TL-MSE2-Net: Transfer learning based nested model for cerebrovascular segmentation with aneurysms.

Cerebrovascular (i.e., cerebral vessel) segmentation is essential for diagnosing and treating brain diseases. Convolutional neural network models, such as U-Net, are commonly used for this purpose. Unfortunately, such models may not be entirely satisfactory in dealing with cerebrovascular segmentation with tumors due to the following issues: (1) Relatively small number of clinical datasets from patients obtained through different modalities such as computed tomography (CT) and magnetic resonance imaging (MRI), leading to inadequate training and lack of transferability in the modeling; (2) Insufficient feature extraction caused by less attention to both convolution sizes and cerebral vessel edges. Inspired by the existence of similar features on cerebral vessels between normal subjects and patients, we propose a transfer learning strategy based on a pre-trained nested model called TL-MSE2-Net. This model uses one of the publicly available datasets for cerebrovascular segmentation with aneurysms. To address issue (1), our transfer learning strategy leverages a pre-trained model that uses a large number of datasets from normal subjects, providing a potential solution to the lack of sufficient clinical datasets. To tackle issue (2), we structure the pre-trained model based on 3D U-Net, comprising three blocks: ResMul, DeRes, and REAM. The ResMul and DeRes blocks enhance feature extraction by utilizing multiple convolution sizes to capture multiscale features, and the REAM block increases the weight of the voxels on the edges of the given 3D volume. We evaluated the proposed model on one small private clinical dataset and two publicly available datasets. The experimental results demonstrated that our MSE2-Net framework achieved an average Dice score of 70.81 % and 89.08 % on the two publicly available datasets, outperforming other state-of-the-art methods. Ablation studies were also conducted to validate the effectiveness of each block. The proposed TL-MSE2-Net yielded better results than MSE2-Net on a small private clinical dataset, with increases of 5.52 %, 3.37 %, 6.71 %, and 0.85 % for the Dice score, sensitivity, Jaccard index, and precision, respectively.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

Analysis of Cell Glycogen with Quantitation and Determination of Branching Using Liquid Chromatography-Mass Spectrometry.

Glycogen is a highly branched biomacromolecule that functions as a glucose buffer. It is involved in multiple diseases such as glycogen storage disorders, diabetes, and even liver cancer, where the imbalance between biosynthetic and catabolic enzymes results in structural alterations and abnormal accumulation of glycogen that can be toxic to cells. Accurate and sensitive glycogen quantification and structural determination are prerequisites for understanding the phenotypes and biological functions of glycogen under these conditions. In this research, we furthered cell glycogen characterization by presenting a highly sensitive method to measure the glycogen content and degree of branching. The method employed a novel fructose density gradient as an alternative to the traditional sucrose gradient to fractionate glycogen from cell mixtures using ultracentrifugation. Fructose was used to avoid the large glucose background, allowing the method to be highly quantitative. The glycogen content was determined by quantifying 1-phenyl-3-methyl-5-pyrazolone (PMP)-derivatized glucose residues obtained from acid-hydrolyzed glycogen using ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/QqQ-MS). The degree of branching was determined through linkage analysis where the glycogen underwent permethylation, hydrolysis, PMP derivatization, and UHPLC/QqQ-MS analysis. The new approach was used to study the effect of insulin on the glycogen phenotypes of human hepatocellular carcinoma (Hep G2) cells. We observed that cells produced greater amounts of glycogen with less branching under increasing insulin levels before reaching the cell's insulin-resistant state, where the trend reversed and the cells produced less but higher-branched glycogen. The advantage of this method lies in its high sensitivity in characterizing both the glycogen level and the structure of biological samples.

Glycomic, Glycoproteomic, and Proteomic Profiling of Philippine Lung Cancer and Peritumoral Tissues: Case Series Study of Patients Stages I-III.

