Dihydroartemisinin-driven TOM70 inhibition leads to mitochondrial destabilization to induce pyroptosis against lung cancer.

Enhancement of malignant cell immunogenicity to relieve immunosuppression of lung cancer microenvironment is essential in lung cancer treatment. In previous study, we have demonstrated that dihydroartemisinin (DHA), a kind of phytopharmaceutical, is effective in inhibiting lung cancer cells and boosting their immunogenicity, while the initial target of DHA's intracellular action is poorly understood. The present in-depth analysis aims to reveal the influence of DHA on the highly expressed TOM70 in the mitochondrial membrane of lung cancer. The affinity of DHA and TOM70 was analyzed by microscale thermophoresis (MST), pronase stability, and thermal stability. The functions and underlying mechanism were investigated using western blots, qRT-PCR, flow cytometry, and rescue experiments. TOM70 inhibition resulted in mtDNA damage and translocation to the cytoplasm from mitochondria due to the disruption of mitochondrial homeostasis. Further ex and in vivo findings also showed that the cGAS/STING/NLRP3 signaling pathway was activated by mtDNA and thereby malignant cells underwent pyroptosis, leading to enhanced immunogenicity of lung cancer cells in the presence of DHA. Nevertheless, DHA-induced mtDNA translocation and cGAS/STING/NLRP3 mobilization were synchronously attenuated when TOM70 was replenished. Finally, DHA was demonstrated to possess potent anti-lung cancer efficacy in vitro and in vivo. Taken together, these data confirm that TOM70 is an important target for DHA to disturb mitochondria homeostasis, which further activates STING and arouses pyroptosis to strengthen immunogenicity against lung cancer thereupon. The present study provides vital clues for phytomedicine-mediated anti-tumor therapy.

Synergistic Therapeutic Effects of D-Mannitol-Cerium-Quercetin (Rutin) Coordination Polymer Nanoparticles on Acute Lung Injury.

Acute lung injury (ALI) remains a significant global health issue, necessitating novel therapeutic interventions. In our latest study, we pioneered the use of D-mannitol-cerium-quercetin/rutin coordination polymer nanoparticles (MCQ/R NPs) as a potential treatment for ALI. The MCQ/R NPs, which integrate rutin and quercetin for their therapeutic potential and D-mannitol for its pulmonary targeting, displayed exceptional efficacy. By utilizing cerium ions for optimal nanoparticle assembly, the MCQ/R NPs demonstrated an average size of less than 160 nm. Impressively, these nanoparticles outperformed conventional treatments in both antioxidative capabilities and biocompatibility. Moreover, our in vivo studies on LPS-induced ALI mice showed a significant reduction in lung tissue inflammation. This groundbreaking research presents MCQ/R NPs as a promising new approach in ALI therapeutics.

Lipotoxicity-polarised macrophage-derived exosomes regulate mitochondrial fitness through Miro1-mediated mitophagy inhibition and contribute to type 2 diabetes development in mice.

AIMS/HYPOTHESIS: Insulin resistance is a major pathophysiological defect in type 2 diabetes and obesity. Numerous experimental and clinical studies have provided evidence that sustained lipotoxicity-induced mitophagy deficiency can exacerbate insulin resistance, leading to a vicious cycle between mitophagy dysfunction and insulin resistance, and thereby the onset of type 2 diabetes. Emerging evidence suggests that exosomes (Exos) from M2 macrophages play an essential role in modulating metabolic homeostasis. However, how macrophages are affected by lipotoxicity and the role of lipotoxicity in promoting macrophage activation to the M1 state have not been determined. The objective of this study was to determine whether M1 macrophage-derived Exos polarised by lipopolysaccharide (LPS) + palmitic acid (PA)-induced lipotoxicity contribute to metabolic homeostasis and impact the development of insulin resistance in type 2 diabetes. METHODS: Lipotoxicity-polarised macrophage-derived M1 Exos were isolated from bone marrow (C57BL/6J mouse)-derived macrophages treated with LPS+PA. Exos were characterised by transmission electron microscopy, nanoparticle tracking analysis and western blotting. Flow cytometry, H&E staining, quantitative real-time PCR, immunofluorescence, glucose uptake and output assays, confocal microscopy imaging, western blotting, GTTs and ITTs were conducted to investigate tissue inflammation, mitochondrial function and insulin resistance in vitro and in vivo. The roles of miR-27-3p and its target gene Miro1 (also known as Rhot1, encoding mitochondrial rho GTPase 1) and relevant pathways were predicted and assessed in vitro and in vivo using specific miRNA mimic, miRNA inhibitor, miRNA antagomir and siRNA. RESULTS: miR-27-3p was highly expressed in M1 Exos and functioned as a Miro1-inactivating miRNA through the miR-27-3p-Miro1 axis, leading to mitochondria fission rather than fusion as well as mitophagy impairment, resulting in NOD-like receptor 3 inflammatory activation and development of insulin resistance both in vivo and in vitro. Inactivation of miR-27-3p induced by M1 Exos prevented type 2 diabetes development in high-fat-diet-fed mice. CONCLUSIONS/INTERPRETATION: These findings suggest that the miR-27-3p-Miro1 axis, as a novel regulatory mechanism for mitophagy, could be considered as a new therapeutic target for lipotoxicity-related type 2 diabetes disease development.

