Exosome nanovesicles: biomarkers and new strategies for treatment of human diseases.

Exosomes are nanoscale vesicles of cellular origin. One of the main characteristics of exosomes is their ability to carry a wide range of biomolecules from their parental cells, which are important mediators of intercellular communication and play an important role in physiological and pathological processes. Exosomes have the advantages of biocompatibility, low immunogenicity, and wide biodistribution. As researchers' understanding of exosomes has increased, various strategies have been proposed for their use in diagnosing and treating diseases. Here, we provide an overview of the biogenesis and composition of exosomes, describe the relationship between exosomes and disease progression, and focus on the use of exosomes as biomarkers for early screening, disease monitoring, and guiding therapy in refractory diseases such as tumors and neurodegenerative diseases. We also summarize the current applications of exosomes, especially engineered exosomes, for efficient drug delivery, targeted therapies, gene therapies, and immune vaccines. Finally, the current challenges and potential research directions for the clinical application of exosomes are also discussed. In conclusion, exosomes, as an emerging molecule that can be used in the diagnosis and treatment of diseases, combined with multidisciplinary innovative solutions, will play an important role in clinical applications.

Carbon monoxide-releasing nanomotors based on endogenous biochemical reactions for tumor therapy.

The lack of selective release ability in the tumor microenvironment and the limited efficacy of monotherapy are important factors that limit the current use of carbon monoxide (CO) donors for tumor therapy. Herein, inspired by endogenous biochemical reactions in vivo, one kind of CO-releasing nanomotor was designed for the multimodal synergistic treatment of tumor. Specifically, glucose oxidase (GOx) and 5-aminolevulinic acid (5-ALA) were co-modified onto metal-organic framework material (MIL-101) to obtain MIL-GOx-ALA nanomotors (M-G-A NMs), which exhibit excellent biocompatibility and degradation ability in tumor microenvironment. Subsequently, the released 5-ALA generates CO in the tumor microenvironment through an endogenous reaction and further acts on mitochondria to release large amounts of reactive oxygen species (ROS), which directly kill tumor cells. Furthermore, the produced ROS and the degradation products of M-G-A NMs can also provide the reaction substrate for the Fenton reaction, thereby enhancing chemodynamic therapy (CDT) and inducing apoptosis of tumor cells. Both in vitro and in vivo experimental data confirm the successful occurrence of the above process, and the combination of CO gas therapy/enhanced CDT can effectively inhibit tumor growth. This CDT-enhancing agent designed based on endogenous biochemical reactions has good prospects for tumor treatment application.

Improved analysis ZIC-HILIC-HCD-Orbitrap method for mapping the glycopeptide by mass spectrometry.

Glycosylation is one of the most common post-translational modifications (PTMs). Protein glycosylation analysis is the bottleneck to deeply understand their functions. At present, the LC-MS analysis of glycosylated post-translational modification is mainly focused on the analysis of glycopeptides. However, the factors affecting the identification of glycopeptides were not fully elucidated. In the paper, we have carefully studied the factors, e.g., HILIC materials, search engines, protein amount, gradient duration, extraction solution, etc. According to the results, HILIC materials were the most important factors affecting the glycopeptides identification, and the amphoteric sulfoalkyl betaine stationary phase enriched glycopeptides 6-fold more compared to the amphiphilic ion-bonded fully porous spherical silica stationary phase. We explored the influence of the extraction solutions on glycan identification. Comparing sodium dodecyl sulfate (SDS) and urea (UA), the results showed that N-glycolylneuraminic acid (NeuGc) type of glycan content was found to be increased 1.4-fold in the SDS compared to UA. Besides, we explored the influence of the search engine on glycopeptide identification. Comparing pGlyco3.0 and MSFragger-Glyco, it was observed that pGlyco3.0 outperformed MSFragger-Glyco in identifying glycopeptides. Then, using our optimized method we found that there was a significant difference in the distribution of monosaccharide types in plasma and brain tissue, e.g., the content of NeuAc in brain was 5-fold higher than that in plasma. To importantly, two glycoproteins (Neurexin-2 and SUN domain-containing protein 2) were also found for the first time by our method. In summary, we have comprehensively studied the factors influencing glycopeptide identification than any previous research, and the optimized method could be widely used for identifying the glycoproteins or glycolpeptides biomarkers for disease detection and therapeutic targets.