Lung cancer is the leading cause of cancer death and non-small cell lung carcinoma (NSCLC) accounting for majority of lung cancers. Thus, it is important to find potential biomarkers, such as glycans and glycoproteins, which can be used as diagnostic tools against NSCLC. Here, the N-glycome, proteome, and N-glycosylation distribution maps of tumor and peritumoral tissues of Filipino lung cancer patients (n = 5) were characterized. We present several case studies with varying stages of cancer development (I-III), mutation status (EGFR, ALK), and biomarker expression based on a three-gene panel (CD133, KRT19, and MUC1). Although the profiles of each patient were unique, specific trends arose that correlated with the role of aberrant glycosylation in cancer progression. Specifically, we observed a general increase in the relative abundance of high-mannose and sialofucosylated N-glycans in tumor samples. Analysis of the glycan distribution per glycosite revealed that these sialofucosylated N-glycans were specifically attached to glycoproteins involved in key cellular processes, including metabolism, cell adhesion, and regulatory pathways. Protein expression profiles showed significant enrichment of dysregulated proteins involved in metabolism, adhesion, cell-ECM interactions, and N-linked glycosylation, supporting the protein glycosylation results. The present case series study provides the first demonstration of a multi-platform mass-spectrometric analysis specifically for Filipino lung cancer patients.

Combining quasi-ZIF-67 hybrid nanozyme and G-quadruplex/hemin DNAzyme for highly sensitive electrochemical sensing.

Zeolitic imidazolate frameworks (ZIFs), a famous subfamily of metal-organic frameworks (MOFs), are considered promising electrocatalysts. Herein, ZIF-67 was selected as an electrocatalyst for designing electrochemical sensors due to having the best electrocatalytic activity in ZIFs. To overcome the insufficient electrocatalytic activity of ZIFs, ZIF-67 derivatives (QZIF-67-X, where X represents calcination time) were obtained by calcining at 250 °C for a certain time. The porous structure of the precursor in QZIF-67-X is maintained, exposing more active centers. QZIF-67-X could accelerate electron transfer and lead to improve the electrocatalytic performance. Moreover, QZIF-67-2 was chosen as an Au nanoparticle-supported nanocarrier to further bind G-quadruplex/hemin DNAzymes with strong catalytic activity due to the best supporting activity of QZIF-67-2 among QZIF-67-X. The synergistic catalysis of QZIF-67-2 and G-quadruplex/hemin DNAzymes effectively amplified the reduction current signal of H2O2. The linear range of the prepared electrochemical sensor was 2 µM-65 mM, and the detection limit was 1.2 µM. Moreover, the real-time detection of H2O2 from HepG2 cells was achieved by the sensor, providing a novel technique for efficient anticancer drug evaluation. These results suggested that QZIF-67 can be utilized as an efficient electrocatalyst for improving the sensitivity of sensors.

Biogeographic and disease-specific alterations in epidermal lipid composition and single-cell analysis of acral keratinocytes.

The epidermis is the outermost layer of skin. Here, we used targeted lipid profiling to characterize the biogeographic alterations of human epidermal lipids across 12 anatomically distinct body sites, and we used single-cell RNA-Seq to compare keratinocyte gene expression at acral and nonacral sites. We demonstrate that acral skin has low expression of EOS acyl-ceramides and the genes involved in their synthesis, as well as low expression of genes involved in filaggrin and keratin citrullination (PADI1 and PADI3) and corneodesmosome degradation, changes that are consistent with increased corneocyte retention. Several overarching principles governing epidermal lipid expression were also noted. For example, there was a strong negative correlation between the expression of 18-carbon and 22-carbon sphingoid base ceramides. Disease-specific alterations in epidermal lipid gene expression and their corresponding alterations to the epidermal lipidome were characterized. Lipid biomarkers with diagnostic utility for inflammatory and precancerous conditions were identified, and a 2-analyte diagnostic model of psoriasis was constructed using a step-forward algorithm. Finally, gene coexpression analysis revealed a strong connection between lipid and immune gene expression. This work highlights (a) mechanisms by which the epidermis is uniquely adapted for the specific environmental insults encountered at different body surfaces and (b) how inflammation-associated alterations in gene expression affect the epidermal lipidome.