Hypoxic Preconditioning Enhances the Efficacy of Mesenchymal Stem Cells-Derived Conditioned Medium in Switching Microglia toward Anti-inflammatory Polarization in Ischemia/Reperfusion.

Activation of pro-inflammatory microglia is an important mechanism of the cerebral ischemia-reperfusion (I/R)-induced neuronal injury and dysfunction. Mesenchymal stem cells (MSCs) together with their paracrine factors demonstrated curative potential in immune disorders and inflammatory diseases, as well as in ischemic diseases. However, it remains unclear whether conditioned medium from MSCs could effectively regulate the activation and polarization of microglia exposed to I/R stimulation. In this study, we investigated the effects of conditioned medium from bone marrow MSCs (BMSCs-CM) on I/R-stimulated microglia and the potential mechanism involved, as well as the way to obtain more effective BMSCs-CM. First, cell model of oxygen-glucose deprivation/reoxygenation (OGD/R) was established in microglia to mimic the I/R. BMSCs-CM from different culture conditions (normoxic: 21% O2; hypoxic: 1% O2; hypoxia preconditioning: preconditioning with 1% O2 for 24 h) was used to treat the microglia. Our results showed that BMSCs-CM effectively promoted the survival and alleviated the injury of microglia. Moreover, in microglia exposed to OGD/R, BMSCs-CM inhibited significantly the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6), CD86 and inducible nitric oxide synthase, whereas upregulated the levels of anti-inflammatory cytokine (IL-10), CD206 and Arginase-1. These results suggested that BMSCs-CM promoted the polarization of anti-inflammatory microglia. In particular, BMSCs-CM from cultures with hypoxia preconditioning was more effective in alleviating cell injury and promoting anti-inflammatory microglia polarization than BMSCs-CM from normoxic cultures and from hypoxic cultures. Furthermore, inhibition of exosomes secretion could largely mitigate these effects of BMSCs-CM. In conclusion, our results suggested that hypoxia preconditioning of BMSCs could enhance the efficacy of BMSCs-CM in alleviating OGD/R-induced injury and in promoting the anti-inflammatory polarization of microglia, and these beneficial effects of BMSCs-CM owed substantially to exosomes.

Celastrol in metabolic diseases: Progress and application prospects.

Metabolic diseases are becoming increasingly common in modern society. Therefore, it is essential to develop effective drugs or new treatments for metabolic diseases. As an active ingredient derived from plants, celastrol has shown great potential in the treatment of a wide variety of metabolic diseases and received considerable attention in recent years. In reported studies, the anti-obesity effect of celastrol resulted from regulating leptin sensitivity, energy metabolism, inflammation, lipid metabolism and even gut microbiota. Celastrol reversed insulin resistance via multiple routes to protect against type 2 diabetes. Celastrol also showed effects on atherosclerosis, cholestasis and osteoporosis. Celastrol in treating metabolic diseases seem to be versatile and the targets or pathways were diverse. Here, we systematically review the mechanism of action, and the therapeutic properties of celastrol in various metabolic diseases and complications. Based on this review, potential research strategies might contribute to the celastrol's clinical application in the future.

