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Endogenous release of HSO 3 - during the enzymatic oxidation of sulfur containing amino acids in mitochondria or insufficiency of sulfite oxidase results in the accumulation of sulfite and thiosulfate in biological fluids affecting mitochondrial homeostasis of brain mitochondria associated with serious clinical symptoms related to neurological disorders. The red fluorescent probe MGQ undergoes self-assembly in water and reveals aggregation induced quenching of fluorescence. MGQ reveals 143-fold and 179-fold increases in fluorescence intensity at 645 nm, respectively, in the presence of HSA and BSA and does not significantly differentiate between two albumins. The detailed studies of MGQ have been performed in the presence of BSA. The presence of other enzymes/proteins and amino acids, viz. pepsin, trypsin, lysozyme, Bromelain, lysine, histidine, hemoglobin, etc. , does not affect the fluorescence of MGQ or MGQ-BSA solutions and points to high selectivity towards BSA. The limit of detection for BSA is 10 nM. In PBS buffer, MGQ in the absence of BSA does not react with HSO 3 - and sluggishly in a 1 : 1 ethanol-water mixture. However, in the confined space of BSA/HSA, MGQ displays a signal amplification, undergoes instantaneous Michael type addition of HSO 3 - and results in a ratiometric change in fluorescence intensity in ≤1.5 min with the decrease of red fluorescence at 645 nm and emergence of green fluorescence at 515 nm. The LOD for the detection of HSO 3 - is 4 nM.

Introduction: Lignin is a promising resource for obtaining aromatic materials, however, its heterogeneous structure poses a challenge for effective utilization. One approach to produce homogeneous aromatic materials from lignin involves the application of microbial catabolism, which is gaining attention. This current study focused on constructing a catabolic pathway in Pseudomonas sp. NGC7 to produce vanillate (VA) from aromatic compounds derived from the chemical depolymerization of sulfite lignin., Results: Alkaline oxidation of sulfite lignin was performed using a hydroxide nanorod copper foam [Cu(OH) 2 /CF]-equipped flow reactor. The flow reactor operated continuously for 50 h without clogging and it yielded a sulfite lignin stream containing acetovanillone (AV), vanillin (VN), and VA as the major aromatic monomers. The catabolic pathway of Pseudomonas sp. NGC7 was optimized to maximize VA production from aromatic monomers in the sulfite lignin stream derived from this oxidation process. Pseudomonas sp. NGC7 possesses four gene sets for vanillate O-demethylase, comprising the oxygenase component (vanA) and its oxidoreductase component (vanB). Among these, the vanA4B4 gene set was identified as the key contributor to VA catabolism. To facilitate the conversion of AV to VA, AV-converting enzyme genes from Sphingobium lignivorans SYK-6 were introduced. The ΔvanA4B4 strain, harboring these AV-converting genes, produced VA from the sulfite lignin stream with 91 mol%. Further disruption of vanA1B1, vanA2B2, vanA3B3, and a vanillin reductase gene, in addition to vanA4B4, and introduction of a 5-carboxyvanillate decarboxylase gene from S. lignivorans SYK-6 to utilize 5-carboxyvanillin and 5-carboxyvanillate from the sulfite lignin stream for VA production achieved a VA yield of 103 mol%., Conclusion: Developing methods to overcome lignin heterogeneity is essential for its use as a raw material. Consolidating continuous alkaline oxidation of lignin in a Cu(OH) 2 /CF-packed flow reactor and biological funneling using an engineered catabolic pathway of Pseudomonas sp. NGC7 is a promising approach to produce VA for aromatic materials synthesis. NGC7 possesses a higher adaptability to various aromatic compounds generated from the alkaline oxidation of lignin and its natural ability to grow on p-hydroxyphenyl, guaiacyl, and syringyl compounds underscores its potential as a bacterial chassis for VA production from a wide range of lignin-derived aromatic compounds., Competing Interests: Declarations Competing interests Y.H, A.Y, N.K, E.M, and T.S. are the inventors on the patent related to this work (PCT/JP2022/13826). The authors declare no competing financial interest., (© 2024. The Author(s).)

Microbial dissimilatory sulfate reduction is a key process in the global sulfur and carbon cycles in anoxic ecosystems. In this anaerobic respiration, sulfate is phosphorylated and reduced to sulfite, which is further reduced to a DsrC-trisulfide by the dissimilatory sulfite reductase DsrAB. DsrD is a small protein that acts as an allosteric activator of DsrAB, increasing the efficiency of sulfite reduction. Here, we report a detailed study of DsrD and its interaction with DsrAB. Sequence similarity analyses show that there are three groups of DsrD in organisms with a reductive-type DsrAB. The protein regions involved in the DsrD-DsrAB interaction and activity-promoting effect were investigated through in vitro and in silico studies, including mutations of conserved DsrD residues. The results reveal that the conserved β-loop of DsrD is involved in the interaction, contributing to a better understanding of its mechanism of action., (© 2024 The Protein Society.)

