Metals/bisulfite system involved generation of 24-sulfonic-25-ene ginsenoside Rg1, a potential quality control marker for sulfur-fumigated ginseng.

Ginseng is a most popular health-promoting food with ginsenosides as its main bioactive ingredients. Illegal sulfur-fumigation causes ginsenosides convert to toxic sulfur-containing derivatives, and reduced the efficacy/safety of ginseng. 24-sulfo-25-ene ginsenoside Rg1 (25-ene SRg1), one of the sulfur-containing derivatives, is a potential quality control marker of fumigated ginseng, but with low accessibility owing to its unknown generation mechanism. In this study, metals/bisulfite system involved generation mechanism was investigated and verified. The generation of 25-ene SRg1 in sulfur-fumigated ginseng is that SO2, formed during sulfur-fumigation, reacted with water and ionized into HSO3-. On the one hand, under the metals/bisulfite system, HSO3- generates HSO5- and free radicals which converted ginsenoside Rg1 to 24,25-epoxide Rg1; on the other hand, as a nucleophilic group, HSO3- reacted with 24,25-epoxide Rg1 and further dehydrated to 25-ene SRg1. This study provided a technical support for the promotion of 25-ene SRg1 as the characteristic quality control marker of sulfur-fumigated ginseng.

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal.

BACKGROUND: 5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. RESULTS: We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by ß-glucosyltransferase (ß-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10-14 to 1 × 10-8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SIGNIFICANCE: This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

Physical impediment to sodium houttuyfonate conversely reinforces ß-glucan exposure stimulated innate immune response to Candida albicans.

Candida albicans is a dimorphic opportunistic pathogen in immunocompromised individuals. We have previously demonstrated that sodium houttuyfonate (SH), a derivative of medicinal herb Houttuynia cordata Thunb, was effective for antifungal purposes. However, the physical impediment of SH by C. albicans ß-glucan may weaken the antifungal activity of SH. In this study, the interactions of SH with cell wall (CW), extracellular matrix (EM), CW ß-glucan, and a commercial ß-glucan zymosan A (ZY) were inspected by XTT assay and total plate count in a standard reference C. albicans SC5314 as well as two clinical fluconazole-resistant strains Z4935 and Z5172. After treatment with SH, the content and exposure of CW ß-glucan, chitin, and mannan were detected, the fungal clearance by phagocytosis of RAW264.7 and THP-1 was examined, and the gene expressions and levels of cytokines TNF-ɑ and IL-10 were also monitored. The results showed that SH could be physically impeded by ß-glucan in CW, EM, and ZY. This impediment subsequently triggered the exposure of CW ß-glucan and chitin with mannan masked in a time-dependent manner. SH-induced ß-glucan exposure could significantly enhance the phagocytosis and inhibit the growth of C. albicans. Meanwhile, the SH-pretreated fungal cells could greatly stimulate the cytokine gene expressions and levels of TNF-ɑ and IL-10 in the macrophages. In sum, the strategy that the instant physical impediment of C. albicans CW to SH, which can induce the exposure of CW ß-glucan may be universal for C. albicans in response to physical deterrent by antifungal drugs.

How to achieve adequate quenching for DBP analysis in drinking water?

