One-pot Effective Approach to 2,5-Diformylfuran From Carbohydrates Using MoS2-decorated Carbonaceous Sugarcane Bagasse.

Exploring the transformation of carbohydrates into valuable chemicals offers a promising and eco-friendly method for utilizing renewable biomass resources. Developing a bi-functional, sustainable heterogeneous catalyst is of utmost importance to attain a high level of selectivity for the desired product, 2,5-diformylfuran (DFF), in this direct conversion process. In this study, we developed a highly effective catalytic system to convert diverse carbohydrates into DFF. Our approach involved utilizing a MoS2 catalyst supported by amorphous carbon derived from sulfonated sugarcane biomass. The MoS2@SBG-SO3H composite was successfully synthesized using a facile and highly efficient method. The transformation of fructose into DFF achieved a significant yield of 70% for 5 h at 160 °C using a one-step and one-pot reaction through dehydration and oxidation with oxygen. The oxidation of 5-hydroxymethylfurfural (HMF) into DFF using MoS2@SBG-SO3H was obtained at 94% DFF within 5 h; the activation energy was 38.3 kJ.mol-1. The catalyst displayed convenient recovery and reusability. The direct synthesis of DFF from various carbohydrates, such as sucrose, glucose, maltose, and lactose, resulted in favorable yields. Our research provides a quick, green, and efficient process for preparing carbon-based solid acid catalysts and DFF.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2).

Comprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

5-Azacytidine Transiently Restores Dysregulated Erythroid Differentiation Gene Expression in TET2-Deficient Erythroleukemia Cells.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations. IMPLICATIONS: TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

A perceptual scaling approach to eyewitness identification.

Eyewitness misidentification accounts for 70% of verified erroneous convictions. To address this alarming phenomenon, research has focused on factors that influence likelihood of correct identification, such as the manner in which a lineup is conducted. Traditional lineups rely on overt eyewitness responses that confound two covert factors: strength of recognition memory and the criterion for deciding what memory strength is sufficient for identification. Here we describe a lineup that permits estimation of memory strength independent of decision criterion. Our procedure employs powerful techniques developed in studies of perception and memory: perceptual scaling and signal detection analysis. Using these tools, we scale memory strengths elicited by lineup faces, and quantify performance of a binary classifier tasked with distinguishing perpetrator from innocent suspect. This approach reveals structure of memory inaccessible using traditional lineups and renders accurate identifications uninfluenced by decision bias. The approach furthermore yields a quantitative index of individual eyewitness performance.

DNA methylation identifies genetically and prognostically distinct subtypes of myelodysplastic syndromes.

Recurrent mutations implicate several epigenetic regulators in the early molecular pathobiology of myelodysplastic syndromes (MDS). We hypothesized that MDS subtypes defined by DNA methylation (DNAm) patterns could enhance our understanding of MDS disease biology and identify patients with convergent epigenetic profiles. Bisulfite padlock probe sequencing was used to measure DNAm of ∼500 000 unique cytosine guanine dinucleotides covering 140 749 nonoverlapping regulatory regions across the genome in bone marrow DNA samples from 141 patients with MDS. Application of a nonnegative matrix factorization (NMF)-based decomposition of DNAm profiles identified 5 consensus clusters described by 5 NMF components as the most stable grouping solution. Each of the 5 NMF components identified by this approach correlated with specific genetic abnormalities and categorized patients into 5 distinct methylation clusters, each largely defined by a single NMF component. Methylation clusters displayed unique differentially methylated regulatory loci enriched for active and bivalent promoters and enhancers. Two clusters were enriched for samples with complex karyotypes, although only one had an increased number of TP53 mutations. Each of the 3 most frequently mutated splicing factors, SF3B1, U2AF1, and SRSF2, was enriched in different clusters. Mutations of ASXL1, EZH2, and RUNX1 were coenriched in the SRSF2-containing cluster. In multivariate analysis, methylation cluster membership remained independently associated with overall survival. Targeted DNAm profiles identify clinically relevant subtypes of MDS not otherwise distinguished by mutations or clinical features. Patients with diverse genetic lesions can converge on common DNAm states with shared pathogenic mechanisms and clinical outcomes.

Using Two-Dimensional Correlation Size Exclusion Chromatography (2D-CoSEC) and EEM-PARAFAC to Explore the Heterogeneous Adsorption Behavior of Humic Substances on Nanoparticles with Respect to Molecular Sizes.