Bone marrow mesenchymal stem cells-derived conditioned medium protects cardiomyocytes from hypoxia/reoxygenation-induced injury through Notch2/mTOR/autophagy signaling.

Bone marrow mesenchymal stem cells (BMSC) can ameliorate ischemic injury of various tissues. However, the molecular mechanisms involved remain to be clarified. In this study, we intend to investigate the effects of BMSC-derived conditioned medium (BMSC-CM) on hypoxia/reoxygenation (H/R)-induced injury of H9c2 myocardial cells, and the potential mechanisms. Cell injury was determined through level of cell viability, lactate dehydrogenase (LDH) release, total intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and cell apoptosis. Autophagic activity of cells was detected through levels of the autophagy-associated proteins and autophagic flux. Results showed that BMSC-CM alleviated H/R-induced injury in H9c2 cells, as demonstrated by increased cell viability and Δψm, decreased ROS production, LDH release, and cell apoptosis. Furthermore, the H/R treatment induced a decrease in autophagic activity and an increase in Notch2 signaling activation in H9c2 cells. In the presence of BMSC-CM, the autophagic activity impaired by the H/R treatment was upregulated with decreased phosphorylation of mTOR, and the activation of Notch2 signaling was downregulated. These effects of BMSC-CM could be replicated by Notch signaling inhibitor. In contrast, inhibitors of cell autophagy including chloroquine (CQ) and 3-methyladenine, diminished the protective effects of BMSC-CM. Taken together results, our study showed that BMSC-CM could protect H9c2 cells from H/R-induced injury potentially through regulating Notch2/mTOR/autophagy signaling. These findings may provide a novel insight into the mechanisms of BMSC-CM in therapy of myocardial ischemia/reperfusion injury as well as other ischemic diseases.

Work Patterns of MamXY Proteins during Magnetosome Formation in Magnetospirillum gryphiswaldense MSR-1.

The bacterium Magnetospirillum gryphiswaldense MSR-1 forms nanosized membrane-enclosed organelles termed magnetosomes. The mamXY operon, part of the magnetosome island (MAI), includes the mamY, mamX, mamZ, and ftsZ-like genes, which initiate gene transcription via the same promoter. We used a combination of molecular biological techniques (targeting of cross-linking reagents) and high-resolution mass spectrometry to investigate the coordinated activity of the four MamXY proteins in magnetite biomineralization. The FtsZ-like protein was shown by confocal laser scanning microscopy to be dispersed in the cytoplasm in the early stage of cell growth and then gradually polymerized along the magnetosome chain. Interactions of various pairs of MamXY proteins were observed using a bacterial two-hybrid system. We constructed a recombinant FtsZ-like-overexpressing strain, examined its growth patterns, and extracted magnetosome membrane proteins using a modified SDS/boiling method with BS2G-d0/d4 reagent, which helped stabilize interactions among MamXY proteins. In liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, MamY expression was detected first and remained highest among the four proteins throughout all stages of cell growth. MamX and MamZ expression was detected subsequently. The four proteins displayed coordinated expression patterns during the magnetosome maturation process. Unique peptides discovered in the MamXY protein sequences appeared to constitute "hidden" interaction sites involved in the formation of MamXY complex that helped control magnetosome shape and size.IMPORTANCEmamXY operon genes play an essential role in magnetite biomineralization, participate in redox reactions, and control magnetosome shape and size. However, mechanisms whereby the four MamXY proteins function together in iron oxidoreduction and transport are poorly understood. We used a combination of targeted cross-linking techniques and high-resolution mass spectrometry to elucidate the coordinated activity patterns of the MamXY proteins during magnetite biomineralization. Our findings indicate that the FtsZ-like protein undergoes polymerization and then recruits MamY, MamX, and MamZ in turn, and that these interactions depend on unique peptides present in the protein sequences. A hypothetical model of the functionalities of these proteins is proposed that accounts for the findings and provides a basis for further studies of coordination among magnetosome island (MAI) gene clusters during the process of magnetosome formation.

A Cell-type-resolved Liver Proteome.