Quenching is an important step to terminate disinfection during preparation of disinfected water samples for the analysis of disinfection byproducts (DBPs). However, an incomplete quenching might result in continued reactions of residual chlorine, whereas an excessive quenching might decompose target DBPs. Therefore, an adequate quenching to achieve simultaneous disinfection termination and DBP preservation is of particular importance. In this study, the two-stage reaction kinetics of chlorine and three commonly used quenching agents (i.e., ascorbic acid, sodium thiosulfate, and sodium sulfite) were determined. Stopping quenching during the first stage prevented interactions of residual chlorine with natural organic matter. Complete quenching was achieved by minimizing the quenching time for ascorbic acid and sodium sulfite, while limiting the quenching time to less than 3 min for sodium thiosulfate. At the optimized quenching times, the molar ratios (MRs) of quenching agent to chlorine were 1.05, 1.10, and 0.75 for ascorbic acid, sodium sulfite, and sodium thiosulfate, respectively. The destructive effects of the three quenching agents on total organic halogen (TOX) followed the rank order of ascorbic acid (33.7-64.8 %) < sodium sulfite (41.6-72.8 %) < sodium thiosulfate (43.3-73.2 %), and the destructive effects on aliphatic DBPs also followed the rank order of ascorbic acid (29.5-44.5 %) < sodium sulfite (34.9-51.9 %) < sodium thiosulfate (46.9-53.2 %). For total organic chlorine (TOCl) and aliphatic DBPs, the quenching behavior itself had more significant destructive effect than the quenching agent type/dose and quenching time, but for total organic bromine (TOBr), the destructive effect caused by quenching agent type/dose and quenching time was more significant. High-dose, long-duration quenching enhanced the reduction of TOX, but had little effect on aliphatic DBPs. Additionally, the three quenching agents reduced the levels of halophenols (except for tribromophenol), while maintained or increased the levels of tribromophenol, halobenzoic/salicylic acids, and halobenzaldehydes/salicylaldehydes. To achieve adequate quenching for overall DBP analysis in chlorinated water samples, it is recommended to use ascorbic acid at a quenching agent-to-chlorine MR of 1.0 for a quenching time of < 0.5 h.

Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

Unveiling the Chemical Composition of Sulfur-Fumigated Herbs: A Triple Synthesis Approach Using UHPLC-LTQ-Orbitrap MS-A Case Study on Steroidal Saponins in Ophiopogonis Radix.

Ophiopogonis Radix (OR) is a traditional Chinese medicine. In recent years, in order to achieve the purpose of drying, bleaching, sterilizing and being antiseptic, improving appearance, and easy storage, people often use sulfur fumigation for its processing. However, changes in the chemical composition of medicinal herbs caused by sulfur fumigation can lead to the transformation and loss of potent substances. Therefore, the development of methods to rapidly reveal the chemical transformation of medicinal herbs induced by sulfur fumigation can guarantee the safe clinical use of medicines. In this study, a combined full scan-parent ions list-dynamic exclusion acquisition-diagnostic product ions analysis strategy based on UHPLC-LTQ-Orbitrap MS was proposed for the analysis of steroidal saponins and their transformed components in sulfur-fumigated Ophiopogonis Radix (SF-OR). Based on precise mass measurements, chromatographic behavior, neutral loss ions, and diagnostic product ions, 286 constituents were screened and identified from SF-OR, including 191 steroidal saponins and 95 sulfur-containing derivatives (sulfates or sulfites). The results indicated that the established strategy was a valuable and effective analytical tool for comprehensively characterizing the material basis of SF-OR, and also provided a basis for potential chemical changes in other sulfur-fumigated herbs.

The Effects of DNA Methylation on Cytoplasmic Male Sterility in Sugar Beet.

DNA methylation is widely found in higher plants and can control gene expression by regulation without changing the DNA sequence. In this study, the whole-genome methylation map of sugar beet was constructed by WGBS (whole-genome bisulfite sequencing) technology, and the results of WGBS were verified by bisulfite transformation, indicating that the results of WGBS technology were reliable. In addition, 12 differential methylation genes (DMGs) were identified, which were related to carbohydrate and energy metabolism, pollen wall development, and endogenous hormone regulation. Quantitative real-time PCR (qRT-PCR) showed that 75% of DMG expression levels showed negative feedback with methylation level, indicating that DNA methylation can affect gene expression to a certain extent. In addition, we found hypermethylation inhibited gene expression, which laid a foundation for further study on the molecular mechanism of DNA methylation at the epigenetic level in sugar beet male sterility.

A FRET-based ratiometric fluorescent probe for SO32- detection in Chinese medicine and living cells.

Chinese herbal medicine is receiving more and more attention at home and abroad as a traditional Chinese clinical medicine. To make herbal medicines can be preserved for a long time, they are usually fumigated with sulfur. However, after the medicinal materials have been fumigated with sulfur, SO2 residues will remain, which, when exposed to water, will create sulfites and bisulfites. Excessive sulfites can cause a variety of severe ailments and diminish the quality and effectiveness of therapeutic plants. Therefore, developing an effective SO32-/HSO3- detection method is important. This study chose coumarin derivatives as fluorescent acceptors and pyridinium acrylonitrile structures as fluorescent donors to create a ratiometric fluorescent probe CPA using the fluorescence resonance energy transfer (FRET) effect. The probe CPA exhibited a fluorescence transition from red to green under excitation at 405 nm with an interval of 149 nm, a reaction time of less than 1 min, a low detection limit of 86 nM, and the probe CPA has good specific recognition of SO32- and is resistant to interference. In addition, CPA has low in vitro cytotoxicity and can successfully detect endogenous sulfites in living cells.