Sulfate is the second most common nonmetallic ion in modern oceans, as its concentration dramatically increased alongside tectonic activity and atmospheric oxidation in the Proterozoic. Microbial sulfate/sulfite metabolism, involving organic carbon or hydrogen oxidation, is linked to sulfur and carbon biogeochemical cycles. However, the coevolution of microbial sulfate/sulfite metabolism and Earth's history remains unclear. Here, we conducted a comprehensive phylogenetic analysis to explore the evolutionary history of the dissimilatory sulfite reduction (Dsr) pathway. The phylogenies of the Dsr-related genes presented similar branching patterns but also some incongruencies, indicating the complex origin and evolution of Dsr. Among these genes, dsrAB is the hallmark of sulfur-metabolizing prokaryotes. Our detailed analyses suggested that the evolution of dsrAB was shaped by vertical inheritance and multiple horizontal gene transfer events and that selection pressure varied across distinct lineages. Dated phylogenetic trees indicated that key evolutionary events of dissimilatory sulfur-metabolizing prokaryotes were related to the Great Oxygenation Event (2.4-2.0 Ga) and several geological events in the "Boring Billion" (1.8-0.8 Ga), including the fragmentation of the Columbia supercontinent (approximately 1.6 Ga), the rapid increase in marine sulfate (1.3-1.2 Ga), and the Neoproterozoic glaciation event (approximately 1.0 Ga). We also proposed that the voluminous iron formations (approximately 1.88 Ga) might have induced the metabolic innovation of iron reduction. In summary, our study provides new insights into Dsr evolution and a systematic view of the coevolution of dissimilatory sulfur-metabolizing prokaryotes and the Earth's environment., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Young coconuts (Cocos nucifera L.) used for export are trimmed to reduce their size and weight to lower transport costs. However, trimmed coconuts have a shorter shelf life due to microbial spoilage and surface discoloration caused by enzymatic browning. To minimize these effects, trimmed coconuts were dipped in an anti-browning agent, sodium metabisulfite (SMB), and stored under ambient conditions. However, there have been no reports on the effects of SMB treatment on metabolome changes in the flesh and water of young coconuts. Hence, this study investigated the metabolite changes in trimmed young coconuts after SMB treatment under different storage conditions using a gas chromatography (GC)/mass spectrometry (MS) metabolomic profiling approach. Tall young coconut samples were trimmed and treated with a 2% SMB solution for 5 min before storage at 25 °C or 4 °C for 2-4 weeks. Coconut flesh and water samples were collected after storage for 0, 2, and 4 weeks, and were subjected to GC-MS analysis. The results showed that the major metabolites affected by coconut deterioration were amino acids, sugars, and sugar alcohols. SMB treatment and/or refrigeration can help prevent metabolite changes in the flesh and water of young coconuts. In the future, improvements in storage conditions based on metabolite profiles should be explored., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Pulsed light illumination stands out as a noteworthy technique for photosynthetic H 2 production, playing a crucial role in eliminating O 2 and activating hydrogenase enzymes. However, further improvements are essential to make H 2 photoproduction suitable for future commercial applications. In our study, we observed a distinct enhancement in pulsed light-induced H 2 photoproduction in the unicellular green alga Chlamydomonas reinhardtii when treated with the optimal concentration of the mild O 2 scavenger Na 2 SO 3 . This improvement was a result of reduced O 2 content, increased hydrogenase enzyme activity, and suppressed H 2 -uptake activity. Furthermore, our findings indicate that exposing Na 2 SO 3 -treated C. reinhardtii to optimal light waveform continues to significantly boost pulsed light-induced H 2 photoproduction, attributed to the alleviation of impaired photosystem II activity. Altogether, the combined application of optimal sulfite concentration and light waveform effectively enhances pulsed light-induced photosynthetic H 2 production in the green alga C. reinhardtii., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant., (© 2024 Wiley-VCH GmbH.)

The production of short-chain fatty acids (SCFAs) is constrained by substrate availability and the increased fractional pressure of H 2 emitted by acidogenic/fermentative bacteria during anaerobic fermentation of waste activated sludge (WAS). This study introduced a novel approach employing zero-valent iron (ZVI)-activated sulfite pretreatment combined with H 2 -consuming sulfate-reducing bacteria (SRB) mediation to improve SCFAs, especially acetate production from WAS fermentation. Experimental results showed that the combined ZVI-activated sulfite and incomplete-oxidative SRB (io-SRB) process achieved a peak SCFAs production of 868.11 mg COD/L, with acetate accounting for 80.55 %, which was 7.90- and 2.18-fold higher than that obtained from raw WAS fermentation, respectively. This could be firstly attributed to the SO 4 - and OH generated by ZVI-activated sulfite, which significantly promoted WAS decomposition, e.g., soluble proteins and carbohydrates increased 14.3- and 10.8-fold, respectively, over those in raw WAS. The biodegradation of dissolved organic matter was subsequently enhanced by the synergistic interaction and H 2 transfer between anaerobic fermentation bacteria (AFB) and io-SRB. The positive and negative correlations among AFB, nitrate-reducing bacteria (NRB) and the io-SRB consortia were revealed by molecular ecological network (MEN) and Mantel test. Moreover, the expression of functional genes was also improved, for instance, in relation to acetate formation, the relative abundances of phosphate acetyltransferase and acetate kinase was 0.002 % and 0.005 % higher than that in the control test, respectively. These findings emphasized the importance of sulfate radicals-based oxidation pretreatment and the collaborative relationships of multifunctional microbes on the value-added chemicals and energy recovery from sludge fermentation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The sulfite-reducing bacterium Bilophila wadsworthia, a common human intestinal pathobiont, is unique in its ability to metabolize a wide variety of sulfonates to generate sulfite as a terminal electron acceptor (TEA). The resulting formation of H 2 S is implicated in inflammation and colon cancer. l-cysteate, an oxidation product of l-cysteine, is among the sulfonates metabolized by B. wadsworthia, although the enzymes involved remain unknown. Here we report a pathway for l-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of l-cysteate to d-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a d-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for d-cysteate over l-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a l-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for d-cysteate. Growth of B. wadsworthia with cysteate as the electron acceptor is accompanied by production of H 2 S and induction of BwCuyA. Close homologs of BwCuyA and BwCuyB are present in diverse bacteria, including many sulfate- and sulfite-reducing bacteria, suggesting their involvement in cysteate degradation in different biological environments., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sulfate/sulfite-reducing microorganisms (SRM) are ubiquitous in nature, driving the global sulfur cycle. A hallmark of SRM is the dissimilatory sulfite reductase encoded by the genes dsrAB. Based on analysis of 950 mainly metagenome-derived dsrAB-carrying genomes, we redefine the global diversity of microorganisms with the potential for dissimilatory sulfate/sulfite reduction and uncover genetic repertoires that challenge earlier generalizations regarding their mode of energy metabolism. We show: (i) 19 out of 23 bacterial and 2 out of 4 archaeal phyla harbor uncharacterized SRM, (ii) four phyla including the Desulfobacterota harbor microorganisms with the genetic potential to switch between sulfate/sulfite reduction and sulfur oxidation, and (iii) the combination as well as presence/absence of different dsrAB-types, dsrL-types and dsrD provides guidance on the inferred direction of dissimilatory sulfur metabolism. We further provide an updated dsrAB database including > 60% taxonomically resolved, uncultured family-level lineages and recommendations on existing dsrAB-targeted primers for environmental surveys. Our work summarizes insights into the inferred ecophysiology of newly discovered SRM, puts SRM diversity into context of the major recent changes in bacterial and archaeal taxonomy, and provides an up-to-date framework to study SRM in a global context., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