Parenchymatous organs consist of multiple cell types, primarily defined as parenchymal cells (PCs) and nonparenchymal cells (NPCs). The cellular characteristics of these organs are not well understood. Proteomic studies facilitate the resolution of the molecular details of different cell types in organs. These studies have significantly extended our knowledge about organogenesis and organ cellular composition. Here, we present an atlas of the cell-type-resolved liver proteome. In-depth proteomics identified 6000 to 8000 gene products (GPs) for each cell type and a total of 10,075 GPs for four cell types. This data set revealed features of the cellular composition of the liver: (1) hepatocytes (PCs) express the least GPs, have a unique but highly homogenous proteome pattern, and execute fundamental liver functions; (2) the division of labor among PCs and NPCs follows a model in which PCs make the main components of pathways, but NPCs trigger the pathways; and (3) crosstalk among NPCs and PCs maintains the PC phenotype. This study presents the liver proteome at cell resolution, serving as a research model for dissecting the cell type constitution and organ features at the molecular level.

The neddylation-cullin 2-RBX1 E3 ligase axis targets tumor suppressor RhoB for degradation in liver cancer.

The neddylation-cullin-RING E3 ligase (CRL) pathway has recently been identified as a potential oncogenic event and attractive anticancer target; however, its underlying mechanisms have not been well elucidated. In this study, RhoB, a well known tumor suppressor, was identified and validated with an iTRAQ-based quantitative proteomic approach as a new target of this pathway in liver cancer cells. Specifically, cullin 2-RBX1 E3 ligase, which requires NEDD8 conjugation for its activation, interacted with RhoB and promoted its ubiquitination and degradation. In human liver cancer tissues, the neddylation-CRL pathway was overactivated and reversely correlated with RhoB levels. Moreover, RhoB accumulation upon inhibition of the neddylation-CRL pathway for anticancer therapy contributed to the induction of tumor suppressors p21 and p27, apoptosis, and growth suppression. Our findings highlight the degradation of RhoB via the neddylation-CRL pathway as an important molecular event that drives liver carcinogenesis and RhoB itself as a pivotal effector for anticancer therapy targeting this oncogenic pathway.

In-Depth Proteomic Analysis of the Hippocampus in a Rat Model after Cerebral Ischaemic Injury and Repair by Danhong Injection (DHI).

Stroke is the second most common cause of death worldwide. A systematic description and characterization of the strokes and the effects induced in the hippocampus have not been performed so far. Here, we analysed the protein expression in the hippocampus 24 h after cerebral ischaemic injury and repair. Drug intervention using Danhong injection (DHI), which has been reported to have good therapeutic effects in a clinical setting, was selected for our study of cerebral ischaemia repair in rat models. A larger proteome dataset and total 4091 unique proteins were confidently identified in three biological replicates by combining tissue extraction for rat hippocampus and LC-MS/MS analysis. A label-free approach was then used to quantify the differences among the four experimental groups (Naive, Sham, middle cerebral artery occlusion (MCAO) and MCAO + DHI groups) and showed that about 2500 proteins on average were quantified in each of the experiment group. Bioinformatics analysis revealed that in total 280 unique proteins identified above were differentially expressed (P < 0.05). By combining the subcellular localization, hierarchical clustering and pathway information with the results from injury and repair phase, 12 significant expressed proteins were chosen and verified with respect to their potential as candidates for cerebral ischaemic injury by Western blot. The primary three signalling pathways of the candidates related may be involved in molecular mechanisms related to cerebral ischaemic injury. In addition, a glycogen synthase kinase-3ß (Gsk-3ß) inhibitor of the candidates with the best corresponding expression trends between western blotting (WB) and label-free quantitative results were chosen for further validation. The results of Western blot analysis of protein expression and 2,3,5- chloride three phenyl tetrazole (TTC) staining of rat brains showed that DHI treatment and Gsk-3ß inhibitor are both able to confer protection against ischaemic injury in rat MCAO model. The observations of the present study provide a novel understanding regarding the regulatory mechanism of cerebral ischaemic injury.

Dual effects of VEGF-B on activating cardiomyocytes and cardiac stem cells to protect the heart against short- and long-term ischemia-reperfusion injury.

