Visible-Light-Promoted Deoxygenative Alkylation of Quinoxalin-2(1H)-ones with Activated Alcohols.

A one-pot strategy for deoxygenative alkylation of alcohols with quinoxalin-2(1H)-ones was developed by using xanthate salts as alcohol-activating groups for radical generation in the presence of tricyclohexylphosphine under visible-light-promoted conditions. The remarkable features of this reaction include a broad substrate scope, excellent functional group tolerance, mild conditions, and simple operation. Moreover, the synthetic utility of this reaction was validated by the success of two-step one-pot reactions, scale-up synthesis, and chemoselective radical monodeoxygenation of diols.

Phototriggered Fluoroalkylation/Cyclization of Unactivated 1-Acryloyl-2-cyanoindoles: Synthesis of RCOCF2-Substituted Pyrrolo[1,2-a]indolediones.

A photochemical approach toward RCOCF2-substituted pyrrolo[1,2-a]indolediones was developed by the radical cascade difluoroalkylation/cyclization reaction of unactivated 1-acryloyl-2-cyanoindoles with ethyl iododifluoroacetate or iododifluoramides under visible-light irradiation. This transition-metal- and photosensitizer-free protocol afforded diverse difluoroalkylated pyrrolo[1,2-a]indolediones in moderate to good yields under mild reaction conditions. Most appealingly, the reaction can proceed smoothly under sunlight irradiation, which opens a new avenue toward difluoroalkylated pyrrolo[1,2-a]indolediones.

Visible-Light-Induced Cascade Cyclization of 1-Acryloyl-2-cyanoindole: Access of Difluoroalkylated Pyrrolo[1,2-a]indolediones.

An effective method for accessing diverse difluoroalkylated pyrrolo[1,2-a]indolediones via visible-light-induced PhI(OAc)2-promoted cascade difluoroalkylation/cyclization reaction under mild conditions has been established. This method is noteworthy for its use of DMSO-H2O as a green medium at room temperature and avoidance of photocatalysts. The reactions are straightforward to execute and convenient to expand on, provide good to excellent yields, and have good functional group tolerance.

Unveiling the potential of machine learning in schizophrenia diagnosis: A meta-analytic study of task-based neuroimaging data.

The emergence of machine learning (ML) techniques has opened up new avenues for identifying biomarkers associated with schizophrenia (SCZ) using task-related fMRI (t-fMRI) designs. To evaluate the effectiveness of this approach, we conducted a comprehensive meta-analysis of 31 t-fMRI studies using a bivariate model. Our findings revealed a high overall sensitivity of 0.83 and specificity of 0.82 for t-fMRI studies. Notably, neuropsychological domains modulated the classification performance, with selective attention demonstrating a significantly higher specificity than working memory (ß = 0.98, z = 2.11, P = 0.04). Studies involving older, chronic patients with SCZ reported higher sensitivity (P <0.015) and specificity (P <0.001) than those involving younger, first-episode patients or high-risk individuals for psychosis. Additionally, we found that the severity of negative symptoms was positively associated with the specificity of the classification model (ß = 7.19, z = 2.20, P = 0.03). Taken together, these results support the potential of using task-based fMRI data in combination with machine learning techniques to identify biomarkers related to symptom outcomes in SCZ, providing a promising avenue for improving diagnostic accuracy and treatment efficacy. Future attempts to deploy ML classification should consider the factors of algorithm choice, data quality and quantity, as well as issues related to generalization.

Temporal and spatial distribution of soil water repellency in grassland soils and its relation to soil moisture, hydrophobic matter, and particle size.

In grassland soils, soil water repellency (SWR) may be one of the triggers of soil erosion and degradation as it can reduce water infiltration and penetration into the soil. Few studies were focusing on the evaluation of soil hydro-physical properties, such as hydrophobicity, and their relation to soil moisture, hydrophobic matter, and particle size in grassland soils. In this study, 800 soil samples were collected from the Xilingol grassland in Inner Mongolia, China, using the water droplet penetration time (WDPT) test to evaluate water repellency and we aimed to investigate the temporal and spatial distribution of SWR in grassland soils using the Kriging and Inverse Distance Weighting (IDW) interpolation methods and determine the physical-chemical properties that trigger the SWR. The results showed that the grassland soils in the studied area were slightly water-repellent and a few portions of the area exhibited strong water-repellency. In April, areas of soils with a depth of 0-5 cm and slight to strong SWR accounted for 80 % of the total studied area, of which 5 % had strong water repellency. Moreover, in August, 90 % of the studied area consisted of soils with slight to strong SWR, of which 60 % accounted for soils with strong SWR. With a soil water content of 10.95 %, the SWR reached its peak, with an average value of 60.32 s. The SWR was positively correlated with total N, available N, and soil organic matter (SOM) contents, and therein the hydrophobic acid matter and the hydrophobic basic matter content had a positive contribution to SWR, and the hydrophilic basic matter and the hydrophilic acidic matter had a negative contribution on SWR. In addition, SWR was found to be negatively related to the soil particle size (r = -0.672). A slight SWR was also observed in the majority of the studied area, particularly in the topsoil and fine soils, especially during the monsoon period; hence, SWR must be also considered to reduce the risk of occurrence of soil erosion and degradation in grasslands.