A proximity labeling method for protein-protein interactions on cell membrane.

Antibodies targeting specific antigens are widely utilized in biological research to investigate protein interactions or to quantify target antigens. Here, we introduce antigen-antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modified with an Fe(iii) catalytic probe. Once bound to their target proteins, Fe(iii)-induced catalytic oxidation occurred in proximity of the antigen's epitope. Oxidative proteomic analysis was then used to determine the degree of oxidation, the site of oxidation within the targeted antigen, and the interacting proteins that were in close proximity to the targeted antigen. An AAPL score was generated for each protein yielding the specificity of the oxidation and proximity of the interacting protein to the target antigen. As a final demonstration of its utility, the AAPL approach was applied to map the interactors of liver-intestine-cadherin (CDH17) in colon cancer cells.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

Direct Electrodeposition of Bimetallic Nanostructures on Co-Based MOFs for Electrochemical Sensing of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) is the most significant reactive oxygen species in biological systems. Here, we reported an electrochemical sensor for the detection of H2O2 on the basis of bimetallic gold-platinum nanoparticles (Au3Pt7 NPs) supported by Co-based metal organic frameworks (Co-MOFs). First, Au3Pt7 NPs, with optimal electrocatalytic activity and accessible active surface, can be deposited on the surface of the Co-MOF-modified glassy carbon electrodes (Au3Pt7/Co-MOFs/GCE) by one-step electrodeposition method. Then, the electrochemical results demonstrated that the two-dimensional (2D) Co-MOF nanosheets as the supporting material displayed better electrocatalytic properties than the 3D Co-MOF crystals for reduction of H2O2. The fabricated Au3Pt7/2D Co-MOF exhibited high electrocatalytic activity, and the catalytic current was linear with H2O2 concentration from 0.1 µM to 5 mM, and 5-60 mM with a low detection limit of 0.02 µM (S/N = 3). The remarkable electroanalytical performance of Au3Pt7/2D Co-MOF can be attributed to the synergistic effect of the high dispersion of the Au3Pt7 NPs with the marvelous electrochemical properties and the 2D Co-MOF with high-specific surface areas. Furthermore, this sensor has been utilized to detect H2O2 concentrations released from the human Hela cells. This work provides a new method for improving the performance of electrochemical sensors by choosing the proper support materials from diverse crystal morphology materials.

Glycan-protein cross-linking mass spectrometry reveals sialic acid-mediated protein networks on cell surfaces.

A cross-linking method is developed to elucidate glycan-mediated interactions between membrane proteins through sialic acids. The method provides information on previously unknown extensive glycomic interactions on cell membranes. The vast majority of membrane proteins are glycosylated with complicated glycan structures attached to the polypeptide backbone. Glycan-protein interactions are fundamental elements in many cellular events. Although significant advances have been made to identify protein-protein interactions in living cells, only modest advances have been made on glycan-protein interactions. Mechanistic elucidation of glycan-protein interactions has thus far remained elusive. Therefore, we developed a cross-linking mass spectrometry (XL-MS) workflow to directly identify glycan-protein interactions on the cell membrane using liquid chromatography-mass spectrometry (LC-MS). This method involved incorporating azido groups on cell surface glycans through biosynthetic pathways, followed by treatment of cell cultures with a synthesized reagent, N-hydroxysuccinimide (NHS)-cyclooctyne, which allowed the cross-linking of the sialic acid azides on glycans with primary amines on polypeptide backbones. The coupled peptide-glycan-peptide pairs after cross-linking were identified using the latest techniques in glycoproteomic and glycomic analyses and bioinformatics software. With this approach, information on the site of glycosylation, the glycoform, the source protein, and the target protein of the cross-linked pair were obtained. Glycoprotein-protein interactions involving unique glycoforms on the PNT2 cell surface were identified using the optimized and validated method. We built the GPX network of the PNT2 cell line and further investigated the biological roles of different glycan structures within protein complexes. Furthermore, we were able to build glycoprotein-protein complex models for previously unexplored interactions. The method will advance our future understanding of the roles of glycans in protein complexes on the cell surface.

