DNA methylation is well-established as a major epigenetic mechanism that can control gene expression and is involved in both normal development and disease. Analysis of high-throughput-sequencing-based DNA methylation data is a step toward understanding the relationship between disease and phenotype. Analysis of CpG methylation at single-base resolution is routinely done by bisulfite sequencing, in which methylated Cs remain as C while unmethylated Cs are converted to U, subsequently seen as T nucleotides. Sequence reads are aligned to the reference genome using mapping tools that accept the C-T ambiguity. Then, various statistical packages are used to identify differences in methylation between (groups of) samples. We have previously developed the Differential Methylation Analysis Pipeline (DMAP) as an efficient, fast, and flexible tool for this work, both for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS). The protocol described here includes a series of scripts that simplify the use of DMAP tools and that can accommodate the wider range of input formats now in use to perform analysis of whole-genome-scale DNA methylation sequencing data in various biological and clinical contexts. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: DMAP2 workflow for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS).

Deoxyribonucleic acid (DNA) methylation plays a key role in gene regulation and is critical for development and human disease. Techniques such as whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) allow DNA methylation analysis at the genome scale, with Illumina NovaSeq 6000 and MGI Tech DNBSEQ-T7 being popular due to their efficiency and affordability. However, detailed comparative studies of their performance are not available. In this study, we constructed 60 WGBS and RRBS libraries for two platforms using different types of clinical samples and generated approximately 2.8 terabases of sequencing data. We systematically compared quality control metrics, genomic coverage, CpG methylation levels, intra- and interplatform correlations, and performance in detecting differentially methylated positions. Our results revealed that the DNBSEQ platform exhibited better raw read quality, although base quality recalibration indicated potential overestimation of base quality. The DNBSEQ platform also showed lower sequencing depth and less coverage uniformity in GC-rich regions than did the NovaSeq platform and tended to enrich methylated regions. Overall, both platforms demonstrated robust intra- and interplatform reproducibility for RRBS and WGBS, with NovaSeq performing better for WGBS, highlighting the importance of considering these factors when selecting a platform for bisulfite sequencing.

Employing chemical pretreatment for waste activated sludge (WAS) fermentation is crucial to achieving sustainable sludge management. This study investigated the feasibility of metabisulfite (MS) pretreatment for enhancing volatile fatty acids (VFAs) production from WAS. The results show that after 24-h MS pretreatment, the content of soluble organic matter and loosely bound extracellular polymeric substances (LB-EPS), especially proteins, increased significantly. During the fermentation, MS pretreatment under alkaline conditions was more efficient, with VFA peaking on the fifth day, showing a 140 % increase compared to the alkaline control group. Correlation analysis suggests that the dosage of MS, rather than pH, is closely related to the levels of soluble protein, polysaccharides, LB-EPS, and subsequential VFAs production, while alkaline conditions facilitate the dissolution of total organic carbon. Furthermore, sulfite radicals (SO3•-) are attributed to cell inactivation and lysis, while alkaline conditions initially reduce the size of the flocs, further promoting MS for attacking flocs, thereby improving the performance of fermentation. The study also found that MS pretreatment reduced microbial community diversity, enriched hydrolytic and fermentation bacteria (Actinobacteriota and Firmicutes), and suppressed methanogens (Methanobacteriaceae and Methanosaetaceae), making it a safe, viable, and cost-effective chemical agent for sustainable sludge management.

In this study, we proposed a moderate oxidation strategy for accelerating the oxidative dissolution of zerovalent iron (ZVI) using sulfite (S(IV)), thereby improving the removal of As(V) and As(III). Results revealed that, in the presence of 2.0 mM S(IV), both As(V) and As(III) were selectively converted into scorodite at pH0 3.0-7.0, while As(III) oxidation and As(V) immobilization were impressed over pH0 8.0-10.0. Batch experiments, radical quenching experiments, and electron spin resonance (ESR) measurements demonstrated that ZVI initially boosted S(IV) activation to generate SO4•-, •OH, and protons, and in turn, ZVI was further oxidized more intensely by these radicals than by oxygen. Concurrently, substantial protons derived from S(IV) oxidation neutralized hydroxyls produced by ZVI oxidation, maintaining an acidic environment conducive to the generation of scorodite rather than iron (hydr)oxides. Characterizations of X-ray diffraction (XRD), Raman, attenuated total reflectance-Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), X-ray absorption fine structure (XAFS), field emission scanning electron microscopy (FESEM), and high-resolution transmission electron microscopy (HRTEM) confirmed that scorodite was formed in situ and then exfoliated from the surface of ZVI, and approximately 75% of ZVI could still be recovered, which contributed to efficient As removal in successive runs and real As-polluted wastewater. The application of S(IV) achieved a balance among ZVI reactivity improvement, As(V)/As(III) removal, and raw material consumption, making it a promising approach for addressing arsenic contamination in wastewater treatment.