Optimized approach for active peptides identification in Cerebrolysin by nanoLC-MS.

Cerebrolysin (CBL) is a peptide-rich preparation made by hydrolysis and purified extraction of porcine brain. CBL contains various neuroprotective peptides, such as neurotrophic factor, nerve growth factor and ciliary neurotrophic factor, which can be used to treat neurodegenerative diseases. However, the active peptides in CBL had not been studied in depth. In this study, the following was carried out in order to investigate the active peptides in CBL. First, CBL samples were treated using organic reagents (acetonitrile and acetone) to precipitate the proteins and different solid phase extraction methods (MCX mixed-mode cartridges, C18 SPE cartridge columns and HILIC sorbent). Then the samples were analyzed using nanoLC-MS, followed by the identification of peptides using different sequence analysis software (PEAKS, pNovo and novor). Finally, bioinformatics analysis was performed to predict peptides with potential neuroprotective functions in CBL, such as anti-inflammatory and antioxidant peptides. Results showed that the number of peptides obtained by the MCX method coupled with PEAKS was the highest and the method was the most stable. Bioinformatic analysis of the detected peptides showed that two anti-inflammatory peptides (LLNLQPPPR and LSPSLRLP) and an antioxidant peptide (WPFPR) might be neuroprotective peptides in CBL. In addition, this study found that some peptides in CBL were present in myelin basic protein and tubulin beta chain. The results of this study for the detection of active peptides in CBL laid the foundation for the subsequent study of its active ingredients.

Efficacy and safety of PLDR-IMRT for the re-irradiation of recurrent NPC: A prospective, single-arm, multicenter trial.

Salvage treatment of locoregionally recurrent nasopharyngeal carcinoma (NPC) requires weighing the benefits of re-irradiation against increased risks of toxicity. Here, we evaluated the outcomes of patients treated with intensity-modulated-based pulsed low-dose-rate radiotherapy (PLDR-IMRT) to enhance the curative effect of salvage treatment and reduce RT-related SAEs. A prospective clinical trial was conducted from March 2018 to March 2020 at multiple institutions. NPC patients who experienced relapse after radical therapy were re-irradiated with a median dose of 60 Gy (50.4-70 Gy)/30 f (28-35 f) using PLDR-IMRT. Thirty-six NPC patients who underwent PLDR-IMRT for locoregional recurrence were identified. With a median follow-up of 26.2 months, the objective response rate (ORR) of the entire cohort was 91.6%. The estimated mPFS duration was 28 months (95% CI: 24.9-31.1), and the estimated mLRFS duration was 30.4 months (95% CI: 25.2-35.5). The overall survival (OS) rate for all patients was 80.6%, the progression-free survival (PFS) rate was 75% and the cancer-specific survival (CSS) rate was 88.9% at 1 year. The LRFS and DMFS rates were 88.9% and 91.7%, respectively, at 1 year. A combination of systematic therapies could provide survival benefits to patients who experience NPC relapse (p < 0.05), and a Karnofsky performance status (KPS) score of ≥90 was a favorable factor for local control (p < 0.05). The incidence of acute SAEs (grade 3+) from PLDR was 22.2%, and the incidence of chronic SAEs was 19.4% among all patients. PLDR-IMRT combined with systematic therapy can effectively treat patients with locoregionally recurrent nasopharyngeal carcinoma and causes fewer adverse events than the rates expected with IMRT.

Movement disorder caused by FRRS1L deficiency may be associated with morphological and functional disorders in Purkinje cells.

