Manganese-induced Photothermal-Ferroptosis for synergistic tumor therapy.

Ferroptosis-related tumor therapy based on nanomedicines has recently gained significant attention. However, the therapeutic performance is still hindered by the tumor's physical barriers such as the fibrotic tumor matrix and elevated interstitial fluid pressure, as well as chemical barriers like glutathione (GSH) overabundance. These physicochemical barriers impede the bioavailability of nanomedicines and compromise the therapeutic efficacy of lipid reactive oxygen species (ROS). Thus, this study pioneers a manganese-mediated overcoming of physicochemical barriers in the tumor microenvironment using organosilica-based nanomedicine (MMONs), which bolsters the synergy of photothermal-ferroptosis treatment. The MMONs display commendable proficiency in overcoming tumor physical barriers, due to their MnO2-mediated shape-morphing and softness-transformation ability, which facilitates augmented cellular internalization, enhanced tumor accumulation, and superior drug penetration. Also, the MMONs possess excellent capability in chemical barrier overcoming, including MnO2-mediated dual GSH clearance and enhanced ROS generation, which facilitates ferroptosis and heat shock protein inhibition. Notably, the resulting integration of physical and chemical barrier overcoming leads to amplified photothermal-ferroptosis synergistic tumor therapy both in vitro and in vivo. Accordingly, the comparative proteomic analysis has identified promoted ferroptosis with a transient inhibitory response observed in the mitochondria. This research aims to improve treatment strategies to better fight the complex defenses of tumors.

Cryo-EM structure of severe fever with thrombocytopenia syndrome virus.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne human-infecting bunyavirus, which utilizes two envelope glycoproteins, Gn and Gc, to enter host cells. However, the structure and organization of these glycoproteins on virion surface are not yet known. Here we describe the structure of SFTSV determined by single particle reconstruction, which allows mechanistic insights into bunyavirus assembly at near-atomic resolution. The SFTSV Gn and Gc proteins exist as heterodimers and further assemble into pentameric and hexameric peplomers, shielding the Gc fusion loops by both intra- and inter-heterodimer interactions. Individual peplomers are associated mainly through the ectodomains, in which the highly conserved glycans on N914 of Gc play a crucial role. This elaborate assembly stabilizes Gc in the metastable prefusion conformation and creates some cryptic epitopes that are only accessible in the intermediate states during virus entry. These findings provide an important basis for developing vaccines and therapeutic drugs.

Deep Profiling of the Proteome Dynamics of Pseudomonas aeruginosa Reference Strain PAO1 under Different Growth Conditions.

As one of the most common bacterial pathogens causing nosocomial infections, Pseudomonas aeruginosa is highly adaptable to survive under various conditions. Here, we profiled the abundance dynamics of 3489 proteins across different growth stages in the P. aeruginosa reference strain PAO1 using data-independent acquisition-based quantitative proteomics. The proteins differentially expressed during the planktonic growth exhibit several distinct patterns of expression profiles and are relevant to various biological processes, highlighting the continuous adaptation of the PAO1 proteome during the transition from the acceleration phase to the stationary phase. By contrasting the protein expressions in a biofilm to planktonic cells, the known roles of T6SS, phenazine biosynthesis, quorum sensing, and c-di-GMP signaling in the biofilm formation process were confirmed. Additionally, we also discovered several new functional proteins that may play roles in the biofilm formation process. Lastly, we demonstrated the general concordance of protein expressions within operons across various growth states, which permits the study of coexpression protein units, and reversely, the study of regulatory components in the operon structure. Taken together, we present a high-quality and valuable resource on the proteomic dynamics of the P. aeruginosa reference strain PAO1, with the potential of advancing our understanding of the overall physiology of Pseudomonas bacteria.

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice.

BACKGROUND: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. METHODS: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson's trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. RESULTS: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. CONCLUSIONS: TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Discovery and validation of bladder cancer related excreted nucleosides biomarkers by dilution approach in cell culture supernatant and urine using UHPLC-MS/MS.

The exploration of nucleoside changes in human biofluids has profound potential for cancer diagnosis. Herein, we developed a rapid methodology to quantify 17 nucleosides by UHPLC-MS/MS. Five pairs of isomers were successfully separated within 8 min. The ME was mostly eliminated by sample dilution folds of 1000 for urine and 40 for CCS. The optimized method was firstly applied to screen potential nucleoside biomarkers in CCS by comprising bladder cancer cell lines (5637 and T24) and normal human bladder cell line SV-HUC-1 together with student's t-test and OPLS-DA. Nucleosides with significant differences in the supernatant of urine samples were also uncovered comparing BCa with the non-tumor group, as well as a comparison of BCa recurrence group with the non-recurrence group. By intersecting the differential nucleosides in CCS and urine supernatant, and then further confirmed using validation sets, the combination of m3C and m1A with AUC of 0.775 was considered as a potential biomarker for bladder cancer diagnosis. A panel of m3C, m1A, m1G, and m22G was defined as potential biomarkers for bladder cancer prognosis with an AUC of 0.819. Above all, this method provided a new perspective for diagnosis and recurrence monitoring of bladder cancer. SIGNIFICANCE: The exploration of nucleoside changes in body fluids has profound potential for the diagnosis and elucidation of the pathogenesis of cancer. In this study, we developed a rapid methodology for the simultaneous quantitative determination of 17 nucleosides in the supernatant of cells and urine samples using UHPLC-MS/MS to discover and validate bladder cancer related excreted nucleoside biomarkers. The results of this paper provide a new strategy for diagnosis and postoperative recurrence monitoring of bladder cancer and provide theoretical support for the exploration of its pathogenesis.