The adsorption behaviors of different constituents within bulk humic substances (HS) on two nanoparticles, TiO2 and ZnO, were examined by using two-dimensional correlation size exclusion chromatography (2D-CoSEC) and excitation emission matrix-parallel factor analysis (EEM-PARAFAC), which separated bulk HS into different size fractions and fluorescent components, respectively. Subtle changes in the size distributions of HS with increasing adsorbents were successfully identified and tracked via the 2D-CoSEC. From adsorption isotherm experiments, three different HS constituent groups with respect to sizes and fluorescence features were identified by the 2D-CoSEC and EEM-PARAFAC, respectively. The chromatographically separated HS size groups presented dissimilar adsorption behaviors in terms of adsorption affinity and isotherm nonlinearity. The sequence orders of adsorption, interpreted from the 2D-CoSEC, was consistent with those of the isotherm model parameters individually calculated for different HS size subfractions, signifying the promising application of 2D-CoSEC in obtaining an insight into the heterogeneous adsorption of HS in terms of molecular sizes. EEM-PARAFAC results also supported the major finding of the 2D-CoSEC as shown by the preferential adsorption of the fluorescent components associated with large molecular sizes.

Large-Scale Targeted DNA Methylation Analysis Using Bisulfite Padlock Probes.

Bisulfite padlock probes (BSPP) are a method for the targeted quantification of DNA methylation in mammalian genomes. They can simultaneously characterize the level of methylcytosine modification in a large number of targeted regions at single-base resolution. A major advantage of BSPP is that it allows the flexible capture of an arbitrary subset of genomic regions (hundreds to hundreds of thousands of genomic loci) in single-tube reactions. Large number of samples can be processed efficiently and converted into multiplexed sequencing libraries with only three enzymatic steps, without the conventional library preparation procedures. BSPP are applicable to clinical studies, screening cell lines, and for quantifying low abundance regions using deep sequencing.

Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA.

Adjacent CpG sites in mammalian genomes can be co-methylated owing to the processivity of methyltransferases or demethylases, yet discordant methylation patterns have also been observed, which are related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that show highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks, after analysis of 61 whole-genome bisulfite sequencing data sets and validation with 101 reduced-representation bisulfite sequencing data sets and 637 methylation array data sets. Using a metric called methylation haplotype load, we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, using methylation haplotypes we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 patients with lung or colorectal cancer.

Non-catalytic and catalytic degradation of effluent dissolved organic matter under UVA-and UVC-irradiation tracked by advanced spectroscopic tools.

Non-catalytic and catalytic photodegradation of effluent dissolved organic matter (EfDOM) was examined under two different light sources (UVA and UVC). The degradation behavior was tracked by dissolved organic carbon (DOC), UV absorbance, and different fluorescent components. Catalytic UV irradiation resulted in much higher degradation rates than those without photocatalysts (TiO2 and ZnO) regardless of the tracking variables. For non-catalytic degradation, the highest removal rates of UV absorbance were found at wavelengths close to the irradiation of either UVC or UVA, while the photocatalytic degradation rates were consistently higher at longer wavelengths. The pseudo first-order rates of UV absorbance individually calculated at several representative wavelengths were very consistent with the sequential orders interpreted from two-dimensional correlation spectroscopy (2D-COS). Excitation emission matrix - parallel factor analysis (EEM-PARAFAC) identified one tryptophan-like (C1) and two humic-like (C2 and C3) components from EfDOM samples. Among those, C1 exhibited the lowest adsorption extent and the highest degradation rates for both photocatalysts, suggesting that the photocatalysis is mainly governed by hydroxyl radicals in aqueous solution. All observed degradation behaviors were well explained by the irradiation wavelengths, the extent of adsorption onto catalysts, and the presumed structure of the tracked component. Our study demonstrated that EEM-PARAFAC and 2D-COS could provide further insights into both non-catalytic and catalytic degradation of EfDOM upon UV-irradiation.

Insight into photocatalytic degradation of dissolved organic matter in UVA/TiO2 systems revealed by fluorescence EEM-PARAFAC.