Click-iG: Simultaneous Enrichment and Profiling of Intact N-linked, O-GalNAc, and O-GlcNAcylated Glycopeptides.

Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e., intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click-iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N-linked, mucin-type O-linked, and O-GlcNAcylated glycopeptides. We demonstrate the utility of Click-iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N-glycosites, 262 intact O-GalNAc glycosites, and 1947 O-GlcNAcylation sites were identified. Click-iG-enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways.

The impaired swim bladder via ROS-mediated inhibition of the Wnt / Hedgehog pathway in zebrafish embryos exposed to eight toxic chemicals and binary chemical mixtures.

To comprehensively explore the potential toxicity of aquatic organisms exposed to chlorinated or brominated flame retardants (BFRs) and metals mixtures, it is necessary to find a common pathway to relate local toxic targeted sites or organs. A key challenge in environmental risk assessment (ERA) is how to clarify the same or different sites or organs of toxic action in a species after exposure to individual chemicals or chemical mixtures. In this study, zebrafish embryo was used to evaluate the sub-lethal toxicity (swim bladder damage) of tris(2,3-dibromo propyl) isocyanurate (TBC), chlorinated paraffins (CPs), hexabromocyclododecane (HBCD), Cu, Cd, Pb, Ag, and Zn through optical microscopy methods, and corresponding sub-lethal molecular levels (inflammation-related enzymes [deiodinase (DIO) enzymes] and transcriptional levels of key genes) in fish through quantitative real-time PCR (qRT-PCR). The tested chemicals all caused failed inflation of the swim bladder, as indicated by activity inhibition of type 2 iodothyronine deiodinase enzyme. Following embryonic exposure to respective TBC + Cu, HBCD + TBC, and Cd + Pb mixtures, as the concentration of the respective Cu, TBC, and Pb increased, the deformity of the swim bladder increased, as also indicated by activity inhibition of type 2 iodothyronine deiodinase enzyme. Additionally, eight chemicals down-regulated Wnt (wnt3, wnt9b, fzd3b, wnt1, fzd5, and fdz1) signaling pathways, which were neurotoxic responses to individual chemical treatments and Hedgehog (ihh, shh, ptc1 and ptc2) signaling pathways. Moreover, excessive ROS induced by eight chemicals effectively induced defects in the swim bladder and Wnt/Hedgehog signaling, which also be proved in respective TBC + Cu, HBCD + TBC, and Cd + Pb mixture treatments. Our results first revealed that eight chemicals caused swim bladder developmental defects via ROS-mediated inhibition of the Wnt and Hedgehog pathways, which revealed the common targeted sites or organs (swim bladders) for further studying the toxic mechanisms underlying the chemical mixtures.

Transgelin promotes lung cancer progression via activation of cancer-associated fibroblasts with enhanced IL-6 release.

Cancer-associated fibroblasts (CAFs), the principal constituent of the heterogenous tumor microenvironment, have been shown to promote tumor progression; however, the underlying mechanism is still less clear. Here, we find that transgelin (TAGLN) protein levels increased in primary CAFs isolated from human lung cancer, compared with those in paired normal fibroblasts. Tumor microarrays (TMAs) revealed that increased stromal TAGLN levels correlates with more lymphatic metastasis of tumor cells. In a subcutaneous tumor transplantation model, overexpression of Tagln in fibroblasts also increased tumor cell spread in mice. Further experiments show that Tagln overexpression promoted fibroblast activation and mobility in vitro. And TAGLN facilitates p-p65 entry into the nucleus, thereby activating the NF-κB signaling pathway in fibroblasts. Activated fibroblasts promote lung cancer progression via enhancing the release of pro-inflammatory cytokines, especially interleukine-6 (IL-6). Our study revealed that the high levels of stromal TAGLN is a predictive risk factor for patients with lung cancer. Targeting stromal TAGLN may present an alternative therapeutic strategy against lung cancer progression.