Photothermal Nanozymatic Nanoparticles Induce Ferroptosis and Apoptosis through Tumor Microenvironment Manipulation for Cancer Therapy.

It is highly desirable to design a single modality that can simultaneously trigger apoptosis and ferroptosis to efficiently eliminate tumor progression. Herein, a nanosystem based on the intrinsic properties of tumor microenvironment (TME) is designed to achieve tumor control through the simultaneous induction of ferroptosis and apoptosis. CuCP molecules are encapsulated in a liposome-based nanosystem to assemble into biocompatible and stable CuCP nanoparticles (CuCP Lipo NPs). This nanosystem intrinsically possesses nanozymatic activity and photothermal characteristics due to the property of Cu atoms and the structure of CuCP Lipo NPs. It is demonstrated that the synergistic strategy increases the intracellular lipid-reactive oxides species, induces the occurrence of ferroptosis and apoptosis, and completely eradicates the tumors in vivo. Proteomics analysis further discloses the key involved proteins (including Tp53, HMOX1, Ptgs2, Tfrc, Slc11a2, Mgst2, Sod1, and several GST family members) and pathways (including apoptosis, ferroptosis, and ROS synthesis). Conclusively, this work develops a strategy based on one nanosystem to synergistically induce ferroptosis and apoptosis in vivo for tumor suppression, which holds great potential in the clinical translation for tumor therapy.

A Novel CpG Methylation Risk Indicator for Predicting Prognosis in Bladder Cancer.

PURPOSE: Bladder cancer (BLCA) is one of the most common cancers worldwide. In a large proportion of BLCA patients, disease recurs and/or progress after resection, which remains a major clinical issue in BLCA management. Therefore, it is vital to identify prognostic biomarkers for treatment stratification. We investigated the efficiency of CpG methylation for the potential to be a prognostic biomarker for patients with BLCA. PATIENTS AND METHODS: Overall, 357 BLCA patients from The Cancer Genome Atlas (TCGA) were randomly separated into the training and internal validation cohorts. Least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) were used to select candidate CpGs and build the methylation risk score model, which was validated for its prognostic value in the validation cohort by Kaplan-Meier analysis. Hazard curves were generated to reveal the risk nodes throughout the follow-up. Gene Set Enrichment Analysis (GSEA) was used to reveal the potential biological pathways associated with the methylation model. Quantitative real-time polymerase chain reaction (PCR) and western blotting were performed to verify the expression level of the methylated genes. RESULTS: After incorporating the CpGs obtained by the two algorithms, CpG methylation of eight genes corresponding to TNFAIP8L3, KRTDAP, APC, ZC3H3, COL9A2, SLCO4A1, POU3F3, and ADARB2 were prominent candidate predictors in establishing a methylation risk score for BLCA (MRSB), which was used to divide the patients into high- and low-risk progression groups (p < 0.001). The effectiveness of the MRSB was validated in the internal cohort (p < 0.001). In the MRSB high-risk group, the hazard curve exhibited an initial wide, high peak within 10 months after treatment, whereas some gentle peaks around 2 years were noted. Furthermore, a nomogram comprising MRSB, age, sex, and tumor clinical stage was developed to predict the individual progression risk, and it performed well. Survival analysis implicated the effectiveness of MRSB, which remains significant in all the subgroup analysis based on the clinical features. A functional analysis of MRSB and the corresponding genes revealed potential pathways affecting tumor progression. Validation of quantitative real-time PCR and western blotting revealed that TNFAIP8L3 was upregulated in the BLCA tissues. CONCLUSION: We developed the MRSB, an eight-gene-based methylation signature, which has great potential to be used to predict the post-surgery progression risk of BLCA.

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

Comparative Analysis of Different N-glycan Preparation Approaches and Development of Optimized Solid-Phase Permethylation Using Mass Spectrometry.

Protein N-glycosylation characterization is challenging due to structural micro- and macro-heterogeneity. Although various N-glycan preparation strategies, including purification and derivatization, have been previously developed prior to mass spectrometric analysis, systematic evaluation still needs to be performed. This study compared the different N-glycan purification strategies, including filter-aided sample preparation, de-N-glycosylated protein precipitation, and trypsin digestion followed by reversed phase-based solid-phase extraction, and derivatization approaches, such as solid-phase permethylation, reductive amination, and reduction. With the comparative analysis, an optimized solid-phase permethylation (OSPP) workflow was developed for mass spectrometric N-glycomics, showing simplified analysis for N-glycan compositions and high yields using etanercept. The N-glycan samples released from trastuzumab and adalimumab were utilized to test OSPP to obtain their N-glycan profiles using mass spectrometry. Based on different standard procedures across laboratories, this study provides the reference for analysts to select an appropriate N-glycan preparation method with their research purposes.