The obligately anaerobic sulfite-reducing bacterium Bilophila wadsworthia is a common human pathobiont inhabiting the distal intestinal tract. It has a unique ability to utilize a diverse range of food- and host-derived sulfonates to generate sulfite as a terminal electron acceptor (TEA) for anaerobic respiration, converting the sulfonate sulfur to H 2 S, implicated in inflammatory conditions and colon cancer. The biochemical pathways involved in the metabolism of the C2 sulfonates isethionate and taurine by B. wadsworthia were recently reported. However, its mechanism for metabolizing sulfoacetate, another prevalent C2 sulfonate, remained unknown. Here, we report bioinformatics investigations and in vitro biochemical assays that uncover the molecular basis for the utilization of sulfoacetate as a source of TEA (STEA) for B. wadsworthia, involving conversion to sulfoacetyl-CoA by an ADP-forming sulfoacetate-CoA ligase (SauCD), and stepwise reduction to isethionate by NAD(P)H-dependent enzymes sulfoacetaldehyde dehydrogenase (SauS) and sulfoacetaldehyde reductase (TauF). Isethionate is then cleaved by the O 2 -sensitive isethionate sulfolyase (IseG), releasing sulfite for dissimilatory reduction to H 2 S. Sulfoacetate in different environments originates from anthropogenic sources such as detergents, and natural sources such as bacterial metabolism of the highly abundant organosulfonates sulfoquinovose and taurine. Identification of enzymes for anaerobic degradation of this relatively inert and electron-deficient C2 sulfonate provides further insights into sulfur recycling in the anaerobic biosphere, including the human gut microbiome., Competing Interests: Conflict of interest The authors declare that there are no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bilophila wadsworthia is one of the prominent sources of hydrogen sulfide (H 2 S) production in appendices, excessive levels of which can result in a weaker colonic mucus barrier, inflammatory bowel disease, and colorectal cancer. Isethionate sulfite-lyase (IslA) enzyme catalyzes H 2 S production by cleaving CS bond in isethionate, producing acetaldehyde and sulfite. In this study, we aimed to identify potential substrate antagonists for IsIA using a structure-based drug design. Initially, pharmacophore-based computational screening of the ZINC20 database yielded 66 hits that were subjected to molecular docking targeting the isethionate binding site of IsIA. Based on striking docking scores, nine compounds showed strong interaction with critical IsIA residues (Arg189, Gln193, Glu470, Cys468, and Arg678), drug-like features, appropriate adsorption, metabolism, excretion, and excretion profile with non-toxicity. Molecular dynamics simulations uncovered the significant impact of binding the compounds on protein conformational dynamics. Finally, binding free energies revealed substantial binding affinity (ranging from -35.23 to -53.88 kcal/mol) of compounds (ZINC913876497, ZINC913856647, ZINC914263733, ZINC914137795, ZINC915757996, ZINC914357083, ZINC913934833, ZINC9143362047, and ZINC913854740) for IsIA. The compounds proposed herein through a multi-faceted computational strategy can be experimentally validated as potential substrate antagonists of B. wadsworthia's IsIA for developing new medications to curb gut-associated illness in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Selenium (Se) is a micronutrient in most eukaryotes, and Se-enriched yeast is the most common selenium supplement. However, selenium metabolism and transport in yeast have remained unclear, greatly hindering the application of this element. To explore the latent selenium transport and metabolism mechanisms, we performed adaptive laboratory evolution under the selective pressure of sodium selenite and successfully obtained selenium-tolerant yeast strains. Mutations in the sulfite transporter gene ssu1 and its transcription factor gene fzf1 were found to be responsible for the tolerance generated in the evolved strains, and the selenium efflux process mediated by ssu1 was identified in this study. Moreover, we found that selenite is a competitive substrate for sulfite during the efflux process mediated by ssu1 , and the expression of ssu1 is induced by selenite rather than sulfite. Based on the deletion of ssu1 , we increased the intracellular selenomethionine content in Se-enriched yeast. This work confirms the existence of the selenium efflux process, and our findings may benefit the optimization of Se-enriched yeast production in the future. IMPORTANCE Selenium is an essential micronutrient for mammals, and its deficiency severely threatens human health. Yeast is the model organism for studying the biological role of selenium, and Se-enriched yeast is the most popular selenium supplement to solve Se deficiency. The cognition of selenium accumulation in yeast always focuses on the reduction process. Little is known about selenium transport, especially selenium efflux, which may play a crucial part in selenium metabolism. The significance of our research is in determining the selenium efflux process in Saccharomyces cerevisiae, which will greatly enhance our knowledge of selenium tolerance and transport, facilitating the production of Se-enriched yeast. Moreover, our research further advances the understanding of the relationship between selenium and sulfur in transport., Competing Interests: The authors declare no conflict of interest.

Microbial transformation of selenite [Se(IV)] to elemental selenium nanoparticles (SeNPs) is known to be an important process for removing toxic soluble selenium (Se) oxyanions and recovery of Se from the environment as valuable nanoparticles. However, the mechanism of selenite uptake by microorganisms, the first step through which Se exerts its cellular function, remains not well studied. In this study, the effects of selenite concentration, time, pH, metabolic inhibitors, and anionic analogues on selenite uptake in Rahnella aquatilis HX2 were investigated. Selenite uptake by R . aquatilis HX2 was concentration- and time-dependent, and its transport activity was significantly dependent on pH. In addition, selenite uptake in R . aquatilis HX2 was significantly inhibited by the aquaporin inhibitor AgNO 3 and sulfite (SO 3 2- ), and partially inhibited by carbonyl cyanide m -chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP) treatments. Three mutants with in-frame deletions of aqp Z, glp F, and nha A genes were constructed. The transport assay showed that the water channel protein AqpZ, and not GlpF, was a key channel of selenite uptake by R . aquatilis HX2, and sulfite and selenite had a common uptake pathway. In addition, the Na + /H + antiporter NhaA is also involved in selenite uptake in R . aquatilis HX2.

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.

Posttranscriptional RNA modifications occur in almost all types of RNA in all life forms. As an abundant RNA modification in mammals, pseudouridine (Ψ) regulates diverse biological functions of different RNA species such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), etc. However, the functional investigation of mRNA pseudouridine (Ψ) has been hampered by the lack of a quantitative method that can efficiently map Ψ transcriptome-wide. We developed bisulfite-induced deletion sequencing (BID-seq), with the optimized bisulfite-based chemical reaction to convert pseudouridine selectively and completely into Ψ-BS adduct without cytosine deamination. The Ψ-BS adduct can be further read out as deletion signatures during reverse transcription. The deletion ratios induced by Ψ sites were used for estimating the modification stoichiometry at each modified site. BID-seq starts with 10-20 ng polyA + RNA and detects thousands of mRNA Ψ sites with stoichiometry information in cell lines and tissues. We uncovered consensus motifs for Ψ in mammalian mRNA and assigned specific 'writer' proteins to individual Ψ deposition. BID-seq also confirmed the presence of Ψ within stop codons of mammalian mRNA. BID-seq set the stage for future investigations of Ψ functions in diverse biological processes., (Copyright © 2023. Published by Elsevier Inc.)