Ferric Chelate Reductase 1 Like (FRRS1L) protein has been identified as an auxiliary regulatory protein for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR). FRRS1L is highly expressed in the cerebellum and other brain regions associated with the control of motor function. Loss of FRRS1L has been shown to lead to impaired synaptic transmission via AMPARs and to movement disorders. We found that deletion of the FRRS1L gene causes hyperactivity, reduced muscle strength, impaired coordination, and ataxia in mice. Deletion also impairs Purkinje cell dendritic spine formation and AMPAR expression in the cerebellum and damages the electrophysiological discharge rhythm of Purkinje cells. Cerebrospinal fluid examination and oleic acid (OA)-induced lipid accumulation monitoring in FRRS1L-knockdown SH-SY5Y cells indicated that FRRS1L deficiency could lead to aberrant metabolism of amino acids, glucose, and lipids. In summary, we found that the deletion of FRRS1L leads to impaired motor coordination and cerebellar ataxia in mice, which might be related to the reduced expression of AMPARs, metabolic deviations, and dysplastic functional defects in Purkinje cells.

Effect of thymoquinone on sepsis-induced cardiac damage via anti-inflammatory and anti-apoptotic mechanisms.

OBJECTIVE: Sepsis is a systemic and deleterious host reaction to severe infection. Cardiac dysfunction is an established serious outcome of multiorgan failure associated with this condition. Therefore, it is important to develop drugs targeting sepsis-induced cardiac damage and inflammation. Thymoquinone (TQ) has anti-inflammatory, anti-oxidant, anti-fibrotic, anti-tumor, and anti-apoptotic effects. This study examined the effects of thymoquinone on sepsis-induced cardiac damage. METHODS: Male BALB/c mice were randomly segregated into four groups: control, TQ, cecal ligation and puncture (CLP), and CLP + TQ groups. CLP was performed after gavaging the mice with TQ for 2 weeks. After 48 hours, we estimated the histopathological changes in the cardiac tissue and the serum levels of cardiac troponin-T. We evaluated the expression of factors associated with inflammation, apoptosis, oxidative stress, and the PI3K/AKT pathway. RESULTS: TQ significantly reduced intestinal histological alterations and inhibited the upregulation of interleukin-6, tumor necrosis factor-α, Bax, NOX4, p-PI3K, and p-AKT. TQ also increased Bcl-2, HO-1, and NRF2 expression. CONCLUSION: These results suggest that TQ effectively modulates pro-inflammatory, apoptotic, oxidative stress, and PI3K/AKT pathways, making it indispensable in the treatment of sepsis-induced cardiac damage.

Cell-cycle and apoptosis related and proteomics-based signaling pathways of human hepatoma Huh-7 cells treated by three currently used multi-RTK inhibitors.

Sorafenib, lenvatinib and regorafenib, the multi-RTK inhibitors with potent anti-angiogenesis effects, are currently therapeutic drugs generally recommended for the patients with advanced hepatocellular carcinoma (HCC). To date, however, there have been no published studies on the mechanism underling differential effects of the three drugs on HCC cell proliferation, and the proteomic analysis in HCC cell lines treated by regorafenib or lenvatinib. The present study for the first time performed a direct comparison of the cell cycle arrest and apoptosis induction in the Huh-7 cells caused by sorafenib, regorafenib and lenvatinib at respective IC50 using flow cytometry technique, as well as their pharmacological interventions for influencing whole cell proteomics using tandem mass tag-based peptide-labeling coupled with the nLC-HRMS technique. Sorafenib, regorafenib and lenvatinib at respective IC50 drove the remaining surviving Huh-7 cells into a G0/G1 arrest, but lenvatinib and regorafenib were much more effective than sorafenib. Lenvatinib produced a much stronger induction of Huh-7 cells into early apoptosis than sorafenib and regorafenib, while necrotic cell proportion induced by regorafenib was 2.4 times as large as that by lenvatinib. The proteomic study revealed 419 proteins downregulated commonly by the three drugs at respective IC50. KEGG pathway analysis of the downregulated proteins indicated the ranking of top six signaling pathways including the spliceosome, DNA replication, cell cycle, mRNA surveillance, P53 and nucleotide excision repair involved in 33 proteins, all of which were directly related to their pharmacological effects on cell cycle and cell apoptosis. Notably, lenvatinib and regorafenib downregulated the proteins of PCNA, Cyclin B1, BCL-xL, TSP1, BUD31, SF3A1 and Mad2 much more strongly than sorafenib. Moreover, most of the proteins in the P53 signaling pathway were downregulated with lenvatinib and regorafenib by more than 36% at least. In conclusion, lenvatinib and regorafenib have much stronger potency against Huh-7 cell proliferation than sorafenib because of their more potent effects on cell cycle arrest and apoptosis induction. The underling mechanism may be at least due to the 33 downregulated proteins centralizing the signal pathways of cell cycle, p53 and DNA synthesis based on the present proteomics study.