Diet affects glycosylation of serum proteins in women at risk for cardiometabolic disease.

BACKGROUND: Glycoproteomics deals with glycoproteins that are formed by post-translational modification when sugars (like fucose and sialic acid) are attached to protein. Glycosylation of proteins influences function, but whether glycosylation is altered by diet is unknown. OBJECTIVE: To evaluate the effect of consuming a diet based on the Dietary Guidelines for Americans on circulating glycoproteins that have previously been associated with cardiometabolic diseases. DESIGN: Forty-four women, with one or more metabolic syndrome characteristics, completed an 8-week randomized controlled feeding intervention (n = 22) consuming a diet based on the Dietary Guidelines for Americans (DGA 2010); the remaining consumed a 'typical American diet' (TAD, n = 22). Fasting serum samples were obtained at week0 (baseline) and week8 (post-intervention); 17 serum proteins were chosen for targeted analyses. Protein standards and serum samples were analyzed in a UHPLC-MS protocol to determine peptide concentration and their glycan (fucosylation or sialylation) profiles. Data at baseline were used in correlational analyses; change in proteins and glycans following intervention were used in non-parametric analyses. RESULTS: At baseline, women with more metabolic syndrome characteristics had more fucosylation (total di-fucosylated proteins: p = 0.045) compared to women with a lesser number of metabolic syndrome characteristics. Dietary refined grain intake was associated with increased total fucosylation (ρ = - 0.530, p < 0.001) and reduced total sialylation (ρ = 0.311, p = 0.042). After the 8-week intervention, there was higher sialylation following the DGA diet (Total di-sialylated protein p = 0.018, poly-sialylated orosomucoid p = 0.012) compared to the TAD diet. CONCLUSIONS: Based on this study, glycosylation of proteins is likely affected by dietary patterns; higher sialylation was associated with a healthier diet pattern. Altered glycosylation is associated with several diseases, particularly cancer and type 2 diabetes, and this study raises the possibility that diet may influence disease state by altering glycosylation. CLINICAL TRIAL REGISTRATION: NCT02298725 at clinicaltrials.gov; https://clinicaltrials.gov/ct2/show/NCT02298725 .

The N-glycome regulates the endothelial-to-hematopoietic transition.

Definitive hematopoietic stem and progenitor cells (HSPCs) arise from the transdifferentiation of hemogenic endothelial cells (hemECs). The mechanisms of this endothelial-to-hematopoietic transition (EHT) are poorly understood. We show that microRNA-223 (miR-223)-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid-myeloid lineages by suppressing the mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in protein N-glycosylation. ECs that lack miR-223 showed a decrease of high mannose versus sialylated sugars on N-glycoproteins such as the metalloprotease Adam10. EC-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied miR-223 mutant defects. Thus, the N-glycome is an intrinsic regulator of EHT, serving as a key determinant of the hematopoietic fate.

A site-specific map of the human plasma glycome and its age and gender-associated alterations.

Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

High-throughput mutagenesis reveals unique structural features of human ADAR1.

Adenosine Deaminases that act on RNA (ADARs) are enzymes that catalyze adenosine to inosine conversion in dsRNA, a common form of RNA editing. Mutations in the human ADAR1 gene are known to cause disease and recent studies have identified ADAR1 as a potential therapeutic target for a subset of cancers. However, efforts to define the mechanistic effects for disease associated ADAR1 mutations and the rational design of ADAR1 inhibitors are limited by a lack of structural information. Here, we describe the combination of high throughput mutagenesis screening studies, biochemical characterization and Rosetta-based structure modeling to identify unique features of ADAR1. Importantly, these studies reveal a previously unknown zinc-binding site on the surface of the ADAR1 deaminase domain which is important for ADAR1 editing activity. Furthermore, we present structural models that explain known properties of this enzyme and make predictions about the role of specific residues in a surface loop unique to ADAR1.