AIMS: To investigate whether vascular endothelial growth factor B (VEGF-B) improves myocardial survival and cardiac stem cell (CSC) function in the ischemia-reperfusion (I/R) heart and promotes CSC mobilization and angiogenesis. METHODS AND RESULTS: One hour after myocardial ischemia and infarction, rats were treated with recombinant human VEGF-B protein following 24 h or 7 days of myocardial reperfusion. Twenty-four hours after myocardial I/R, VEGF-B increased pAkt and Bcl-2 levels, reduced p-p38MAPK, LC3-II/I, beclin-1, CK, CK-MB and cTnt levels, triggered cardiomyocyte protection against I/R-induced autophagy and apoptosis, and contributed to the decrease of infarction size and the improvement of heart function during I/R. Simultaneously, an in vitro hypoxia-reoxygenation (H/R)-induced H9c2 cardiomyocyte injury model was used to mimic I/R injury model in vivo; in this model, VEGF-B decreased LDH release, blocked H/R-induced apoptosis by inhibiting cell autophagy, and these special effects could be abolished by the autophagy inducer, rapamycin. Mechanistically, VEGF-B markedly activated the Akt signaling pathway while slightly inhibiting p38MAPK, leading to the blockade of cell autophagy and thus protecting cardiomyocyte from H/R-induced activation of the intrinsic apoptotic pathway. Seven days after I/R, VEGF-B induced the expression of SDF-1α and HGF, resulting in the massive mobilization and homing of c-Kit positive cells, triggering further angiogenesis and vasculogenesis in the infracted heart and contributing to the improvement of I/R heart function. CONCLUSION: VEGF-B could contribute to a favorable short- and long-term prognosis for I/R via the dual manipulation of cardiomyocytes and CSCs.

Correction: Structure, function, self-assembly, and applications of bottlebrush copolymers.

Correction for 'Structure, function, self-assembly, and applications of bottlebrush copolymers' by Rafael Verduzco et al., Chem. Soc. Rev., 2015, 44, 2405-2420.

Structure, function, self-assembly, and applications of bottlebrush copolymers.

Bottlebrush polymers are a type of branched or graft polymer with polymeric side-chains attached to a linear backbone, and the unusual architectures of bottlebrushes provide a number of unique and potentially useful properties. These include a high entanglement molecular weight, enabling rapid self-assembly of bottlebrush block copolymers into large domain structures, the self-assembly of bottlebrush block copolymer micelles in a selective solvent even at very low dilutions, and the functionalization of bottlebrush side-chains for recognition, imaging, or drug delivery in aqueous environments. This review article focuses on recent developments in the field of bottlebrush polymers with an emphasis on applications of bottlebrush copolymers. Bottlebrush copolymers contain two (or more) different types of polymeric side-chains. Recent work has explored the diverse properties and functions of bottlebrush polymers and copolymers in solutions, films, and melts, and applications explored include photonic materials, bottlebrush films for lithographic patterning, drug delivery, and tumor detection and imaging. We provide a brief introduction to bottlebrush synthesis and physical properties and then discuss work related to: (i) bottlebrush self-assembly in melts and bulk thin films, (ii) bottlebrushes for photonics and lithography, (iii) bottlebrushes for small molecule encapsulation and delivery in solution, and (iv) bottlebrush micelles and assemblies in solution. We briefly discuss three potential areas for future research, including developing a more quantitative model of bottlebrush self-assembly in the bulk, studying the properties of bottlebrushes at interfaces, and investigating the solution assembly of bottlebrush copolymers.

Proteomic investigation of signatures for geniposide-induced hepatotoxicity.