Sulfur (S) is a crucial component in the environment and living organisms. This work is the first attempt to provide an overview and critical discussion on the roles, mechanisms, and environmental applications of sulfur-oxidizing bacteria (SOB). The findings reveal that key enzymes of SOB embarked on oxidation of sulfide, sulfite, thiosulfate, and elemental S. Conversion of reduced S compounds was oxidatively catalyzed by various enzymes (e.g. sulfide: quinone oxidoreductase, flavocytochrome c-sulfide dehydrogenase, dissimilatory sulfite reductase, heterodisulfide reductase-like proteins). Environmental applications of SOB discussed include detoxifying hydrogen sulfide, soil bioremediation, and wastewater treatment. SOB producing S 0 engaged in biological S soil amendments (e.g. saline-alkali soil remediation, the oxidation of sulfide-bearing minerals). Biotreatment of H 2 S using SOB occurred under both aerobic and anaerobic conditions. Sulfide, nitrate, and sulfamethoxazole were removed through SOB suspension cultures and S 0 -based carriers. Finally, this work presented future perspectives on SOB development, including S 0 recovery, SOB enrichment, field measurement and identification of sulfur compounds, and the development of mathematical simulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Background: ATP sulfurylase (ATPS) is a crucial enzyme for the selenate assimilation pathway in plants., Results: In this study, genome-wide and comparative analyses of ATPS in Cardamine hupingshanensis, including sequence and structural analyses, were performed. The expression of ChATPS gene family members in C. hupingshanensis under selenium (Se) stress was also investigated, and our results suggest that ChATPS1-2 play key roles in the response to Se stress. Nine ATPS genes were found from C. hupingshanensis, which share highly conserved sequences with ATPS from Arabidopsis thaliana. In addition, we performed molecular docking of ATP sulfurylase in complex with compounds ATP, selenate, selenite, sulfate, and sulfite. ChAPS3-1 was found to have stronger binding energies with all compounds tested. Among these complexes, amino acid residues Arg, Gly, Ser, Glu, and Asn were commonly present., Conclusion: Our study reveals the molecular mechanism of C. hupingshanensis ATP sulfurylase interacting with selenate, which is essential for understanding selenium assimilation. This information will guide further studies on the function of the ChATPS gene family in the selenium stress response and lay the foundation for the selenium metabolic pathway in higher plants., (© 2022. The Author(s).)

The removal of environmental pollutants is important for a sustainable ecosystem and human health. Shewanella oneidensis (S. oneidensis) has diverse electron transfer pathways and can use a variety of contaminants as electron acceptors or electron donors. This paper reviews S. oneidensis's function in removing environmental pollutants, including heavy metals, inorganic non-metallic ions (INMIs), and toxic organic pollutants. S. oneidensis can mineralize o-xylene (OX), phenanthrene (PHE), and pyridine (Py) as electron donors, and also reduce azo dyes, nitro aromatic compounds (NACs), heavy metals, and iodate by extracellular electron transfer (EET). For azo dyes, NACs, Cr(VI), nitrite, nitrate, thiosulfate, and sulfite that can cross the membrane, S. oneidensis transfers electrons to intracellular reductases to catalyze their reduction. However, most organic pollutants cannot be directly degraded by S. oneidensis, but S. oneidensis can remove these pollutants by self-synthesizing catalysts or photocatalysts, constructing bio-photocatalytic systems, driving Fenton reactions, forming microbial consortia, and genetic engineering. However, the industrial-scale application of S. oneidensis is insufficient. Future research on the metabolism of S. oneidensis and interfacial reactions with other materials needs to be deepened, and large-scale reactors should be developed that can be used for practical engineering applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sulfur dioxide (SO 2 ) and its derivatives are known to be hazardous but their common application in food, especially the grape industry, is conditionally allowed. Potential hazards to consumers and the environment could occur upon the control-lost SO 2 during grape logistics and storage. Researchers have usually focused on the anti-pathogen role of SO 2 whereas limited efforts were conducted on the sulfur (S) absorption, assimilation patterns, and sulfite detoxification. In this study, short-term, room-temperature, and SO 2 -stored grapes were investigated, whose S flux of various forms was quantified through an estimation model. Accordingly, the additional accumulated S (0.50-0.86%) in pulps from atmospheric SO 2 was considered mainly through rachis transport compared to across skin surfaces and the usage arrangement of the absorbed S was included. The first quantitative evidence of induced S assimilation under SO 2 was also provided, which challenged the previous knowledge. In addition, sulfite oxidase and reductase (SiO and SiR) played major roles in sulfite detoxification, being effectively stimulated at multiple levels. The induced S metabolism associated with enhanced reactive oxygen species (ROS) scavenging capacity and alleviated senescence contributed to quality maintenance. Overall, these findings provide novel insights and are valuable supports for developing SO 2 -controlling strategies to avoid potential hazards., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zisheng Luo reports financial support was provided by Ministry of Science and Technology of the People’s Republic of China. Zisheng Luo reports financial support was provided by National Natural Science Foundation of China. Declaration of Competing Interest The authors declare that there are no known competing financial interests or conflicted personal relationships related to this work., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: DNA methylation is an important epigenetic modification that is known to regulate gene expression. Whole-genome bisulfite sequencing (WGBS) is a powerful method for studying cytosine methylation in a whole genome. However, it is difficult to obtain methylation profiles using the WGBS raw reads and is necessary to be proficient in all types of bioinformatic tools for the study of DNA methylation. In addition, recent end-to-end pipelines for DNA methylation analyses are not sufficient for addressing those difficulties., Results: Here we present msPIPE, a pipeline for DNA methylation analyses with WGBS data seamlessly connecting all the required tasks ranging from data pre-processing to multiple downstream DNA methylation analyses. The msPIPE can generate various methylation profiles to analyze methylation patterns in the given sample, including statistical summaries and methylation levels. Also, the methylation levels in the functional regions of a genome are computed with proper annotation. The results of methylation profiles, hypomethylation, and differential methylation analysis are plotted in publication-quality figures. The msPIPE can be easily and conveniently used with a Docker image, which includes all dependent packages and software related to DNA methylation analyses., Conclusion: msPIPE is a new end-to-end pipeline designed for methylation calling, profiling, and various types of downstream DNA methylation analyses, leading to the creation of publication-quality figures. msPIPE allows researchers to process and analyze the WGBS data in an easy and convenient way. It is available at https://github.com/jkimlab/msPIPE and https://hub.docker.com/r/jkimlab/mspipe ., (© 2022. The Author(s).)