BACKGROUND: Bisulfite (HSO3-) serves as a bleaching agent, antioxidant, antimicrobial, and regulator of enzymatic reactions in biosystem. However, abnormal levels of bisulfite can be detrimental to health. Hypochlorous acid (HOCl), which acts as bioactive small molecules, is crucial for maintaining normal biological functions in living organisms. Disruption of its equilibrium can lead to oxidative stress and various diseases. Therefore, it\'s essential to monitor the fluctuations of HOCl and HSO3- at cellular and in vivo levels to study their physiological and pathological functions.
RESULTS: This study constructed a novel NIR bifunctional colorimetric fluorescent probe using thienocoumarin-indanedione structures to identify hypochlorite (ClO-) and bisulfite (HSO3-). By using CSO-IO to recognize HSO3- and HOCl, two distinct products were generated, displaying green and blue fluorescence, respectively. This property effectively allows for the simultaneous dual-functional detection of HSO3- (LOD: 113 nM) and HOCl (LOD: 43 nM).
CONCLUSIONS: In this work, the biocompatible molecule CSO-IO has been effectively designed to detect HOCl/HSO3- in living cells and zebrafish. As a result, the dual-functional fluorescent probe has the potential to be utilized as a molecular tool to detect HSO3- derived compounds and HOCl simultaneously within the complex biological system.

In this work, a benzofuranone-derived fluorescent probe BFSF was developed for imaging the sulphite level in living hypoxia pulmonary cells. Under the excitation of 510 nm, BFSF showed a strong fluorescence response at 570 nm when reacted with sulphite. In the solution system, the constructed hypercapnia and serious hypercapnia conditions did not affect the fluorescence response. In comparison with the recently reported probes, BFSF suggested the advantages including rapid response, steady signal reporting, high specificity and low cytotoxicity upon living lung cells. Under a normal incubation atmosphere, BFSF realized the imaging of both exogenous and endogenous sulphite in living pulmonary cells. In particular, BFSF achieved imaging the decrease of the sulphite level under severe hypoxia as well as the recovery of the sulphite level with urgent oxygen supplement. With the imaging capability for the sulphite level in living pulmonary cells under hypoxia conditions, BFSF together with the information herein was meaningful for investigating the anaesthesia-related biological indexes.

SO2 derivatives, sulfite/bisulfite, are widely employed in both the food processing and drug synthesis industries. Despite their widespread application, excessive levels of sulfite/bisulfite can negatively impact human health. Most probes for detecting sulfite/bisulfite are restricted by their fluorescence within the visible spectrum range and poor solubility in aqueous solution, which limit their use in food testing and biological imaging. Herein, a near-infrared probe comprising of the cyanopyridine cyanine skeleton, 4-((Z)-2-((E)-2-chloro-3-(2-cyano-2-(1-methylpyridine-4(1H)-ylidene)ethylidene)cyclohex-1-en-1-yl)-1-cyanovinyl)-1-methylpyridin-1-ium (abbreviated as CCP), was developed. This probe enables precise quantification of bisulfite (HSO3-) in almost pure buffered solutions, showing a near-infrared fluorescence emission at 784 nm with an impressively low detection limit of 0.32 μM. The probe stands out for its exceptional selectivity, minimal susceptibility to interference, and strong adaptability. The probe CCP utilizes the CC bond to trigger a near-infrared fluorescence quenching reaction with HSO3- via nucleophilic addition, which effectively disrupts the large delocalization within the molecule for accurate HSO3- identification. Moreover, the probe has been successfully applied in detecting HSO3- in various food products and living cells, simplifying the measurement of HSO3- content in water samples. This advancement not only enhances the analytical capabilities but also contributes to ensuring food safety and environmental protection. ENVIRONMENTAL IMPLICATION: SO2 derivatives including sulfite/bisulfite, serving dual roles as preservatives and antioxidants, have widespread application across various sectors including food preservation, water sanitation, and the pharmaceutical industry. Despite their widespread application, excessive levels of sulfite/bisulfite can affect human health. Developing methods for precisely and sensitively detecting sulfite/bisulfite in food products and biological samples is important for ensuring food safety and environmental protection. Here, a sensitive near-infrared and multifunctional fluorescent probe in a 99.9 % buffered solution, along with water gel encapsulation, has been successfully applied for the detection of bisulfite in food, authentic water samples, and biological cells.