Evaluating the safety of traditional medicinal herbs and their major active constituents is critical for their widespread usage. Geniposide, a major active constituent with a defined structure from the traditional medicinal herb Gardenia jasminoides ELLIS fruit, exhibits remarkable anti-inflammatory, antiapoptotic, and antifibrotic properties and has been used in a variety of medical fields, mainly for the treatment of liver diseases. However, geniposide-induced hepatotoxicity and methods for the early detection of hepatotoxicity have yet to be reported. In this study, geniposide-induced hepatotoxicity was investigated. In addition, candidate biomarkers for the earlier detection of geniposide-induced hepatotoxicity were identified using a label-free quantitative proteomics approach on a geniposide overdose-induced liver injury in a rat model. Using an accurate intensity-based, absolute quantification (iBAQ)-based, one-step discovery and verification approach, a candidate biomarker panel was easily obtained from individual samples in response to different conditions. To determine the biomarkers' early detection abilities, five candidate biomarkers were selected and tested using enzyme-linked immunosorbent assays (ELISAs). Two biomarkers, glycine N-methyltransferase (GNMT) and glycogen phosphorylase (PYGL), were found to indicate hepatic injuries significantly earlier than the current gold standard liver biomarker. This study provides a first insight into geniposide-induced hepatotoxicity in a rat model and describes a method for the earlier detection of this hepatotoxicity, facilitating the efficient monitoring of drug-induced hepatotoxicity.

Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry.

Core fucosylation (CF) is a special glycosylation pattern of proteins that has a strong relationship with cancer. The Food and Drug Administration (FDA) has approved the core fucosylated α-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value. The major method for core fucosylated glycoprotein/glycopeptide analysis is neutral loss-based MS(3) scanning under collision-induced dissociation (CID) by ion trap mass spectrometry. However, due to the limited speed and low resolution of the MS(3) scan mode, it is difficult to achieve high-throughput, with only dozens of core fucosylated proteins identified in a single run. In this work, we developed a novel strategy for the identification of CF glycopeptides at a large scale, integrating the stepped fragmentation function, one novel feature of quadrupole-orbitrap mass spectrometry, with "glycan diagnostic ion"-based spectrum optimization. By using stepped fragmentation, we were able to obtain both highly accurate glycan and peptide information of a simplified CF glycopeptide in one spectrum. Moreover, the spectrum could be recorded with the same high speed as the conventional MS(2) scan. By using the "glycan diagnostic ion"-based spectrum refinement method, the efficiency of the CF glycopeptide discovery was significantly improved. We demonstrated the feasibility and reproducibility of our method by analyzing CF glycoproteomes of mouse liver tissue and HeLa cell samples spiked with standard CF glycoprotein. In total, 1364 and 856 CF glycopeptides belonging to 702 and 449 CF glycoproteins were identified, respectively, within a 78-min gradient analysis, which was approximately a 7-fold increase in the identification efficiency of CF glycopeptides compared to the currently used method. In this work, we took core fucosylated glycopeptides as a practical example to demonstrate the great potential of our novel method for use in glycoproteome analysis, and we also anticipate using the flexible novel method in other research fields.

Thermoresponsive PNIPAAM bottlebrush polymers with tailored side-chain length and end-group structure.

We explore the phase behaviour, solution conformation, and interfacial properties of bottlebrush polymers with side-chains comprised of poly(N-isopropylacrylamide) (PNIPAAM), a thermally responsive polymer that exhibits a lower critical solution temperature (LCST) in water. PNIPAAM bottlebrush polymers with controlled side-chain length and side-chain end-group structure are prepared using a "grafting-through" technique. Due to reduced flexibility of bottlebrush polymer side-chains, side-chain end-groups have a disproportionate effect on bottlebrush polymer solubility and phase behaviour. Bottlebrush polymers with a hydrophobic end-group have poor water solubilities and depressed LCSTs, whereas bottlebrush polymers with thiol-terminated side-chains are fully water-soluble and exhibit an LCST greater than that of PNIPAAM homopolymers. The temperature-dependent solution conformation of PNIPAAM bottlebrush polymers in D2O is analyzed by small-angle neutron scattering (SANS), and data analysis using the Guinier-Porod model shows that the bottlebrush polymer radius decreases as the temperature increases towards the LCST for PNIPAAM bottlebrush polymers with relatively long 9 kg mol(-1) side-chains. Above the LCST, PNIPAAM bottlebrush polymers can form a lyotropic liquid crystal phase in water. Interfacial tension measurements show that bottlebrush polymers reduce the interfacial tension between chloroform and water to levels comparable to PNIPAAM homopolymers without the formation of microemulsions, suggesting that bottlebrush polymers are unable to stabilize highly curved interfaces. These results demonstrate that bottlebrush polymer side-chain length and flexibility impact phase behavior, solubility, and interfacial properties.