Salvia chinensia Benth induces autophagy in esophageal cancer cells via AMPK/ULK1 signaling pathway.

Salvia chinensia Benth (Shijianchuan in Chinese, SJC) has been used as a traditional anti-cancer herb. SJC showed good anti-esophageal cancer efficacy based on our clinical application. However, the current research on SJC is minimal, and its anti-cancer effect lacks scientific certification. This study aims to clarify the inhibitory effect of SJC on esophageal cancer and explore its underlying mechanism. Q-Orbitrap high-resolution LC/MS was used to identify the primary chemical constituents in SJC. Cell proliferation and colony formation assays showed that SJC could effectively inhibit the growth of esophageal tumor cells in vitro. To clarify its mechanism of action, proteomic and bioinformatic analyses were carried out by combining tandem mass labeling and two-dimensional liquid chromatography-mass spectrometry (LC-MS). Data are available via ProteomeXchange with identifier PXD035823. The results indicated that SJC could activate AMPK signaling pathway and effectively promote autophagy in esophageal cancer cells. Therefore, we further used western blotting to confirm that SJC activated autophagy in esophageal cancer cells through the AMPK/ULK1 signaling pathway. The results showed that P-AMPK and P-ULK1 were significantly up-regulated after the treatment with SJC. The ratio of autophagosomes marker proteins LC3II/I was significantly increased. In addition, the expression of the autophagy substrate protein P62 decreased with the degradation of autophagosomes. Using lentiviral transfection of fluorescent label SensGFP-StubRFP-LC3 protein and revalidation of LC3 expression before and after administration by laser confocal microscopy. Compared with the control group, the fluorescence expression of the SJC group was significantly enhanced, indicating that it promoted autophagy in esophageal cancer cells. Cell morphology and the formation of autophagosomes were observed by transmission electron microscopy. Our study shows that the tumor suppressor effect of SJC is related to promoting autophagy in esophageal tumor cells via the AMPK/ULK1 signaling pathway.

Brain-wide projection reconstruction of single functionally defined neurons.

Reconstructing axonal projections of single neurons at the whole-brain level is currently a converging goal of the neuroscience community that is fundamental for understanding the logic of information flow in the brain. Thousands of single neurons from different brain regions have recently been morphologically reconstructed, but the corresponding physiological functional features of these reconstructed neurons are unclear. By combining two-photon Ca2+ imaging with targeted single-cell plasmid electroporation, we reconstruct the brain-wide morphologies of single neurons that are defined by a sound-evoked response map in the auditory cortices (AUDs) of awake mice. Long-range interhemispheric projections can be reliably labelled via co-injection with an adeno-associated virus, which enables enhanced expression of indicator protein in the targeted neurons. Here we show that this method avoids the randomness and ambiguity of conventional methods of neuronal morphological reconstruction, offering an avenue for developing a precise one-to-one map of neuronal projection patterns and physiological functional features.

UnaG as a reporter in adeno-associated virus-mediated gene transfer for biomedical imaging.