Epigenetic regulation of HBV-specific tumor-infiltrating T cells in HBV-related HCC.

BACKGROUND AND AIMS: HBV shapes the T-cell immune responses in HBV-related HCC. T cells can be recruited to the nidus, but limited T cells participate specifically in response to the HBV-related tumor microenvironment and HBV antigens. How epigenomic programs regulate T-cell compartments in virus-specific immune processes is unclear. APPROACH AND RESULTS: We developed Ti-ATAC-seq. 2 to map the T-cell receptor repertoire, epigenomic, and transcriptomic landscape of αß T cells at both the bulk-cell and single-cell levels in 54 patients with HCC. We deeply investigated HBV-specific T cells and HBV-related T-cell subsets that specifically responded to HBV antigens and the HBV + tumor microenvironment, respectively, characterizing their T-cell receptor clonality and specificity and performing epigenomic profiling. A shared program comprising NFKB1/2-, Proto-Oncogene, NF-KB Sub unit, NFATC2-, and NR4A1-associated unique T-cell receptor-downstream core epigenomic and transcriptomic regulome commonly regulated the differentiation of HBV-specific regulatory T-cell (Treg) cells and CD8 + exhausted T cells; this program was also selectively enriched in the HBV-related Treg-CTLA4 and CD8-exhausted T cell-thymocyte selection associated high mobility subsets and drove greater clonal expansion in HBV-related Treg-CTLA4 subset. Overall, 54% of the effector and memory HBV-specific T cells are governed by transcription factor motifs of activator protein 1, NFE2, and BACH1/2, which have been reported to be associated with prolonged patient relapse-free survival. Moreover, HBV-related tumor-infiltrating Tregs correlated with both increased viral titer and poor prognosis in patients. CONCLUSIONS: This study provides insight into the cellular and molecular basis of the epigenomic programs that regulate the differentiation and generation of HBV-related T cells from viral infection and HBV + HCC unique immune exhaustion.

Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis.

T helper 17 (Th17) cells are a subset of CD4+ T helper cells involved in the inflammatory response in autoimmunity. Th17 cells secrete Th17 specific cytokines, such as IL-17A and IL17-F, which are governed by the master transcription factor RoRγt. However, the epigenetic mechanism regulating Th17 cell function is still not fully understood. Here, we reveal that deletion of RNA 5-methylcytosine (m5C) methyltransferase Nsun2 in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates Th17 cell-induced colitis pathogenesis. Mechanistically, RoRγt can recruit Nsun2 to chromatin regions of their targets, including Il17a and Il17f, leading to the transcription-coupled m5C formation and consequently enhanced mRNA stability. Our study demonstrates a m5C mediated cell intrinsic function in Th17 cells and suggests Nsun2 as a potential therapeutic target for autoimmune disease.

Cancer Serum Atlas-Supported Precise Pan-Targeted Proteomics Enable Multicancer Detection.

The wide dynamic range of serum proteome restrained discovery of clinically interested proteins in large cohort studies. Herein, we presented a high-sensitivity, high-throughput, and precise pan-targeted serum proteomic strategy for highly efficient cancer serum proteomic research and biomarker discovery. We constructed a resource of over 2000 cancer-secreted proteins, and the standard MS assays and spectra of at least one synthetic unique peptide per protein were acquired and documented (Cancer Serum Atlas, www.cancerserumatlas.com). Then, the standard peptide-anchored parallel reaction monitoring (SPA-PRM) method was developed with support of the Cancer Serum Atlas, achieving precise quantification of cancer-secreted proteins with high throughput and sensitivity. We directly quantified 325 cancer-related serum proteins in 288 serums of four cancer types (liver, stomach, lung, breast) and controls with the pan-targeted strategy and discovered considerable potential biomarker benefits for early detection of cancer. Finally, a proteomic-based multicancer detection model was built, demonstrating high sensitivity (87.2%) and specificity (100%), with 73.8% localization accuracy for an independent test set. In conclusion, the Cancer Serum Atlas provides a wide range of potential biomarkers that serve as targets and standard assays for systematic and highly efficient serological studies of cancer. The Cancer Serum Atlas-supported pan-targeted proteomic strategy enables highly efficient biomarker discovery and multicancer detection and thus can be a powerful tool for liquid biopsy.

Particle size-dependent effects of silver nanoparticles on swim bladder damage in zebrafish larvae.