Proteomics of the corpus callosum to identify novel factors involved in hypomyelinated Niemann-Pick Type C disease mice.

Hypomyelination in the central nerves system (CNS) is one of the most obviously pathological features in Niemann-Pick Type C disease (NPC), which is a rare neurodegenerative disorder caused by mutations in the NPC intracellular cholesterol transporter 1 or 2 (Npc1 or Npc2). Npc1 plays key roles in both neurons and oligodendrocytes during myelination, however, the linkage between the disturbed cholesterol transport and inhibited myelination is unrevealed. In this study, mass spectrometry (MS)-based differential quantitative proteomics was applied to compare protein composition in the corpus callosum between wild type (WT) and NPC mice. In total, 3009 proteins from both samples were identified, including myelin structural proteins, neuronal proteins, and astrocyte-specific proteins. In line to hypomyelination, our data revealed downregulation of myelin structural and indispensable proteins in Npc1 mutant mice. Notably, the reduced ceramide synthase 2 (Cers2), UDP glycosyltransferase 8 (Ugt8), and glycolipid transfer protein (Gltp) indicate the altered sphingolipid metabolism in the disease and the involvement of Gltp in myelination. The identification of most reported myelin structural proteins and proteins from other cell types advocates the use of the corpus callosum to investigate proteins in different cell types that regulate myelination.

Structural and In Vitro Functional Comparability Analysis of Altebrel™, a Proposed Etanercept Biosimilar: Focus on Primary Sequence and Glycosylation.

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. "Bottom-up" mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.

Preparation of low molecular weight heparins from bovine and ovine heparins using nitrous acid degradation.

Low molecular weight heparins (LMWHs) are important anticoagulant drugs. Nitrous acid degradation is a major approach to produce LMWHs, such as dalteparin. Due to the foreseeable shortage of porcine intestinal mucosa heparin and other potential risks, expansion of other animal tissues for heparin preparation is necessary. Heparins from different tissues differ in structure and bioactivity potency, and these variations may be carried over to the LMWH products. Sophisticated analytical techniques have been applied to compare various versions of dalteparins produced from porcine intestinal, bovine lung and ovine intestinal heparins to elucidate the effects of different animal tissues starting materials and processing conditions on the properties of final dalteparin products. With adjusted depolymerization conditions, versions of dalteparins that qualify under the European Pharmacopeia (EP) specifications were manufactured using non-porcine heparins. Dissimilarities among the three interspecies animal tissue heparin-derived dalteparins regarding fine structures are also disclosed, and their origins are discussed.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Lung Heparin and Porcine Intestine Heparin.

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin-derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin-derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain.

From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins.

O-glycosylation-site characterization of individual glycoproteins is a major challenge because of the heterogeneity of O-glycan core structures. In proteomic studies, O-glycosylation-site analysis is even more difficult because of the complexity of the sample. In this work, we designed a rapid and convenient workflow for characterizing the O-glycosylation sites of individual proteins and the human-plasma proteome. A mixture of exoglycosidases was used to partially remove O-glycan chains and leave an N-acetylgalacosamine (GalNAc) residue attached to the Ser or Thr residues. The O-glycosylated peptides could then be identified by using liquid chromatography-tandem mass spectrometry (LC-MS-MS) to detect the 203 Da mass increase. Jacalin was used to selectively isolate O-GalNAc glycopeptides before LC-MS-MS analysis, which is optional for individual proteins and necessary for complex human-plasma proteins. Bovine fetuin and human chorionic gonadotropin (hCG) were used to test the analytical workflow. The workflow indicated superior sensitivity by not only covering most previously known O-glycosylation sites but also discovering several novel sites. Using only one drop of blood, a total of 49 O-GalNAc-linked glycopeptides from 36 distinctive glycoproteins in human plasma were identified unambiguously. The approach described herein is simple, sensitive, and global for site analysis of core 1 through core 4 O-glycosylated proteins.

Nested fiber ring resonator enhanced Mach-Zehnder interferometer for temperature sensing.

We numerically investigate the properties of the nested fiber ring resonator coupled Mach-Zehnder interferometer as a sensor. By introducing the phase bias of 0.5π in the reference arm, the two output intensities exhibit sharp asymmetric line shapes around the resonance wavelength. Utilizing the intensity interrogation, we analyze the effect of parameters on the sensitivity and the detection limit. For the 30 dB signal-noise system, the sensitivity and the detection limit can achieve 4.0866/°C and 7.341×10(-3)°C, respectively; the results indicate that this structure is suitable for high-sensitivity measurements.