Adeno-associated virus (AAV) is one of the most common gene transfer vectors, but it has a limited capacity. A smaller fluorescent protein is urgently needed since it is more suitable to act as a reporter in AAV. In this study, a bilirubin-dependent reporter smaller than EGFP, termed UnaG, was found to have the ability to label the neurons of a mouse brain as clearly as EGFP without the addition of exogenous bilirubin. We also found that UnaG's pH tolerance is better than that of EGFP; however, its fluorescence recovery after protonated quenching is not as good as that of EGFP. In addition, UnaG preserved its fluorescence better than EGFP in SeeDB clearing. Taken together, this study demonstrates that UnaG can act as a small intrinsically fluorescent reporter in the mouse brain without an additional ligand, thus providing an alternative over EGFP for AAV-mediated neuron labeling in mammals.

Repression of deoxynivalenol-triggered cytotoxicity and apoptosis by mannan/ß-glucans from yeast cell wall: Involvement of autophagy and PI3K-AKT-mTOR signaling pathway.

Deoxynivalenol (DON) is the most common trichothecene distributed in food and feed. So far, much work has focused on investigating the cytotoxicity of DON, while there is few researches aimed at intervening in the toxic impacts on humans and livestock posed by DON. The objective of this study is to investigate the underlying mechanism of biomacromolecules mannan/ß-glucans from yeast cell wall (BYCW) for their potency to impede the cytotoxicity and apoptosis caused by DON with porcine jejunum epithelial cell lines (IPEC-J2) used as a cell injury model. We analyzed the cell morphology, cell activity, oxidative stress, fluorescence intensity and expressions of proteins relevant to autophagy, apoptosis and PI3K-AKT-mTOR signaling pathway by using inverted microscopy, MTS, reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) assay, Annexin V-FITC / propidium iodide (PI) double staining and Western blot assay. The consequent data demonstrated that in the presence of BYCW, the cell morphology and activity were relatively ameliorated and that the oxidation damage was attenuated with DON-induced autophagy concomitantly decreased, which, furthermore, was found involved in the positive regulation on PI3K-AKT-mTOR signaling pathway by BYCW. In a word, BYCW possess an ability to repress the cytotoxicity and apoptosis induced by DON through the inhibition of autophagy via activating PI3K-AKT-mTOR signaling pathway.

Protective effects of mannan/ß-glucans from yeast cell wall on the deoxyniyalenol-induced oxidative stress and autophagy in IPEC-J2 cells.

The aim of this study was to investigate the effects of biomacromolecules mannan/ß-glucans from yeast cell wall (BYCW) to alleviate Deoxynivalenol(DON)-induced injury. Considering that DON has strong oxidizing effect and stimulates autophagy and apoptosis, we examined the effects of BYCW on consequent oxidative stress damage indicators, cells autophagy and apoptosis induced by DON using the porcine jejunum epithelial cell lines (IPEC-J2) as a cell culture model. The results showed that application of BYCW could reverse the decrease of cell viability by DON significantly, and suppress the levels of tumor necrosis factor-α (TNF-α) and interleukin-8 and -6 (IL-8 and IL-6), except IL-1ß. Further experiments revealed that BYCW treatment counteracted the DON-induced down-regulation of intracellular glutathione (GSH) and up-regulation of reactive oxygen species (ROS) and malondialdehyde (MDA). Through western blot analysis, we observed that BYCW treatment was able to down-regulate the expression of autophagy protein LC3-II and up-regulate the expression of P62 protein against DON, which suggested that autophagy induced by DON may be suppressed. Altogether, these results indicated a potential ability of supplementation of BYCW to improve cell growth and metabolism as well as the preventive properties of BYCW against the DON-induced cell damage by activating antioxidant system.

Protective role of fenofibrate in sepsis-induced acute kidney injury in BALB/c mice.