Multivalent hydrazide-functionalized magnetic nanoparticles for glycopeptide enrichment and identification.

Among the common approaches for global glycopeptide enrichment, hydrazide chemistry is well recognized. However, conventional hydrazide-functionalized products are composed of a single layer of hydrazide functional groups. Due to the limited specific surface area of such a structure, the loading amount of hydrazide groups immobilized on these materials is restricted. Therefore, these materials can only provide a limited reaction rate with glycopeptides in complex protein samples, which is exacerbated by the microheterogeneities of glycosylation. Here, we introduce a new functionalized magnetic nanoparticle coating with hydrazide-modified non-crosslinked polymer chains. The multivalent hydrazide-functionalized particles were synthesized by the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. The density of the hydrazide groups on the surface of these nanoparticles was three-fold higher than that of conventional single-layered materials. The new particles enabled the highly sensitive and selective enrichment of glycopeptides from a digestion mixture of fetuin, even from a background mixture of non-glycosylated protein that was 100-fold more abundant. The recovery ratio of glycopeptides was determined to be 77.8%, and the glycopeptide binding capacity of the materials was determined to be 25 µg mg(-1). Finally, the novel multivalent hydrazide-functionalized particles were applied in the enrichment of N-linked glycopeptides from mouse liver tissues, which resulted in the assignment of 511 unique glycopeptides belonging to 372 different glycoproteins. The results further demonstrated the potential of the multivalent particles for glycopeptide enrichment in complex proteomics samples.

Angong Niuhuang Wan inhibit ferroptosis on ischemic and hemorrhagic stroke by activating PPARγ/AKT/GPX4 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Angong Niuhuang Wan (AGNHW) is a prescription from traditional Chinese medicine (TCM) that has been used for centuries to treat ischemic stroke (IS) and hemorrhagic stroke (HS). According to a recent study, targeting ferroptosis might be effective in the management of IS and HS. However, the ferroptosis-related effects and mechanisms of AGNHW have not yet been reported. AIM OF THE STUDY: This research examines the anti-ferroptosis mechanisms of AGNHW in the treatment of IS and HS. MATERIALS AND METHODS: A system pharmacological approach including in vivo experiment, UHPLC-Q-Orbitrap HRMS, network pharmacology, molecular docking, microscale thermophoresis, and in vitro experiment was utilized to study the anti-ferroptosis mechanisms of AGNHW against IS and HS. RESULTS: In vivo experiments indicated that AGNHW enhanced nerve function, decreased cerebral infarct volume, ameliorated histological brain injuries, improved the structural integrity of the blood-brain barrier, ameliorated the mitochondrial dysfunction and morphology disruption, and inhibits ROS, LPO and Fe2+ accumulations in IS and HS rats. Using UHPLC-Q-Orbitrap HRMS, the key ingredients of AGNHW-containing serum were identified as bilirubin, berberine, baicalin, and wogonoside. According to the network pharmacology analyses, AGNHW could inhibit ferroptosis by modulating the PPAR and PI3K/AKT signaling pathways. The core targets are PPARγ, AKT, and GPX4. Molecular docking and microscale thermophoresis experiments further revealed that the key ingredients have strong interactions with ferroptosis-regulating core proteins. Moreover, in vitro experiment results showed that AGNHW alleviated ferroptosis injury induced by erastin in PC12 cells, increased cell viability, reduced the LPO and Fe2+ levels, and up-regulated mRNA expressions of PPARγ, AKT, and GPX4. AGNHW also up-regulated protein expressions of PPARγ, p-AKT/AKT, and GPX4 in IS and HS rats. CONCLUSIONS: AGNHW attenuated ferroptosis in treating IS and HS by targeting the PPARγ/AKT/GPX4 pathway. This work reveals AGNHW's anti-ferroptosis mechanism against IS and HS, but it also develops an integrated approach to demonstrate the common characteristics of drugs in treating different diseases.

In-depth analysis of the treatment effect and synergistic mechanism of TanReQing injection on clinical multi-drug resistant Pseudomonas aeruginosa.