Adaptive Laboratory Evolution Reveals the Selenium Efflux Process To Improve Selenium Tolerance Mediated by the Membrane Sulfite Pump in Saccharomyces cerevisiae.

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.

Sulfite Impairs Bioenergetics and Redox Status in Neonatal Rat Brain: Insights into the Early Neuropathophysiology of Isolated Sulfite Oxidase and Molybdenum Cofactor Deficiencies.

Isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies are genetic diseases biochemically characterized by the toxic accumulation of sulfite in the tissues of patients, including the brain. Neurological dysfunction and brain abnormalities are commonly observed soon after birth, and some patients also have neuropathological alterations in the prenatal period (in utero). Thus, we investigated the effects of sulfite on redox and mitochondrial homeostasis, as well as signaling proteins in the cerebral cortex of rat pups. One-day-old Wistar rats received an intracerebroventricular administration of sulfite (0.5 µmol/g) or vehicle and were euthanized 30 min after injection. Sulfite administration decreased glutathione levels and glutathione S-transferase activity, and increased heme oxygenase-1 content in vivo in the cerebral cortex. Sulfite also reduced the activities of succinate dehydrogenase, creatine kinase, and respiratory chain complexes II and II-III. Furthermore, sulfite increased the cortical content of ERK1/2 and p38. These findings suggest that redox imbalance and bioenergetic impairment induced by sulfite in the brain are pathomechanisms that may contribute to the neuropathology of newborns with ISOD and MoCD. Sulfite disturbs antioxidant defenses, bioenergetics, and signaling pathways in the cerebral cortex of neonatal rats. CII: complex II; CII-III: complex II-III; CK: creatine kinase; GST: glutathione S-transferase; HO-1: heme oxygenase-1; SDH: succinate dehydrogenase; SO32-: sulfite.

Uptake of Selenite by Rahnella aquatilis HX2 Involves the Aquaporin AqpZ and Na+/H+ Antiporter NhaA.

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 AgNO3 and sulfite (SO32-), 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 aqpZ, glpF, and nhaA 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.

ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR.

MOTIVATION: Nowadays, epigenetic gene regulations are studied in each part of the biology, from embryonic development to diseases such as cancers and neurodegenerative disorders. Currently, to quantify and compare CpG methylation levels of a specific region of interest, the most accessible technique is the bisulfite sequencing PCR (BSP). However, no existing user-friendly tool is able to analyze data from all approaches of BSP. Therefore, the most convenient way to process results from the direct sequencing of PCR products (direct-BSP) is to manually analyze the chromatogram traces, which is a repetitive and prone to error task. RESULTS: Here, we implement a new R-based tool, called ABSP for analysis of bisulfite sequencing PCR, providing a complete analytic process of both direct-BSP and cloning-BSP data. It uses the raw sequencing trace files (.ab1) as input to compute and compare CpG methylation percentages. It is fully automated and includes a user-friendly interface as a built-in R shiny app, quality control steps and generates publication-ready graphics. AVAILABILITY AND IMPLEMENTATION: The ABSP tool and associated data are available on GitHub at https://github.com/ABSP-methylation-tool/ABSP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparison of Alkaline Sulfite Pretreatment and Acid Sulfite Pretreatment with Low Chemical Loading in Saccharification of Poplar.