Particle size-dependent biological effects of silver nanoparticles (AgNPs) are of great interest; however, the mechanism of action of silver ions (Ag+) released from AgNPs concerning AgNP particle size remains unclear. Thus, we evaluated the influence of particle size (20, 40, 60, and 80 nm) on the acute 96-h bioaccumulation and toxicity (swim bladder damage) of AgNPs in zebrafish (Danio rerio) larvae, with a focus on the mechanism of action of Ag+ released from differently sized AgNPs. The 40- and 60-nm AgNPs were more toxic than the 20- and 80-nm versions in terms of inflammation and oxidative damage to the swim bladder, as indicated by inhibition of type 2 iodothyroxine deiodinase enzyme activity, mitochondrial injury, and reduced 30-50% adenosine triphosphate content. Furthermore, up-regulation and down-regulation of swim bladder development-related gene expression was not observed for pbx1a and anxa5, but up-regulation expression of shha and ihha was observed with no statistical significance. That 20-nm AgNPs were less toxic was attributed to their rapid elimination from larvae in comparison with the elimination of 40-, 60-, and 80-nm AgNPs; thus, less Ag+ was released in 20-nm AgNP-exposed larvae. Failed inflation of swim bladders was affected by released Ag+ rather than AgNPs themselves. Overall, we reveal the toxicity contribution of Ag+ underlying the observed size-dependent effects of AgNPs and provide a scientific basis for comprehensively assessing the ecological risk and biosafety of AgNPs.

pGlycoQuant with a deep residual network for quantitative glycoproteomics at intact glycopeptide level.

Large-scale intact glycopeptide identification has been advanced by software tools. However, tools for quantitative analysis remain lagging behind, which hinders exploring the differential site-specific glycosylation. Here, we report pGlycoQuant, a generic tool for both primary and tandem mass spectrometry-based intact glycopeptide quantitation. pGlycoQuant advances in glycopeptide matching through applying a deep learning model that reduces missing values by 19-89% compared with Byologic, MSFragger-Glyco, Skyline, and Proteome Discoverer, as well as a Match In Run algorithm for more glycopeptide coverage, greatly expanding the quantitative function of several widely used search engines, including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. Further application of pGlycoQuant to the N-glycoproteomic study in three different metastatic HCC cell lines quantifies 6435 intact N-glycopeptides and, together with in vitro molecular biology experiments, illustrates site 979-core fucosylation of L1CAM as a potential regulator of HCC metastasis. We expected further applications of the freely available pGlycoQuant in glycoproteomic studies.

Overexpressing HPGDS in adipose-derived mesenchymal stem cells reduces inflammatory state and improves wound healing in type 2 diabetic mice.

BACKGROUND: In diabetes, delayed wound healing was considered as the result of excessive recruitment and retention of pro-inflammatory cells and factors. Hematopoietic prostaglandin D synthase (HPGDS) was identified from differently expressed genes of diabetic human foot skin. HPGDS is responsible for the production of prostaglandin D2 (PGD2), an inflammatory mediator. Therefore, we aim to explore whether HPGDS could be a therapeutic target in the diabetic wound (DW). METHOD: In this study, we compared gene expression profilings of diabetic human foot skin and non-diabetic human foot skin from the Gene Expression Omnibus database. We detected the characteristics of immune components in diabetic mice wound and investigated the role and underlying mechanism of the differently expressed Hpgds for the diabetic wound healing. For in vivo studies, we engineered ADSC to overexpress Hpgds (ADSCHpgds) and evaluated its effects on diabetic wound healing using a full-thickness skin wound model. For in vitro studies, we evaluated the role of ADSCHpgds conditioned medium and PGD2 on Lipopolysaccharide (LPS) induced macrophage. RESULTS: Hpgds was significantly down-regulated in type 2 diabetic mice wound and its deficiency delayed normal wound healing. ADSCHpgds accelerated DW healing by reducing neutrophil and CD8T cell recruitment, promoting M2 macrophage polarization and increasing the production of growth factors. ADSCHpgds conditioned medium showed superior capability in promoting M2 macrophage transition than conditioned medium derived from ADSC alone. CONCLUSION: Our results demonstrated that Hpgds is required for wound healing, and ADSCHpgds could accelerate DW healing by improving anti-inflammatory state and normalizing the proliferation phase of wound healing in mice. These findings provide a new insight in the therapeutic strategy of diabetic wound.

Comprehensive comparison of sample preparation workflows for proteomics.

