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.

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.

This study describes the development of a resource module that is part of a learning platform named \'NIGMS Sandbox for Cloud-based Learning\' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module is designed to facilitate interactive learning of whole-genome bisulfite sequencing (WGBS) data analysis utilizing cloud-based tools in Google Cloud Platform, such as Cloud Storage, Vertex AI notebooks and Google Batch. WGBS is a powerful technique that can provide comprehensive insights into DNA methylation patterns at single cytosine resolution, essential for understanding epigenetic regulation across the genome. The designed learning module first provides step-by-step tutorials that guide learners through two main stages of WGBS data analysis, preprocessing and the identification of differentially methylated regions. And then, it provides a streamlined workflow and demonstrates how to effectively use it for large datasets given the power of cloud infrastructure. The integration of these interconnected submodules progressively deepens the user\'s understanding of the WGBS analysis process along with the use of cloud resources. Through this module, we can enhance the accessibility and adoption of cloud computing in epigenomic research, speeding up the advancements in the related field and beyond. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning\'\' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Pseudouridine (Ψ) is a widespread RNA modification found in various RNA species, including rRNA, tRNA, snRNA, mRNA, and long noncoding RNA (lncRNA). Understanding the function of Ψ in these RNA types requires a robust method for the detection and quantification of the Ψ level at single-nucleotide resolution. A previously used method utilizes Ψ labeling by N-cyclohexyl-N\'-β-(4-methylmorpholinium)ethylcarbodiimide (CMC). The quantification of Ψ is based on the stop ratio after reverse transcription. However, the use of CMC followed by strong alkaline treatment causes severe RNA degradation, often requiring a large amount of RNA. The removal of CMC and recovery of RNA by ethanol precipitation are also time-consuming. Here, we introduce a Bisulfite Incorporation Hindered ligation-based method (BIHIND), which can detect and quantify Ψ sites on rRNA, mRNA, and noncoding RNA. BIHIND can be coupled with quantitative PCR (BIHIND-qPCR) for quantitative detection of Ψ fraction at individual modification sites, as well as with next-generation sequencing (BIHIND-seq) for high-throughput sequencing of Ψ without requiring reverse transcription. We validated the robustness of BIHIND with the elucidation of Ψ dynamics following pseudouridine synthase depletion.

DNA methylation is an important epigenetic modification that regulates chromatin structure and the cell-type-specific expression of genes. The association of aberrant DNA methylation with many diseases, as well as the increasing interest in modifying the methylation mark in a directed manner at genomic sites using epigenome editing for research and therapeutic purposes, increases the need for easy and efficient DNA methylation analysis methods. The standard approach to analyze DNA methylation with a single-cytosine resolution is bisulfite conversion of DNA followed by next-generation sequencing (NGS). In this chapter, we describe a robust, powerful, and cost-efficient protocol for the amplification of target regions from bisulfite-converted DNA, followed by a second PCR step to generate libraries for Illumina NGS. In the two consecutive PCR steps, first, barcodes are added to individual amplicons, and in the second PCR, indices and Illumina adapters are added to the samples. Finally, we describe a detailed bioinformatics approach to extract DNA methylation levels of the target regions from the sequencing data. Combining barcodes with indices enables a high level of multiplexing allowing to sequence multiple pooled samples in the same sequencing run. Therefore, this method is a robust, accurate, quantitative, and cheap approach for the readout of DNA methylation patterns at defined genomic regions.

DNA methylation is a covalent modification of DNA that plays important roles in processes such as the regulation of gene expression, transcription factor binding, and suppression of transposable elements. The use of whole-genome bisulfite sequencing (WGBS) enables the genome-wide identification and quantification of DNA methylation patterns at single-base resolution and is the gold standard for the analysis of DNA methylation. However, the computational analysis of WGBS data can be particularly challenging, as many computationally intensive steps are required. Here, we outline step-by-step an approach for the analysis and interpretation of WGBS data. First, sequencing reads must be trimmed, quality-checked, and aligned to the genome. Second, DNA methylation levels are estimated at each cytosine position using the aligned sequence reads of the bisulfite-treated DNA. Third, regions of differential cytosine methylation between samples can be identified. Finally, these data need to be visualized and interpreted in the context of the biological question at hand.

Whole-genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at a single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high-quality libraries amplified with fewer polymerase chain reaction (PCR) cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3\'-adenylated, and ligated to adaptors with fewer clean-up steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with a few PCR cycles to reach the yield needed for sequencing.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.