Synthetic Biology Speeds Up Drug Target Discovery.

As a rising emerging field, synthetic biology intends to realize precise regulations of cellular network by constructing artificial synthetic circuits, and it brings great opportunities to treat diseases and discover novel drug targets. Depending on the combination mode of different logic gates, various synthetic circuits are created to carry out multilevel regulations. In given synthetic circuits, drugs often act as inputs to drive circuits operation. It is becoming available to construct drug-responsive gene circuits for experimentally treating various disease models, including metabolic disease, immunity disease, cancer and bacterial infection. Synthetic biology works well in association with the CRISPR system for drug target functional screening. Remarkably, more and more well-designed circuits are developed to discover novel drug targets and precisely regulate drug therapy for diseases.

Determination of the glycoprotein specificity of lectins on cell membranes through oxidative proteomics.

The cell membrane is composed of a network of glycoconjugates including glycoproteins and glycolipids that presents a dense matrix of carbohydrates playing critical roles in many biological processes. Lectin-based technology has been widely used to characterize glycoconjugates in tissues and cell lines. However, their specificity toward their putative glycan ligand and sensitivity in situ have been technologically difficult to study. Additionally, because they recognize primarily glycans, the underlying glycoprotein targets are generally not known. In this study, we employed lectin proximity oxidative labeling (Lectin PROXL) to identify cell surface glycoproteins that contain glycans that are recognized by lectins. Commonly used lectins were modified with a probe to produce hydroxide radicals in the proximity of the labeled lectins. The underlying polypeptides of the glycoproteins recognized by the lectins are oxidized and identified by the standard proteomic workflow. As a result, approximately 70% of identified glycoproteins were oxidized in situ by all the lectin probes, while only 5% of the total proteins were oxidized. The correlation between the glycosites and oxidation sites demonstrated the effectiveness of the lectin probes. The specificity and sensitivity of each lectin were determined using site-specific glycan information obtained through glycomic and glycoproteomic analyses. Notably, the sialic acid-binding lectins and the fucose-binding lectins had higher specificity and sensitivity compared to other lectins, while those that were specific to high mannose glycans have poor sensitivity and specificity. This method offers an unprecedented view of the interactions of lectins with specific glycoproteins as well as protein networks that are mediated by specific glycan types on cell membranes.

Novel Metabolites from the Endophytic Fungus Chaetomium subaffine L01.

One natural p-terphenyl glycoside, gliocladinin C, and two furano-polyene derivatives, chaetominins A and B, were isolated from potato endophytic fungus Chaetomium subaffine. The absolute configurations of these compounds were elucidated by HR-ESI-MS, NMR, the DP4+ probabilities and electronic circular dichroism (ECD) spectra. Furthermore, gliocladinin C and chaetominin A showed cytotoxic activity against two selected human tumor cell lines (Hep-2 and HepG-2).

An iron-carbon-activated carbon and zeolite composite filter, anaerobic-aerobic integrated denitrification device for nitrogen removal in low C/N ratio sewage.

In this study, we use an anaerobic-aerobic integrated denitrification (Fe/C-ZACID) device with an iron-carbon-activated carbon and zeolite composite filter to remove nitrogen from simulated low carbon-nitrogen ratio (C/N) sewage. The impacts of dissolved oxygen (DO) level, hydraulic retention time (HRT), C/N and nitrate recirculation ratio on denitrification performance were studied. The results show that when HRT was 6 h, DO was 3 ± 0.1 mg/L, influent C/N was 3, and nitrate recirculation ratio was 100%, and removal rates of 95% for ammonia and 85% for total nitrogen (TN) were achieved. A beaker comparison test demonstrated that this synergistic denitrification system included heterotrophic denitrification, physicochemical denitrification, iron autotrophic denitrification and hydrogen autotrophic denitrification, etc. The Fe/C-ZACID device has a high-efficiency nitrogen removal effect for low C/N ratio sewage and strong shock resistance, which provides technical support and a theoretical basis for advanced denitrification of rural domestic sewage.