Mass spectrometry-based proteomics experiments can be subject to a large variability, which forms an obstacle to obtaining deep and accurate protein identification. Here, to obtain an optimal sample preparation workflow for in-depth proteome identification in human tissues, we systematically compared typical procedures in the four key steps during sample preparation, including two lysis buffers (5% SDS and 7M urea/2M thiourea), acetone precipitation, two proteolytic enzyme methods (in-solution digestion and FASP), and two pre-fractionation methods (SDS-PAGE and hi-pH RPLC). Compared with other methods, the procedure, including urea/thiourea as the lysis buffer, in-solution digestion, followed by hi-pH RPLC, yields an increase in proteome coverage (+15%), matched peptides (+42.4%), and significantly high protein concentrations. We also used combinations of these sample preparation methods to demonstrate a high identification rate in the range of low molecular weight (LMW), and the performance of sample preparation workflows varied between different groups of proteins. Importantly, 3 proteins defined as missing proteins (MPs) following the Human Proteome Project (HPP) guidelines were found in our data set. Overall, our findings provide an optimal sample preparation workflow for highly efficient and unbiased global proteomic analysis in human tissues.

Shotgun lipidomics combined targeted MRM reveals sphingolipid signatures of coronary artery disease.

Sphingolipids (SPLs) are bioactive lipids that manifest structural diversity and complexity in eukaryotes. However, the distributions and functions of these molecules in mammalian tissues/cells have not been systematically investigated. Herein, we integrated shotgun lipidomics with targeted LC-MRM/MS approach to comprehensively analyze SPL species in various biological samples with high accuracy. Preliminarily, 1311 SPL molecules were identified in 18 kinds of mammalian samples, including 3 groups of human sera, 10 mouse tissues and 5 cell lines via 26 sphingoid long-chain bases scanning. The sphingolipidome compositions and distributions were systematically characterized and distinct qualitative and quantitative profiles were clearly exhibited in various samples, indicating unique biological functions of the sphingolipidomes. Next, targeted SPLs analysis by LC-MRM/MS with critical criteria monitoring two characteristic fragments of one precursor was applied to human serum samples from 24 coronary artery disease (CAD) patients and 12 healthy controls, which successfully quantified 170 SPL molecules. Ten novel SPL molecules were discovered as a potential diagnostic panel for CAD patients via multivariate exploratory receiver operating characteristic curve-based biomarker analysis. The diagnostic panel with the 10 SPL molecules achieved 97.2% accuracy, with a favorable auxiliary diagnostic value (AUC = 1.000), for the detection of CAD. These results clearly support the sphingolipidomic approach in application to discovering disease biomarker panel as well as deep investigation of biological functions of complex SPLs in mammalian samples.

Proteome-centric cross-omics characterization and integrated network analyses of triple-negative breast cancer.

We report a comprehensive proteomic study of a 90-case cohort of paired samples of triple-negative breast cancer (TNBC) in quantification, phosphorylation, and DNA-binding capacity. Four integrative subtypes (iP-1-4) are stratified on the basis of global proteome and phosphoproteome, each of which exhibits distinct molecular and pathway features. Scaffold and co-expression network analyses of three proteomic datasets, integrated with those from genome and transcriptome of the same cohort, reveal key pathways and master regulators that, characteristic of TNBC subtypes, play important regulatory roles within and between scaffold sub-structures and co-expression communities. We find that NAE1 is a potential drug target for subtype iP-1, and a series of key molecules in fatty acid metabolism, such as AKT1/FASN, are plausible targets for subtype iP-2. Libraries of proteins, pathways and networks of TNBC provide a valuable molecular infrastructure for further clinical exploration and in-depth studies of the molecular mechanisms of the disease.

Establishment of an inducible cell line for Hepatitis B virus genotype C2 and its pharmacological responses to interferons.

Hepatitis B virus (HBV) genotype C is closely associated with poor prognosis, contributing greatly to heavy chronic hepatitis B (CHB)-related liver disease burden in China and worldwide. However, the mechanistic studies on genotype C of HBV remain largely limited, partially because of a long-term lack of genotype C HBV-based stable cellular tools. According to a bioinformatics analysis on the sub-genotype C2 HBV that is predominantly endemic in China, we selected 17.3 strain as a representative isolate. With a Tet-off gene expression system, an inducible viral replication and virion DNA production of genotype C2 HBV were achieved in a cell line carrying persistent rcDNA-cccDNA recycling, termed HepG2-17.3, can be useful for virological studies. Additionally, this cell line has been formatted into cell-based assay that permits particular pharmacological screening of drug candidates, such as interferon regimens, for evaluations of the inhibitory effects on genotype C2 HBV replication.