The Terrestrial Miscellaneous Euryarchaeota Group has been identified in various environments, and the single genome investigated thus far suggests that these archaea are anaerobic sulfite reducers. We assemble 35 new genomes from this group that, based on genome analysis, appear to possess aerobic and facultative anaerobic lifestyles and may oxidise rather than reduce sulfite. We propose naming this order (representing 16 genera) "Lutacidiplasmatales" due to their occurrence in various acidic environments and placement within the phylum Thermoplasmatota. Phylum-level analysis reveals that Thermoplasmatota evolution had been punctuated by several periods of high levels of novel gene family acquisition. Several essential metabolisms, such as aerobic respiration and acid tolerance, were likely acquired independently by divergent lineages through convergent evolution rather than inherited from a common ancestor. Ultimately, this study describes the terrestrially prevalent Lutacidiciplasmatales and highlights convergent evolution as an important driving force in the evolution of archaeal lineages., (© 2022. The Author(s).)

Sulfite is a potent toxic substance causing harm to multi-organ in human. Despite toxicity, it is widely used as preservative, anti-browning and anti-oxidant in foods, beverages, and pharmaceuticals, which cause easy admission of sulfite in human. Sulfite is also produced endogenously during the catabolism of cysteine and methionine. In vivo, the serum sulfite level at physiological range is strictly maintained by a molybdenum dependent sulfite oxidase (SO), which catalyzes sulfite to sulfate oxidation via a two-electron oxidation pathway. The loss of SO activity causes high serum sulfite level that fosters several diseases, including asthma, neurological dysfunction, birth defects, and heart diseases. The cytotoxicity of (bi)sulfite is implicated as sulfite radicals, which are generated by mainly heme-peroxidases via a one-electron oxidation pathway. On the other hand, the toxic sulfite radicals are neutralized to sulfite by heme-globins. The enzymatic reduction of sulfite to sulfide is catalyzed by sulfite reductase, which contains an unusual metal cofactor, siroheme-[4Fe4S]-cluster. Overall, the interaction of sulfite with various metalloproteins in vivo is a close relation with human health. Therefore, this review describes the metabolic conversion of (bi)sulfite to sulfate, sulfite radical or sulfide via oxidation or reduction pathways by various metalloproteins (specially SOs, peroxidases, heme-globins, and sulfite reductases), and the potential applications of sulfite in biosensors/biofuel cells, anti-browning, and advance oxidation process., (© 2022 Wiley-VCH GmbH.)

The roles of cytosolic O-acetylserine-(thiol)-lyase A (OASTLA), chloroplastic OASTLB, and mitochondrial OASTLC in plant selenate resistance were studied in Arabidopsis. Impairment in OASTLA and OASTLB resulted in reduced biomass, chlorophyll and soluble protein content compared with selenate-treated OASTLC-impaired and wild-type plants. The generally lower total selenium (Se), protein-Se, organic-sulfur and protein-sulfur (S) content in oastlA and oastlB compared with wild-type and oastlC leaves indicated that Se accumulation was not the main cause for the stress symptoms in these mutants. Notably, the application of selenate positively induced S-starvation markers and the OASTLs, followed by increased sulfite reductase, sulfite oxidase activities, and increased sulfite and sulfide concentrations. Taken together, our results indicate a futile anabolic S-starvation response that resulted in lower glutathione and increased oxidative stress symptoms in oastlA and oastlB mutants. In-gel assays of l-cysteine and l-seleno-cysteine, desulfhydrase activities revealed that two of the three OASTL activity bands in each of the oastl single mutants were enhanced in response to selenate, whereas the impaired proteins exhibited a missing activity band. The absence of differently migrated activity bands in each of the three oastl mutants indicates that these OASTLs are major components of desulfhydrase activity, degrading l-cysteine and l-seleno-cysteine in Arabidopsis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii . In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO 3 2- ). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

Saccharomyces uvarum is one of the few fermentative species that can be used in winemaking, but its weak sulfite tolerance is the main reason for its further use. Previous studies have shown that the expression of the methionine synthase gene ( MET4 ) is upregulated in FZF1 (a gene encoding a putative zinc finger protein, which is a positive regulator of the transcription of the cytosolic sulfotransferase gene SSU1 ) overexpression transformant strains, but its exact function is unknown. To gain insight into the function of the MET4 gene, in this study, a MET4 overexpression vector was constructed and transformed into S. uvarum strain A9. The MET4 transformants showed a 20 mM increase in sulfite tolerance compared to the starting strain. Ninety-two differential genes were found in the transcriptome of A9- MET4 compared to the A9 strain, of which 90 were upregulated, and two were downregulated. The results of RT-qPCR analyses confirmed that the expression of the HOMoserine requiring gene ( HOM3 ) in the sulfate assimilation pathway and some fermentation-stress-related genes were upregulated in the transformants. The overexpression of the MET4 gene resulted in a significant increase in sulfite tolerance, the upregulation of fermentation-stress-related gene expression, and significant changes in the transcriptome profile of the S. uvarum strain.