Sulfite pretreatment is a productive process for lignin dissolution in lignocelluloses and to reduce the hydrophobicity of lignin by sulfonation, thus promoting the hydrolyzability of the substrate. Previously, sulfite pretreatment needs high dosages of chemicals and thus results in the high cost of the pretreatment and the great pressure of environmental pollution. To overcome these problems, it was crucial to research whether alkaline sulfite pretreatment (ALS) and acid sulfite pretreatment (ACS) with low chemical loading could enhance the saccharification of poplar. In this work, the results indicated that with low loading of chemicals in sulfite pretreatment, ALS pretreatment (1.6% Na2SO3 and 0.5% NaOH) at 180 °C removed more lignin, resulted in lower hydrophobicity and higher cellulase adsorption capacity of poplar than ACS pretreatment (1.6% Na2SO3 and 0.5% H2SO4) at 180 °C. A satisfying glucose yield of 84.9% and a xylose yield of 76.0% were obtained from poplar after ALS pretreatment with 1.6% Na2SO3 and 0.5% NaOH at 180 °C for 1 h using 10 FPU cellulase/g dry matter, saving sodium sulfite by 60.0% compared to the loading of sulfite in traditional sulfite pretreatment. The strategy developed in this work reduced chemical loading and cellulase loading in alkali sulfite pretreatment for the saccharification of poplar.

Efficient degradation of atrazine through in-situ anchoring NiCo2O4 nanosheets on biochar to activate sulfite under neutral condition.

Sulfite (S(IV)) is a promising substitute for sulfate radical-based advanced oxidation processes. Here, a composite of in-situ anchoring NiCo2O4 nanosheets on biochar (BC) was firstly employed as a heterogeneous activator for sulfite (NiCo2O4@BC-sulfite) to degrade atrazine (ATZ) in the neutral environment. The synergistic coupling of BC and NiCo2O4 endows the resulting composite excellent catalytic activity. 82% of the degradation ratio of ATZ (1 mg/L) could be achieved within 10 min at initial concentrations of 0.6 g/L NiCo2O4@BC, 3.0 mmol/L sulfite in neutral environment. When further supplementing sulfite into the system at 20 min (considering the depletion of sulfite), outstanding degradation efficiency (∼ 100%) were achieved in the next 10 min without any other energy input by the NiCo2O4@BC-sulfite system. The features of the prepared catalysts and the effects of some key parameters on ATZ degradation were systematically examined. A strong inner-sphere complexation (Co2+/Ni2+-SO32-) was explored between sulfite and the metal sites on the NiCo2O4@BC surface. The redox cycle of the surface metal efficiently mediated sulfite activation and triggered the series radical chain reactions. The generated radicals, in particular the surface-bound radicals were involved in ATZ degradation. High performance liquid chromatography-tandem mass spectrometry (LC-MS) technique was used to detect the degradation intermediates. Density functional theory (DFT) calculations were performed to illustrate the possible degradation pathways of ATZ. Finally, an underlying mechanism for ATZ removal was proposed. The present study offered a low-cost and sustainable catalyst for sulfite activation to remove ATZ in an environmentally friendly manner from wastewater.

Development of a responsive probe for colorimetric and fluorescent detection of bisulfite in food and animal serum samples in 100% aqueous solution.

Bisulfite (HSO3-) has the functions of bleaching, antiseptic, antioxidant, inhibiting bacterial growth, and controlling enzymatic reactions in food. However, long-term consumption of foods containing excessive amounts of bisulfite can be harmful to health. In addition, large doses of sulfur dioxide (SO2) can cause diarrhea, hypotension, allergic and asthmatic reactions in susceptible individuals. Therefore, it is urgent and essential to explore some rapid, reliable, and convenient tools to detect HSO3- in food and SO2 gas. Herein, we exploited a fluorescent probe, NPO, to detect HSO3- in 100 % aqueous solution. The probe has the advantages of easy synthesis, excellent water solubility, significant colorimetric change, good selectivity, high sensitivity, and fast response (within 1 min). Probe NPO was successfully applied for testing strips to visualize the behavior of HSO3- and SO2 gas. Moreover, the probe has been used to monitor the behavior of HSO3- in real food samples and animal serum samples.

Gene Identification, expression analysis and molecular docking of ATP sulfurylase in the selenization pathway of Cardamine hupingshanensis.

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.

Simple and sensitive determination of sulfites in Chinese herbal teas by ultrahigh-performance liquid chromatography tandem mass spectrometry.