Intracellular lipid droplets (LDs) are important organelles regulating intracellular redox processes. Endogenous bisulfite/sulfite (HSO3-/SO32-) is one of the metabolites of thiol metabolism. The variation in HSO3-/SO32- content around LDs is closely related to cellular homeostasis. However, there is currently no effective method to visualize and quantify the dynamic changes in HSO3-/SO32- content around LDs. In this work, a fluorescent probe MC-BEN utilizing a triphenylamine basic framework was developed to selectively recognize HSO3-/SO32- via a nucleophilic addition reaction. The probe exhibits excellent anti-interference capability, short response time, outstanding photostability, and a low fluorescence detection limit (6.1 μM) for HSO3-/SO32- recognition. More interesting, there is a trend of accelerated contact between LDs and lysosomes after MC-BEN targeting LDs and reacting with endogenous/exogenous HSO3-/SO32-, which may provide new ideas for the study of intracellular lysosomal lipophagy.

DNA methylation is an epigenetic modification that plays a crucial role in various biological processes. Aberrant DNA methylation is closely associated with the onset of diseases, and the specific localization of methylation sites in the genome offers further insight into the connection between methylation and diseases. Currently, there are numerous methods available for site-specific methylation detection. Electrochemical biosensors have garnered significant attention due to their distinct advantages, such as rapidity, simplicity, high sensitivity, low cost, and the potential for miniaturization. In this paper, we present a systematic review of the primary sensing strategies utilized in the past decade for analyzing site-specific methylation and their applications in electrochemical sensors, from a novel perspective focusing on the localization analysis of site-specific methylation. These strategies include bisulfite treatment, restriction endonuclease treatment, other sensing strategies, and deamination without direct bisulfite treatment. We hope that this paper can offer ideas and references for establishing site-specific methylation electrochemical analysis in clinical practice.

Ultrafiltration (UF) is a highly efficient technique for algal-rich water purification, but it is heavily contaminated due to the complex water characteristics. To solve this problem, potassium permanganate (KMnO4) oxidation enhanced with sodium sulfite (Na2SO3) was proposed as a pretreatment means. The results showed that the end-normalized flux was elevated from 0.10 to 0.91, and the reversible fouling resistance was reduced by 99.95%. The membrane fouling mechanism also changed obviously, without the generation of cake filtration. Regarding the properties of algal-rich water, the zeta potential was decreased from -29.50 to -5.87 mV after KMnO4/Na2SO3 pretreatment, suggesting that the electrostatic repulsion was significantly reduced. Meanwhile, the fluorescent components in algal-rich water were significantly eliminated, and the removal of dissolved organic carbon was increased to 67.46%. In the KMnO4/Na2SO3 process, reactive manganese species (i.e., Mn(V), Mn(III) and MnO2) and reactive oxygen species (i.e., SO4•- and •OH) played major roles in purifying algal-rich water. Specifically, SO4•-, •OH, Mn(V) and Mn(III) could effectively oxidize algal pollutants. Simultaneously, the in-situ adsorption and coagulation of MnO2 could accelerate the formation of flocs by decreasing the electrostatic repulsion between cells, and protect the algal cells from being excessive oxidized. Overall, the KMnO4/Na2SO3 process showed significant potential for membrane fouling alleviation in purifying algal-rich water.