Background: The commercial preservation of table grapes largely depends on the application of sulfur dioxide (SO 2 ). However, little is known about whether SO 2 participates in sulfur metabolism to improve the postharvest quality of table grapes. In this study, the contents of sulfur-containing compounds, activities of enzymes, and expression of genes involved in sulfur metabolism in table grapes (Vitis vinifera cv. Thompson Seedless) were evaluated., Results: The results indicated that SO 2 treatment maintained the postharvest quality of table grapes. The sulfite content in rachises and berries, but not the sulfate content, increased in response to SO 2 treatment. SO 2 caused high activities of sulfite reductase, O-acetylserine (thiol)-lyase, and γ-glutamylcysteine synthetase, thereby increasing the contents of cysteine, hydrogen sulfide, and glutathione in the rachises and berries. The expression of VvSURTL, VvATPS1, VvATPS2, and VvAPR3 decreased in response to SO 2 treatment; however, the transcript levels of VvSiR1 and VvOASTL exhibited the opposite tendency., Conclusion: These findings indicated that the sulfite converted from SO 2 participated in sulfur metabolism and maintained the postharvest quality of table grapes by modulating the contents of metabolites, activities of enzymes, and expression of genes related to sulfur metabolism. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

The mechanism of the addition of a methyl group to cytosine has been identified as one of several heritable epigenetic mechanisms. In plants, DNA methylation is involved in mediating response to stress, plant development, polyploidy, and domestication through regulation of gene expression. The correlation of epigenetic variation to phenotypic traits expands our understanding toward plant evolution, and provides new source for targeted manipulation in crop improvement. To address the increasing interest to map methylation landscape in plant species, this chapter describes methods to analyze bisulfite sequencing data and identify epigenetic variation between samples. We also detailed guidelines to highlight possible optimizations, as well as ways to tailor parameters according to data and biological variability., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Bilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H 2 S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves the glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethanesulfonate) into acetaldehyde and sulfite., Results: We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis., Conclusions: We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H 2 S production in the human gut., (© 2021. The Author(s).)

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VIIXVI and XIXVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments., Competing Interests: The authors have declared that no competing interests exist.

To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO 2 . Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO 2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO 2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO 2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO 2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20 , which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO 2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO 2 , an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO 2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO 2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

This paper explored the potential mediating role of hydrogen sulfide (H 2 S) and the oxytocin (OT) systems in hemorrhagic shock (HS) and/or traumatic brain injury (TBI). Morbidity and mortality after trauma mainly depend on the presence of HS and/or TBI. Rapid "repayment of the O 2 debt" and prevention of brain tissue hypoxia are cornerstones of the management of both HS and TBI. Restoring tissue perfusion, however, generates an ischemia/reperfusion (I/R) injury due to the formation of reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, pre-existing-medical-conditions (PEMC's) can aggravate the occurrence and severity of complications after trauma. In addition to the "classic" chronic diseases (of cardiovascular or metabolic origin), there is growing awareness of psychological PEMC's, e.g., early life stress (ELS) increases the predisposition to develop post-traumatic-stress-disorder (PTSD) and trauma patients with TBI show a significantly higher incidence of PTSD than patients without TBI. In fact, ELS is known to contribute to the developmental origins of cardiovascular disease. The neurotransmitter H 2 S is not only essential for the neuroendocrine stress response, but is also a promising therapeutic target in the prevention of chronic diseases induced by ELS. The neuroendocrine hormone OT has fundamental importance for brain development and social behavior, and, thus, is implicated in resilience or vulnerability to traumatic events. OT and H 2 S have been shown to interact in physical and psychological trauma and could, thus, be therapeutic targets to mitigate the acute post-traumatic effects of chronic PEMC's. OT and H 2 S both share anti-inflammatory, anti-oxidant, and vasoactive properties; through the reperfusion injury salvage kinase (RISK) pathway, where their signaling mechanisms converge, they act via the regulation of nitric oxide (NO).

Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p, and a low affinity transporter, Soa1p. Sulfate contributes to H 2 S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H 2 S are highly desirable. We report on the use of toxic analogues of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H 2 S and SO 2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K and E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology and shown to reduce accumulation of H 2 S and do not result in increased SO 2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H 2 S production as well as the potential use of these strains for winemaking and in yeast breeding programmes., (© 2021 John Wiley & Sons, Ltd.)

In this study, we investigated the extracellular reactive sulfur species produced by Shewanella oneidensis MR-1 during growth. The results showed that sulfite is the major extracellular sulfur metabolite released to the growth medium under both aerobic and anaerobic growth conditions. Exogenous sulfite at physiological concentrations protected S. oneidensis MR-1 from hydrogen peroxide toxicity and enhanced tolerance to the beta-lactam antibiotics cefazolin, meropenem, doripenem and ertapenem. These findings suggest that the release of extracellular sulfite is a bacterial defence mechanism that plays a role in the mitigation of environmental stress., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Sulfite oxidase (SO) deficiency is a disorder caused either by isolated deficiency of SO or by defects in the synthesis of its molybdenum cofactor. It is characterized biochemically by tissue sulfite accumulation. Patients present with seizures, progressive neurological damage, and basal ganglia abnormalities, the pathogenesis of which is not fully established. Treatment is supportive and largely ineffective. To address the pathophysiology of sulfite toxicity, we examined the effects of intrastriatal administration of sulfite in rats on antioxidant defenses, energy transfer, and mitogen-activated protein kinases (MAPK) and apoptosis pathways in rat striatum. Sulfite administration decreased glutathione (GSH) concentration and glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and glutathione reductase activities in striatal tissue. Creatine kinase (CK) activity, a crucial enzyme for cell energy transfer, was also decreased by sulfite. Superoxide dismutase-1 (SOD1) and catalase (CAT) proteins were increased, while heme oxygenase-1 (HO-1) was decreased. Additionally, sulfite altered phosphorylation of MAPK by decreasing of p38 and increasing of ERK. Sulfite further augmented the content of GSK-3β, Bok, and cleaved caspase-3, indicating increased apoptosis. JP4-039 is a mitochondrial-targeted antioxidant that reaches higher intramitochondrial levels than other traditional antioxidants. Intraperitoneal injection of JP4-039 before sulfite administration preserved activity of antioxidant enzymes and CK. It also prevented or attenuated alterations in SOD1, CAT, and HO-1 protein content, as well as changes in p38, ERK, and apoptosis markers. In sum, oxidative stress and apoptosis induced by sulfite injection are prevented by JP4-039, identifying this molecule as a promising candidate for pharmacological treatment of SO-deficient patients., (© 2020 SSIEM.)

R-loops are three-stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation is associated with genome instability and is observed in neurodevelopmental diseases and cancers. Genome-wide profiling of R-loops in normal and diseased cells can help identify locations of pathogenic R-loops and advance efforts to attenuate them. We present an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce genome-wide profiles that retain strand information. BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. In mouse embryonic stem cells, we apply BisMapR to find that gene promoters form R-loops in both directions and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression., Competing Interests: PW No competing interests declared, KS Reviewing editor, eLife, (© 2021, Wulfridge and Sarma.)

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (m 5 C) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of m 5 C in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.