Sulfites are used widely in food and beverage production to prevent browning or oxidation. However, the overingestion of sulfites is harmful to human health and may cause medical complications. Chinese herbal teas have been widely consumed for centuries. However, sulfite levels in Chinese herbal teas are rarely investigated and reported. Here, we present a simple, sensitive, and quantitative method to determine sulfites in Chinese herbal teas using ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) coupled with dispersive solid phase extraction. The method utilized a SeQuant ZIC-HILIC column for separation, and the optimal gradient eluents consisted of acetonitrile and aqueous solution with 0.1% acetic acid and 10 mM ammonium acetate. Porous chitosan/partially reduced graphene oxide/diatomite (CS/prGO/DM) composites were used as efficient dispersive solid phase extraction adsorbents for sample preparation. Several parameters were investigated during the extraction process, including sample-to-extraction solvent volume ratios, the extraction procedure and dosage of the adsorbent. Under the optimum conditions, the developed method gave a good determination coefficient (r2 > 0.99), low detection limits (0.51-12.1 µg kg-1) and high recoveries in the range of 83.8-102.7% at different spiked levels. The method has the great advantages of being time saving, good reproducibility and much lower detection limits when compared to titration methods. The method was further applied to analyze real herbal tea samples collected from the local market, demonstrating that our developed method is robust and useful for determining sulfites in practical application.

From Methylome to Integrative Analysis of Tissue Specificity.

DNA methylation is the most studied epigenetic mark in both plants and animals. The gold standard for assaying genome-wide DNA methylation at single-base resolution is whole-genome bisulfite sequencing (WGBS). Here, we describe an improved procedure for WGBS and original bioinformatic workflows applied to unravel tissue-specific variations of the methylome in relation to gene expression and accumulation of secondary metabolites in the medicinal plant Catharanthus roseus.

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches.

BACKGROUND: Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional software, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and to understand the DNA methylome in the context of the genetic background. The confounding effect of bisulfite conversion however can be conceptually resolved by observing differences in allele counts on a per-strand basis, whereby artificial mutations are reflected by non-complementary base pairs. RESULTS: Herein, we present a computational pre-processing approach for adapting sequence alignment data, thus indirectly enabling downstream analysis on a per-strand basis using conventional variant calling software such as GATK or Freebayes. In comparison to specialised tools, the method represents a marked improvement in precision-sensitivity based on high-quality, published benchmark datasets for both human and model plant variants. CONCLUSION: The presented "double-masking" procedure represents an open source, easy-to-use method to facilitate accurate variant calling using conventional software, thus negating any dependency on specialised tools and mitigating the need to generate additional, conventional sequencing libraries alongside bisulfite sequencing experiments. The method is available at https://github.com/bio15anu/revelio and an implementation with Freebayes is available at https://github.com/EpiDiverse/SNP.

A novel design of in-line static mixer for permanganate/bisulfite process: Numerical simulations and pilot-scale testing.

An increasing number of chemical technologies to wipe out contaminants within an incredibly short period of time have been developed recently, while their application was always hindered by the inefficient or improper mixing of reactants. To address this issue, the present work proposed a new static mixer named Tai-Chi which consists of blade, fin, and spoiler elements. Tai-Chi mixer can slice and divert the solutions inside and generate high shear flow to promote mixing process. Numerical simulations helped to determine the optimal operating conditions for Tai-Chi mixer, including laying its components anterior to the injection nozzles and keeping the velocity rate ratio of main pipe to branch pipe within the range of 0.5 to 1. Numerical simulations further proved that Tai-Chi mixer could strike a great balance between mixing performance (coefficient of variation [CoV] reaches 0.1 within 5 to 7 pipe diameters downstream) and head loss (nearly a half of other high shear static mixer in the market). Data of pilot-scale testing by Tai-Chi mixer confirm that 80% sulfamethoxazole could be eliminated in permanganate/bisulfite process within 8 pipe diameters, as well as showed the superiority of Tai-Chi's mixing performance in early stage compared with other static mixers in the market. PRACTITIONER POINTS: A Tai-Chi static mixer with blade, fin, and spoiler elements is devised. The optimal condition of flow rate and installment of Tai-Chi mixer is determined. Ultra-fast mixing is achieved by Tai-Chi (CoV < 0.1 within 5-7 pipe diameters). Pilot-scale test verifies the mixing efficiency of Tai-Chi mixer.