degree in the DOM caused by vermicomposting contributed to the increased mobility of Zn in the substrate residues and helped Zn migrate into the earthworms or DOM.

Comparative adsorption of Pb2+ and Cd2+ by cow manure and its vermicompost.

Organic waste has great potential for use as an amendment to immobilize heavy metals in the environment. Therefore, this study investigates various properties of cow manure (CM) and its derived vermicompost (CV), including the pH, cationic exchangeable capacity (CEC), elemental composition and surface structure, to determine the potential of these waste products to remove Pb2+ and Cd2+ from solution. The results demonstrate that CV has a much higher pH, CEC and more irregular pores than CM and is enriched with minerals and ash content but has a lower C, H, O and N content. Adsorption isotherms studies shows that the adsorption of Pb2+ and Cd2+ onto either CM or CV follows a Langmuir model and presents maximum Pb2+ and Cd2+ adsorption capacities of 102.77 mg g-1 and 38.11 mg g-1 onto CM and 170.65 and 43.01 mg g-1 onto CV, respectively. Kinetic studies show that the adsorption of Pb2+ onto CM and CV fits an Elovich model, whereas the adsorption of Cd2+ onto CM and CV fits a pseudo-second-order model. Desorption studies indicate that CV is more effective than CM in removing Pb2+ and Cd2+. FTIR analysis demonstrates that the adsorption of Pb2+ and Cd2+ onto CM mainly depends on existed aliphatic alcohol, aromatic acid as well as new produced carbonates, whereas that onto CV may be contributed by the existed aliphatic alcohol, aromatic acids as well as some carbonates and phosphates. Thus, vermicomposting disposal of cow manure with destination mineral addition may broaden the way of its recycle and environmental usage.

[Adsorption of Pb2+ and Cd2+ from Aqueous Solution Using Vermicompost Derived from Cow Manure and Its Biochar].

Using vermicompost (CV) as raw material, its biochar (CVC350) was prepared at 350℃ and then their physio-biochemical properties were characterized. Furthermore, adsorption studies were performed in a batch system for removing Pb2+ and Cd2+ ions from solution. The characterization results revealed much higher surface area, smaller pore size, greater aromaticity and nonpolarity of CVC350 as compared to CV. Batch adsorption experiments revealed that both the adsorption of Pb2+ and Cd2+ onto CV or CVC350 fitted Langmuir isotherm model very well, and the maximum adsorption capacity of Pb2+ was in the order of CVC350>CV, but no difference was observed for the adsorption capacity of Cd2+ between CV and CVC350. The desorption studies showed that both CV and CVC350 had much higher adsorption rate for Pb2+ than that for Cd2+, and the Cd2+ adsorbed could be more easily desorbed from CV and CVC350 compared with that for the Pb2+ adsorbed. Both the dynamic adsorption process of Pb2+ onto CV and CVC350 was a rapid process, however, the adsorption process of Cd2+ onto CV and CVC350 could be separated into the first rapid step and the second slower step. The adsorption capacity of Pb2+ or Cd2+ onto CV and CVC350 was only affected by the much lower initial pH of the solution, besides, the adsorption capacity of Cd2+ onto CV and CVC350 was relatively more influenced by the initial pH compared with that of Pb2+. Moreover, FTIR analysis showed that the adsorption of Pb2+ and Cd2+on CV depended on the active sites such as aliphatic alcohol, aliphatic acid,carbonates as well as phosphate while that on CVC350 mainly relied on aromatic alcohol, aromatic acid and carbonates.