DNA methylation is an important epigenetic modification and has been shown to be involved in the response to abiotic stress. However, there are few studies on DNA methylation in insect response to environmental signals. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles between two silkworm strains with significantly different resistance to heat and humidity by whole-genome bisulfite sequencing (WGBS). We identified, in total, 2934 differentially methylated regions (DMRs) between RT_48h (resistant strain with high-temperature/humidity treatment for 48 h) and ST_48h (sensitive strain with high-temperature/humidity treatment for 48 h) under cytosine context (CG), which corresponded to 1230 DMR-related genes (DMGs), and the DMRs were primarily located in the gene body (exon and intron) region. Gene ontology (GO) and KEGG analysis showed that these DMGs were most significantly enriched in binding, cellular metabolic process, and RNA transport pathways. Moreover, 10 DMGs have been revealed to be involved in the heat-humidity stress response in the silkworm. The results of this study indicated that DNA methylation plays crucial roles in silkworm response to environmental stressors and provides important clues to identify key resistance genes in silkworm under high-temperature/humidity stress response., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The metabolism of sulfur-containing amino acids (SAAs) requires an orchestrated interplay among several dozen enzymes and transporters, and an adequate dietary intake of methionine (Met), cysteine (Cys), and B vitamins. Known human genetic disorders are due to defects in Met demethylation, homocysteine (Hcy) remethylation, or cobalamin and folate metabolism, in Hcy transsulfuration, and Cys and hydrogen sulfide (H2S) catabolism. These disorders may manifest between the newborn period and late adulthood by a combination of neuropsychiatric abnormalities, thromboembolism, megaloblastic anemia, hepatopathy, myopathy, and bone and connective tissue abnormalities. Biochemical features include metabolite deficiencies (e.g. Met, S-adenosylmethionine (AdoMet), intermediates in 1-carbon metabolism, Cys, or glutathione) and/or their accumulation (e.g. S-adenosylhomocysteine, Hcy, H2S, or sulfite). Treatment should be started as early as possible and may include a low-protein/low-Met diet with Cys-enriched amino acid supplements, pharmacological doses of B vitamins, betaine to stimulate Hcy remethylation, the provision of N-acetylcysteine or AdoMet, or experimental approaches such as liver transplantation or enzyme replacement therapy. In several disorders, patients are exposed to long-term markedly elevated Met concentrations. Although these conditions may inform on Met toxicity, interpretation is difficult due to the presence of additional metabolic changes. Two disorders seem to exhibit Met-associated toxicity in the brain. An increased risk of demyelination in patients with Met adenosyltransferase I/III (MATI/III) deficiency due to biallelic mutations in the MATIA gene has been attributed to very high blood Met concentrations (typically >800 μmol/L) and possibly also to decreased liver AdoMet synthesis. An excessively high Met concentration in some patients with cystathionine β-synthase deficiency has been associated with encephalopathy and brain edema, and direct toxicity of Met has been postulated. In summary, studies in patients with various disorders of SAA metabolism showed complex metabolic changes with distant cellular consequences, most of which are not attributable to direct Met toxicity., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Due to an extreme rarity of 5-carboxylcytosine (5caC) in the mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental and computational resources, increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high-resolution mapping of 5caCGs. The method uses an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC, generating unmodified CGs, which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supports the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflect their distinct demethylation dynamics and demonstrate an association of 5caC with gene expression. Generally, we demonstrate that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Circulating cell-free DNA (cfDNA) methylation has been demonstrated to be a promising approach for non-invasive cancer diagnosis. However, the high cost of whole genome bisulfite sequencing (WGBS) hinders the clinical implementation of a methylation-based cfDNA early detection biomarker. We proposed a novel strategy in low-pass WGBS (~ 5 million reads) to detect methylation changes in circulating cell-free DNA (cfDNA) from patients with liver diseases and hepatocellular carcinoma (HCC)., Methods: The effective small sequencing depth were determined by 5 pilot cfDNA samples with relative high-depth WGBS. CfDNA of 51 patients with hepatitis, cirrhosis, and HCC were conducted using low-pass WGBS. The strategy was validated in an independent WGBS cohort of 32 healthy individuals and 26 early-stage HCC patients. Fifteen paired tumor tissue and buffy coat samples were used to characterize the methylation of hepatitis B virus (HBV) integration regions and genome distribution of cfDNA., Results: A significant enrichment of cfDNA in intergenic and repeat regions, especially in previously reported HBV integration sites were observed, as a feature of cfDNA and the bias of cfDNA release. Methylation profiles nearby HBV integration sites were a better indicator for hypomethylation of tumor genome comparing to Alu and LINE (long interspersed nuclear element) repeats, and were able to facilitate the cfDNA-based HCC prediction. Hypomethylation nearby HBV integration sites (5 kb flanking) was detected in HCC patients, but not in patients with hepatitis and cirrhosis (Methyl HBV5k , median:0.61 vs 0.72, P = 0.0003). Methylation levels of integration sites certain candidate regions exhibited an area under the receiver operation curve (AUC) value > 0.85 to discriminate HCC from non-HCC samples. The validation cohort achieved the prediction performance with an AUC of 0.954., Conclusions: Hypomethylation around viral integration sites aids low-pass cfDNA WGBS to serve as a non-invasive approach for early HCC detection, and inspire future efforts on tumor surveillance for oncovirus with integration activity.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Ca v 3.2 T-type Ca 2+ channel activity by H 2 S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H 2 S/Ca v 3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca 2+ channels or CSE, and genetic deletion of Ca v 3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X 4 /P2X 7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X 7 /P2X 4 , and caused HMGB1 release via P2X 7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H 2 S-targeted Ca v 3.2-dependent nociceptor excitation, resulting in bladder pain.

The atypical bacterial pathogen Mycoplasma pneumoniae is a leading etiological agent of community-acquired pneumonia in humans; infections are often recalcitrant, recurrent and resistant to antibiotic treatment. These characteristics suggest a mechanism that facilitates long-term colonization in hosts. In an in vitro setting, M. pneumoniae forms biofilms that are unusual in that motility plays no more than a very limited role in their formation and development. Given the unusual nature of M. pneumoniae biofilms, open questions remain concerning phenotypes associated with persistence, such as what properties might favour the bacteria while minimizing host damage. M. pneumoniae also produces several cytotoxic molecules including community-acquired respiratory distress syndrome (CARDS) toxin, H 2 S and H 2 O 2 , but how it deploys these agents during growth is unknown. Whereas several biochemical techniques for biofilm disruption were ineffective, sonication was required for disruption of M. pneumoniae biofilms to generate individual cells for comparative studies, suggesting unusual physical properties likely related to the atypical cell envelope. Nonetheless, like for other bacteria, biofilms were less susceptible to antibiotic inhibition and complement killing than dispersed cells, with resistance increasing as the biofilms matured. CARDS toxin levels and enzymatic activities associated with H 2 S and H 2 O 2 production were highest during early biofilm formation and decreased over time, suggesting attenuation of virulence in connection with chronic infection. Collectively, these findings result in a model of how M. pneumoniae biofilms contribute to both the establishment and propagation of M. pneumoniae infections, and how both biofilm towers and individual cells participate in persistence and chronic disease.