Antibiotic resistance is a recognized and concerning public health issue. Gram-negative bacilli, such as Pseudomonas aeruginosa (P. aeruginosa), are notorious for their rapid development of drug resistance, leading to treatment failures. TanReQing injection (TRQ) was chosen to explore its pharmacological mechanisms against clinical multidrug-resistant P. aeruginosa (MDR-PA), given its antibacterial and anti-inflammatory properties. We revealed the expression of proteins and genes in P. aeruginosa after co-culture with TRQ. This study developed an assessment method to evaluate clinical resistance of P. aeruginosa using MALDI-TOF MS identification and Biotyper database searching techniques. Additionally, it combined MIC determination to investigate changes in MDR-PA treated by TRQ. TRQ effectively reduced the MICs of ceftazidime and cefoperazone and enhanced the confidence scores of MDR-PA as identified by mass spectrometry. Using this evaluation method, the fingerprints of standard P. aeruginosa and MDR-PA were compared, and the characteristic peptide sequence (Seq-PA No. 1) associated with flagellum was found. The phenotypic experiments were conducted to confirm the effect of TRQ on the motility and adhesion of P. aeruginosa. A combination of co-immunoprecipitation and proteome analysis was employed, and 16 proteins were significantly differentially expressed and identified as potential candidates for investigating the mechanism of inhibiting resistance in P. aeruginosa treated by TRQ. The candidates were verified by quantitative real-time PCR analysis, and TRQ may affect these core proteins (MexA, MexB, OprM, OprF, OTCase, IDH, and ASL) that influence resistance of P. aeruginosa. The combination of multiple methods helps elucidate the synergistic mechanism of TRQ in overcoming resistance of P. aeruginosa.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen closely associated with various life-threatening acute and chronic infections. The presence of antimicrobial resistance and multidrug resistance in P. aeruginosa infections significantly complicates antibiotic treatment. The expression of ß-lactamase, efflux systems such as MexAB-OprM, and outer membrane permeability are considered to have the greatest impact on the sensitivity of P. aeruginosa. The study used a method to assess the clinical resistance of P. aeruginosa using matrix-assisted laser desorption ionization time of flight mass spectrometry identification and Biotyper database search techniques. TanReQing injection (TRQ) effectively reduced the MICs of ceftazidime and cefoperazone in multidrug-resistant P. aeruginosa (MDR-PA) and improved the confidence scores for co-cultured MDR-PA. The study found a characteristic peptide sequence for distinguishing whether P. aeruginosa is resistant. Through co-immunoprecipitation and proteome analysis, we explored the mechanism of TRQ overcoming resistance of P. aeruginosa.

Xinshubao tablet rescues cognitive dysfunction in a mouse model of vascular dementia: Involvement of neurogenesis and neuroinflammation.

Vascular dementia (VaD) represents a severe cognitive dysfunction syndrome closed linked to cardiovascular function. In the present study, we assessed the potential of Xinshubao tablet (XSB), a traditional Chinese prescription widely used for cardiovascular diseases, to mitigate neuropathological damage in a mouse model of VaD and elucidated the underlying mechanisms. Our findings revealed that oral administration of XSB rescued the cardiac dysfunction resulting from bilateral common carotid artery stenosis (BCAS), improved the cerebral blood flow (CBF) and cognitive function, reduced white matter injury, inhibited excessive microglial and astrocytic activation, stimulated hippocampal neurogenesis, and reduced neural apoptosis in the brains of BCAS mice. Mechanistically, RNA-seq analysis indicated that XSB treatment was significantly associated with neuroinflammation, vasculature development, and synaptic transmission, which were further confirmed by q-PCR assays. Western blot results revealed that XSB treatment hindered the nuclear translocation of nuclear factor-κB (NF-κB), thereby suppressing the NF-κB signaling pathway. These results collectively demonstrated that XSB could ameliorate cognitive dysfunction caused by BCAS through regulating CBF, reducing white matter lesions, suppressing glial activation, promoting neurogenesis, and mitigating neuroinflammation. Notably, the NF-κB signaling pathway emerged as a pivotal player in this mechanism.