Photocatalytic degradation of dissolved organic matter (DOM) using TiO2 as a catalyst and UVA as a light source was examined under various experimental settings with different TiO2 doses, solution pH, and the light intensities. The changes in UV absorbance and fluorescence with the irradiation time followed a pseudo-first order model much better than those of dissolved organic carbon. In general, the degradation rates were increased by higher TiO2 doses and light intensities. However, the exact photocatalytic responses of DOM to the irradiation were affected by many other factors such as aggregation of TiO2, light scattering, hydroxyl radicals produced, and DOM sorption on TiO2. Fluorescence excitation-emission matrix (EEM) coupled with parallel factor analysis (PARAFAC) revealed that the DOM changes in fluorescence could be described by the combinations of four dissimilar components including one protein-like, two humic-like, and one terrestrial humic-like components, each of which followed well the pseudo-first order model. The photocatalytic degradation rates were higher for protein-like versus humic-like component, whereas the opposite order was displayed for the degradation rates in the absence of TiO2, suggesting different dominant mechanisms operating between the systems with and without TiO2. Our results based on EEM-PARAFAC provided new insights into the underlying mechanisms associated with the photocatalytic degradation of DOM as well as the potential environmental impact of the treated water. This study demonstrated a successful application of EEM-PARAFAC for photocatalytic systems via directly comparing the kinetic rates of the individual DOM components with different compositions.

Advances in the profiling of DNA modifications: cytosine methylation and beyond.

Chemical modifications of DNA have been recognized as key epigenetic mechanisms for maintenance of the cellular state and memory. Such DNA modifications include canonical 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Recent advances in detection and quantification of DNA modifications have enabled epigenetic variation to be connected to phenotypic consequences on an unprecedented scale. These methods may use chemical or enzymatic DNA treatment, may be targeted or non-targeted and may utilize array-based hybridization or sequencing. Key considerations in the choice of assay are cost, minimum sample input requirements, accuracy and throughput. This Review discusses the principles behind recently developed techniques, compares their respective strengths and limitations and provides general guidelines for selecting appropriate methods for specific experimental contexts.

Spatial variability in chromophoric dissolved organic matter for an artificial coastal lake (Shiwha) and the upstream catchments at two different seasons.

Selected water quality parameters and spectroscopic characteristics of dissolved organic matter (DOM) were examined during two different seasons for an artificial coastal lake (Shiwha Lake in South Korea), which are affected by seawater exchange due to the operation of a tidal power plant and external organic loadings from the upstream catchments. The coastal lake exhibited much lower concentrations of organic matter and nutrients than the upstream sources. The spectroscopic properties of the lake DOM were easily distinguished from those of the catchment sources as revealed by a lower absorption coefficient, lower degree of humification, and higher spectral slopes. The observed DOM properties suggest that the lake DOM may be dominated by smaller molecular size and less condensed structures. For the lake and the upper streams, higher absorption coefficients and fluorescence peak intensities but lower spectral slopes and humification index were found for the premonsoon versus the monsoon season. However, such seasonal differences were less pronounced for the industrial channels in the upper catchments. Three distinctive fluorophore groups including microbial humic-like, tryptophan-like, and terrestrial humic-like fluorescence were decomposed from the fluorescence excitation-emission matrix (EEM) of the DOM samples by parallel factor analysis (PARAFAC) modeling. The microbial humic-like component was the most abundant for the industrial channels, suggesting that the component may be associated with anthropogenic organic pollution. The terrestrial humic-like component was predominant for the upper streams, and its relative abundance was higher for the rainy season. Our principal component analysis (PCA) results demonstrated that exchange of seawater and seasonally variable input of allochthonous DOM plays important roles in determining the characteristics of DOM in the lake.

Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics.

TET dioxygenases successively oxidize 5-methylcytosine (5mC) in mammalian genomes to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired to regenerate unmodified cytosines by thymine-DNA glycosylase (TDG) and base excision repair (BER) pathway, but it is unclear to what extent and at which part of the genome this active demethylation process takes place. Here, we have generated genome-wide distribution maps of 5hmC/5fC/5caC using modification-specific antibodies in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). In wild-type mouse ESCs, 5fC/5caC accumulates to detectable levels at major satellite repeats but not at nonrepetitive loci. In contrast, Tdg depletion in mouse ESCs causes marked accumulation of 5fC and 5caC at a large number of proximal and distal gene regulatory elements. Thus, these results reveal the genome-wide view of iterative 5mC oxidation dynamics and indicate that TET/TDG-dependent active DNA demethylation process occurs extensively in the mammalian genome.