Macrophage-mediated mechanisms of lung injury in the sensitization reaction to Echinococcus granulosus.

Objective: In this study, the impact of inhibiting the PI3K/AKT/NF-κB pathway on lung oxidative damage induced by Echinococcus granulosus cyst fluid was investigated. Methods: Twenty-four mice were randomly assigned to four groups. Three months after inoculation with hydatid cyst segments, mice in group A were treated with intraperitoneal and intratracheal saline injections; mice in group B were administered a caudal vein injection of a PI3K inhibitor, followed by cyst fluid sensitization; mice in group C received an AKT inhibitor via caudal vein, followed by cyst fluid sensitization; and mice in group D were subjected to cyst fluid sensitization without any inhibitor treatment. Cellular changes in lung tissues across all groups were evaluated, including pathological section analysis. Analysis of pulmonary tissue and serum from these mice included the assessment of PI3K/AKT/NF-κB pathway proteins, inflammatory factors, and related mRNA levels. Results: Mice in groups B and C exhibited a higher proportion of M2-type macrophages and significantly lower levels of PI3K/AKT/NF-κB pathway proteins, inflammatory factors (interleukin-6 [IL-6]/tumor necrosis factor-α [TNF-α]), and oxidative markers in lung tissues compared to mice in group D (P < 0.05). Conclusion: Our results in this study indicate that activation of the PI3K/AKT/NF-κB pathway contributed to an increase in the M1 macrophage phenotype, leading to enhanced secretion of peroxidases and inflammatory factors. This mechanism plays a crucial role in the oxidative and inflammatory lung damage associated with allergic reactions to E. granulosus cyst fluid.

Detection of Pancreatic Ductal Adenocarcinoma-Associated Proteins in Serum.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types, partly because it is frequently identified at an advanced stage, when surgery is no longer feasible. Therefore, early detection using minimally invasive methods such as blood tests may improve outcomes. However, studies to discover molecular signatures for the early detection of PDAC using blood tests have only been marginally successful. In the current study, a quantitative glycoproteomic approach via data-independent acquisition mass spectrometry was utilized to detect glycoproteins in 29 patient-matched PDAC tissues and sera. A total of 892 N-linked glycopeptides originating from 141 glycoproteins had PDAC-associated changes beyond normal variation. We further evaluated the specificity of these serum-detectable glycoproteins by comparing their abundance in 53 independent PDAC patient sera and 65 cancer-free controls. The PDAC tissue-associated glycoproteins we have identified represent an inventory of serum-detectable PDAC-associated glycoproteins as candidate biomarkers that can be potentially used for the detection of PDAC using blood tests.

Muscone promotes functional recovery by facilitating microglia polarization into M2 phenotype through PPAR-γ pathway after ischemic stroke.

Exploring regimens to facilitate microglia transformation from M1 to M2 phenotype is a feasible strategy to suppress neuroinflammation, therefore reinforcing functional recovery after ischemic stroke. Muscone easily crosses the blood brain barrier (BBB) and distributes throughout the brain. Here, the results illustrated the administration of 8 mg/kg muscone promoted functional recovery through reducing the infarct volume by 2,3,5-triphenyltetrazolium chloride (TTC) staining after ischemic stroke in mice. Then, the expression of pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), was significantly decreased, whereas the level of anti-inflammatory agents including C-X-C Motif Chemokine Ligand 1 (CXCL1), transforming growth factor-ß (TGF-ß) and interleukin-10 (IL-10) was obviously elevated in penumbra with the treatment of 8 mg/kg muscone using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), western blot and enzyme-linked immunosorbent assay (ELISA) tests. Subsequently, the results showed the application of muscone upregulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) to facilitate microglia transformation into M2 phenotype using RT-qPCR, western blot and immunofluorescence analysis. Collectively, the present study provides evidence for our hypothesis that muscone intensifies microglia transformation into M2 phenotype via activating PPAR-γ signaling pathway in penumbra after ischemic stroke. These findings demonstrate muscone is a promising candidate for the treatment of ischemic stroke.

Optimized data-independent acquisition approach for proteomic analysis at single-cell level.

BACKGROUND: Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. METHODS: We report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. RESULTS: We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. CONCLUSIONS: Our results demonstrate that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

Core fucosylation of N-linked glycoproteins has been linked to the functions of glycoproteins in physiological and pathological processes. However, quantitative characterization of core fucosylation remains challenging due to the complexity and heterogeneity of N-linked glycosylation. Here we report a mass spectrometry-based method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to analyze site-specific core fucosylation of glycoproteins. The STAGE method utilizes Endo F3 followed by PNGase F treatment to generate mass signatures for glycosites that are formerly modified by core fucosylated N-linked glycans. We benchmark the STAGE method and use it to characterize site specific core fucosylation of glycoproteins from human hepatocellular carcinoma and pancreatic ductal adenocarcinoma, resulting in the identification of 1130 and 782 core fucosylated glycosites, respectively. These results indicate that our STAGE method enables quantitative characterization of core fucosylation events from complex protein mixtures, which may benefit our understanding of core fucosylation functions in various diseases.