In wine production, sulfites are widely used as antimicrobials and antioxidants, whereas copper is associated with fungicides and wine fining treatments. Therefore, wine yeasts are constantly exposed to these agents. Copper tolerance is related to the copy number of the CUP1 gene, encoding for a metallothionein involved in copper detoxification. In wine yeasts, sulfite resistance mainly depends on the presence of the translocation t(XVI;VIII) in the promoter region of the SSU1 gene. This gene encodes for a plasma membrane sulfite pump involved in sulfite metabolism and detoxification. Recently, a new translocation, t(XVI;VIII), was identified. In this work, 253 Saccharomyces cerevisiae strains, representing three vineyard populations from two different continents, were analyzed, along with 20 industrial starters. Copper and sulfites tolerance as well as distribution of CUP1 gene copy-number, t(XVI;VIII)and t(XVI;XV) of SSU1 gene were studied to evaluate the impact of these genomic variations on population phenotypes. The CUP1 gene copy-number was found to be highly variable, ranging from zero to 79 per strain. Moreover it differently impacted the copper tolerance in the populations of the two continents. The diffusion of t(XVI;VIII) and, for the first time, t(XVI;XV) was determined in the three vineyard populations. The correlation between the presence of the translocation and strain sulfite tolerance levels was significant only for the t(XVI;VIII)., (© FEMS 2020.)

Hemicellulose-rich substrates produced in the lignocellulose biorefinery context can yield macromolecular xylan structures with assorted application in the chemical industry. Xylan presents natural affinity to cellulose and its incorporation onto fibers increases the physical processability of pulp; however, current studies diverge on how molar mass affects xylan interaction with cellulose. In the current work, xylans with varied structural characteristics were prepared from alkaline-sulfite pretreated sugarcane bagasse with aid of an alkaline-active xylanase and selective precipitations using different ethanol concentrations. Prepared xylan fractions, containing low levels of lignin contamination (4-9%) and molar masses ranging from 2.3 kDa to 34 kDa, were incorporated onto eucalyptus pulp fibers up to 4.7 g xylan/100 g pulp. The efficiency of xylan incorporation onto cellulosic fibers was dependent on the xylan structures, where low molar mass and low substitution degree favored high incorporation levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pulp and paper factories produce several residues that can be explored and valorized through polyhydroxyalkanoate (PHA) production via a three-step process. The objective of this work was focused on the selection step. Acidified hardwood spent sulfite liquor (HSSL), a fermented waste stream from a pulp and paper factory, was used to select a mixed microbial culture (MMC) in a sequencing batch reactor (SBR) operated for 156 days under different operational conditions. The MMC adapted to the imposed conditions, revealing its robustness whenever the operational parameters were changed. Feast-to-Famine ratio was kept below or equal to 0.2, with constant production of a copolymer of P(3HB-co-3 HV), and with storage contents values over 30 %. Changes in the operational conditions, namely cycle length, and organic load rate (OLR), successfully led to the selection of an MMC with a stable accumulation capacity and an increased biomass concentration. Next Generation Sequencing analysis was performed on samples collected during the SBR operational period. The analysis of the microbial composition of the MMC showed a rise in PHA-accumulating bacteria over time. Acidovorax and Comamonas species were found mainly to drive the PHA storage process during the first two periods of operation. After an increase in the OLR, in the last period, a shift towards Comamonas dominance occurred, suggesting a higher tolerance to the inhibitory compounds of the HSSL for this genus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Stress has been defined as any environmental factor that impairs the growth of a living organism. High concentrations of ethanol, sugars and SO 2 as well as temperature variations occurring during winemaking processes are some recognized stress factors that yeasts must overcome in order to avoid stuck or sluggish fermentations. At least two of these factors -sugar and ethanol concentrations- are strongly influenced by the global warming, which become them a worry for the future years in the winemaking industry. One of the most interesting strategies to face this complex situation is the generation of hybrids possessing, in a single yeast strain, a broader range of stress factors tolerance than their parents. In the present study, we evaluated four artificial hybrids generated with S. cerevisiae, S. uvarum and S. eubayanus using a non-GMO-generating method, in their tolerance to a set of winemaking stress factors. Their capacity to overcome specific artificial winemaking situations associated with global warming was also analyzed. All four hybrids were able to grow in a wider temperature range (8-37 °C) than their parents. Hybrids showed intermediate tolerance to higher ethanol, sugar and sulphite concentrations than their parents. Additionally, the hybrids showed an excellent fermentative behaviour in musts containing high fructose concentrations at low temperature as well as under a condition mimicking a stuck fermentation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The origin(s) of dissimilatory sulfate and/or (bi)sulfite reducing organisms (SRO) remains enigmatic despite their importance in global carbon and sulfur cycling since at least 3.4 Ga. Here, we describe novel, deep-branching archaeal SRO populations distantly related to other Diaforarchaea from two moderately acidic thermal springs. Dissimilatory (bi)sulfite reductase homologs, DsrABC, encoded in metagenome assembled genomes (MAGs) from spring sediments comprise one of the earliest evolving Dsr lineages. DsrA homologs were expressed in situ under moderately acidic conditions. MAGs lacked genes encoding proteins that activate sulfate prior to (bi)sulfite reduction. This is consistent with sulfide production in enrichment cultures provided sulfite but not sulfate. We suggest input of volcanic sulfur dioxide to anoxic spring-water yields (bi)sulfite and moderately acidic conditions that favor its stability and bioavailability. The presence of similar volcanic springs at the time SRO are thought to have originated (>3.4 Ga) may have supplied (bi)sulfite that supported ancestral SRO. These observations coincide with the lack of inferred SO 4 2- reduction capacity in nearly all organisms with early-branching DsrAB and which are near universally found in hydrothermal environments.