Acute kidney injury (AKI) is a severe complication of sepsis, which largely contributes to the associated high mortality rate. Fenofibrate, a peroxisome proliferator activated receptor α (PPARα) agonist, has received considerable attention because of its effects related to renal damage-related energy metabolism and inflammation. The present study investigated the effects of fenofibrate on sepsis-associated AKI in BALB/c mice subjected to caecal ligation and puncture (CLP). Eight-week-old male BALB/c mice were divided into four groups: control group, fenofibrate group, caecal ligation and puncture (CLP) group, and fenofibrate + CLP group. CLP was performed after mice were gavaged with fenofibrate for 2 weeks. After 48 hours, we measured the histopathological alterations of the kidney tissue and plasma levels of serum creatinine (CRE), neutrophil gelatinase-associated lipocalin (NGAL), reactive oxygen species (ROS), ATP, and ADP. We evaluated PPARα and P53 protein levels as well as interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α mRNA levels. Our results showed that administering fenofibrate significantly reduced kidney histological alterations caused by CLP. Fenofibrate inhibited the plasma levels of ROS, CRE, NGAL, and increased the ATP/ADP ratio. Fenofibrate significantly inhibited elevations in P53, IL-1ß, IL-6, and tumour necrosis factor-α expression. The results suggest that fenofibrate administration effectively modulates energy metabolism and may be a novel approach to treat sepsis-induced renal damage.

Functional interaction of CD154 protein with α5ß1 integrin is totally independent from its binding to αIIbß3 integrin and CD40 molecules.

In addition to its classical CD40 receptor, CD154 also binds to αIIbß3, α5ß1, and αMß2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbß3, and α5ß1 receptors. We found that the binding affinity of CD154 for αIIbß3 is ∼4-fold higher than for α5ß1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbß3 and show that CD154 residues involved in its binding to CD40 or αIIbß3 are distinct from those implicated in its interaction to α5ß1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5ß1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5ß1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.

Radiolabeling of folic acid-modified chitosan with (99m)Tc as potential agents for folate-receptor-mediated targeting.

The feasibility of chitosan (CS) as a backbone for the design of (99m)Tc-labeled targeting agent was evaluated in this study. Chitosan-folate conjugate (CSFA) and chitosan-folate dithiocarbamate (CSFADTC) were synthesized, characterized and radiolabeled with (99m)Tc as model compounds for folate-receptor (FR) targeting. (99m)Tc-complexes were prepared with high radiochemical purity and high stability. The hydrophilicities of these (99m)Tc-complexes were determined by partition coefficient experiments. The results of biodistribution in normal mice showed that the folic-acid modified agents ((99m)Tc-CSFA and (99m)TcN-CSFADTC) had obviously higher uptake in FR-positive kidney and much lower liver and spleen uptakes than that of non-folic-acid modified (99m)Tc-agent, and the kidney uptakes of FA-modified agents could be blocked significantly by the corresponding cold ligand. Furthermore in vitro and in vivo specific studies will be done in cell line and tumor bearing mice to confirm the usefulness of this chitosan backbone for FR targeting agent design.

HLA-DR signaling inhibits Fas-mediated apoptosis in A375 melanoma cells.

Although melanocytes are devoid of the human major histocompatibility complex class II (HLA II) molecules, melanomas often display constitutive expression of these molecules, particularly HLA-DR. This constitutive expression of HLA-DR molecules is associated with tumor progression and poor prognosis but the molecular basis for this association remains poorly understood. Within the hypothesis of a role in immune escape, we analyzed the regulation of Fas-mediated apoptosis by HLA-DR signaling in the HLA-DR-positive malignant melanoma cell line A375. Our study demonstrates that engagement of HLA-DR molecules with anti-HLA-DR-specific monoclonal antibody L243 significantly reduces Fas-mediated apoptosis; DNA fragmentation and cell death were decreased by 50% and 40%, respectively. We found that while HLA-DR signaling does not affect Fas receptor expression, it significantly reduces Fas-induced activation of caspase-8 and Bid. Furthermore, inhibition studies and expression of dominant negative form of Mek-1 demonstrated that HLA-DR-mediated inhibition of caspase-8/Bid activation and apoptosis are dependent on the activation of the MAPK/Erk pathway. Together, our results provide evidence that HLA-DR signaling activates the MAPK/Erk pathway in A375 melanoma cells, which has a functional role in the resistance of these cells to Fas-mediated apoptosis. These observations underline the potential importance that HLA-DR signaling might have in melanoma immune escape and tumor progression.