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma.

We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.

Proteomic signatures of 16 major types of human cancer reveal universal and cancer-type-specific proteins for the identification of potential therapeutic targets.

BACKGROUND: Proteomic characterization of cancers is essential for a comprehensive understanding of key molecular aberrations. However, proteomic profiling of a large cohort of cancer tissues is often limited by the conventional approaches. METHODS: We present a proteomic landscape of 16 major types of human cancer, based on the analysis of 126 treatment-naïve primary tumor tissues, 94 tumor-matched normal adjacent tissues, and 12 normal tissues, using mass spectrometry-based data-independent acquisition approach. RESULTS: In our study, a total of 8527 proteins were mapped to brain, head and neck, breast, lung (both small cell and non-small cell lung cancers), esophagus, stomach, pancreas, liver, colon, kidney, bladder, prostate, uterus and ovary cancers, including 2458 tissue-enriched proteins. Our DIA-based proteomic approach has characterized major human cancers and identified universally expressed proteins as well as tissue-type-specific and cancer-type-specific proteins. In addition, 1139 therapeutic targetable proteins and 21 cancer/testis (CT) antigens were observed. CONCLUSIONS: Our discoveries not only advance our understanding of human cancers, but also have implications for the design of future large-scale cancer proteomic studies to assist the development of diagnostic and/or therapeutic targets in multiple cancers.

Chromatographic determination and in-situ cell imaging of thiol compounds based on a fluorigenic probe.

This study combines a high-performance liquid chromatography-fluorescent detection method (HPLC-FLD) with in-situ cell imaging for the sensitive analysis of glutathione (GSH), cysteine (Cys) and homocysteine (Hcys), using BODIPY®507/545 IA as a labeling reagent. The analytical potential of BODIPY®507/545 IA in cell imaging was deeply explored, concerning fluorescent response, selectivity, cell-permeability, biotoxicity and so on. It is demonstrated that BODIPY®507/545 IA has good biocompatibility and the fluorescence intensity is enhanced remarkably after reacting with thiols. The best derivative condition was obtained in boric acid buffer (0.05 mmol/L, pH 9.5) at 45 °C for 15 min. For chromatographic method, two sensitive methods, HPLC-FLD and capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) were both developed, validated, and compared. The detection limits for the thiols ranged from 5 to 10 nmol/L with HPLC-FLD and 0.5 nmol/L for the CE-LIF method. Finally, HPLC-FLD is adopted to quantify the thiols in HepG2 cell samples after cell imaging.

The role of unfolded protein response and ER-phagy in quantum dots-induced nephrotoxicity: an in vitro and in vivo study.

Unfolded protein response (UPR) and endoplasmic reticulum (ER)-phagy are essential for cell homeostasis. Quantum dots (QDs), which have been widely used for biomedical applications, can accumulate in the kidney tissues and may cause renal dysfunction. However, the molecular mechanism of QDs-induced nephrotoxicity is still obscure. The present study was aimed to elucidate the role and mechanism of UPR and ER-phagy in QDs-induced nephrotoxicity. Herein, human embyronic kidney (HEK) cells were exposed to 15, 30, 45, and 60 nM cadmium telluride (CdTe)-QDs for 12 and 24 h. And CdTe-QDs (30-60 nM) inhibited the HEK cell viability. The clathrin-dependent endocytosis was determined as the main pathway of CdTe-QDs cellular uptake. Within cells, CdTe-QDs disrupted ER ultrastructure and induced UPR and FAM134B-dependent ER-phagy. Blocking UPR with inhibitors or siRNA rescued the FAM134B-dependent ER-phagy, which was triggered by CdTe-QDs. Moreover, suppression of UPR or FAM134B-dependent ER-phagy restored the cell vability. In vivo, mice were intravenously injected with 8 and 16 nmol/kg body weight CdTe-QDs for 24 h. Kidney was shown as one of highest distributed organs of CdTe-QDs, resulting in renal dysfunction, as well as UPR and FAM134B-dependent ER-phagy in it. Thus, for the first time, we demonstrated that ER-phagy can be triggered by nanomaterials both in vitro and in vivo. In addition, blocking of UPR and ER-phagy showed protective effects against CdTe-QDs-induced toxicity in kideny cells. Notably, a secreted alkaline phosphatase reporter gene system has been developed as a sensitive and rapid method for evaluating the ER quality under the exposure of nanomaterials.

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein.

HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.

N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry.

N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

A novel zwitterionic HILIC stationary phase based on "thiol-ene" click chemistry between cysteine and vinyl silica.

A novel zwitterionic stationary phase with high hydrophilicity was facilely synthesized based on the "thiol-ene" click reaction between cysteine and vinyl silica, which exhibited great potential in the separation of oligosaccharides, peptides and basic compounds, as well as in the enrichment of glycopeptides.

Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes.

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.