Production and characterization of novel monoclonal antibodies against pathological human TDP-43 proteins.

The RNA/DNA-binding protein TDP-43 plays a pivotal role in the ubiquitinated inclusions characteristic of TDP-43 proteinopathies, including most cases of frontotemporal lobar degeneration (FTLD-TDP) and Alzheimer disease (AD). To understand the mechanisms of pathological TDP-43 processing and identify potential biomarkers, we generated novel phosphorylation-independent monoclonal antibodies (MAbs) using bacteria-expressed human full-length recombinant TDP-43. Remarkably, we identified a distinctive MAb, No. 9, targeting an epitope in amino acid (aa) region 311-360 of the C-terminus. This antibody showed preferential reactivity for pathological TDP-43 inclusions, with only mild reactivity for normal nuclear TDP-43. MAb No. 9 revealed more pathology in FTLD-TDP type A and type B brains and in AD brains compared to the commercial p409/410 MAb. Using synthetic phosphorylated peptides, we also obtained MAbs targeting the p409/410 epitope. Interestingly, MAb No. 14 was found to reveal additional pathology in AD compared to the commercial p409/410 MAb, specifically, TDP-43-immunopositive deposits with amyloid plaques in AD brains. These unique immunopositivities observed with MAbs No. 9 and No. 14 are likely attributed to their conformation-dependent binding to TDP-43 inclusions. We expect that this novel set of MAbs will prove valuable as tools for future patient-oriented investigations into TDP-43 proteinopathies.

Zinc deficiency drives ferroptosis resistance by lactate production in esophageal squamous cell carcinoma.

Trace metal zinc is involved in key processes of solid tumors by its antioxidant properties, while the role of zinc at the onset of esophageal squamous cell carcinoma (ESCC) remains controversial. This study aimed to determine whether zinc is associated with the ESCC and underlying molecular events involving malignant progression. Based on a case-control study, we found serum and urine zinc were decreased and correlated with ESCC progression. Thus, an in vitro model for zinc deficiency (ZD) was established, and we found that ZD contributed to the proliferation, migration, and invasion of EC109 cells. Untargeted metabolomics identified 59 upregulated metabolites and 6 downregulated metabolites, among which glycolysis and ferroptosis-related oxidation of chain fatty acids might play crucial steps in ZD-treated molecular events. Interestingly, ZD disrupted redox homeostasis and enhanced cytosolic Fe2+ of EC109 cells, while lipid peroxidation, the key marker of ferroptosis occurrence, was decreased after ZD treatment. The mechanism underlying these changes may involve ZD-enhanced ESCC glycolysis and lactate production, which confer ferroptosis resistance by inhibiting of p-AMPK and leading to the upregulation of SREBP1 and SCD1 to enhance the production of anti-ferroptosis monounsaturated fatty acids.

FAM76B regulates PI3K/Akt/NF-κB-mediated M1 macrophage polarization by influencing the stability of PIK3CD mRNA.

Macrophage polarization is closely related to inflammation development, yet how macrophages are polarized remains unclear. In our study, the number of M1 macrophages was markedly increased in Fam76b knockout U937 cells vs. wild-type U937 cells, and FAM76B expression was decreased in M1 macrophages induced from different sources of macrophages. Moreover, Fam76b knockout enhanced the mRNA and protein levels of M1 macrophage-associated marker genes. These results suggest that FAM76B inhibits M1 macrophage polarization. We then further explored the mechanism by which FAM76B regulates macrophage polarization. We found that FAM76B can regulate PI3K/Akt/NF-κB pathway-mediated M1 macrophage polarization by stabilizing PIK3CD mRNA. Finally, FAM76B was proven to protect against inflammatory bowel disease (IBD) by inhibiting M1 macrophage polarization through the PI3K/Akt/NF-κB pathway in vivo. In summary, FAM76B regulates M1 macrophage polarization through the PI3K/Akt/NF-κB pathway in vitro and in vivo, which may inform the development of future therapeutic strategies for IBD and other inflammatory diseases.

Role of mast cells activation in the tumor immune microenvironment and immunotherapy of cancers.

The mast cell is an important cellular component that plays a crucial role in the crosstalk between innate and adaptive immune responses within the tumor microenvironment (TME). Recently, numerous studies have indicated that mast cells related to tumors play a dual role in regulating cancers, with conflicting results seemingly determined by the degranulation medium. As such, mast cells are an ignored but very promising potential target for cancer immunotherapy based on their immunomodulatory function. In this review, we present a comprehensive overview of the roles and mechanisms of mast cells in diverse cancer types. Firstly, we evaluated the infiltration density and location of mast cells on tumor progression. Secondly, mast cells are activated by the TME and subsequently release a range of inflammatory mediators, cytokines, chemokines, and lipid products that modulate their pro-or anti-tumor functions. Thirdly, activated mast cells engage in intercellular communication with other immune or stromal cells to modulate the immune status or promote tumor development. Finally, we deliberated on the clinical significance of targeting mast cells as a therapeutic approach to restrict tumor initiation and progression. Overall, our review aims to provide insights for future research on the role of mast cells in tumors and their potential as therapeutic targets for cancer treatment.

Expression and functional analysis of fam76b in zebrafish.

FAM76B is nuclear speckle-localized protein with a molecular weight of 39 kDa. The amino sequence of FAM76B protein is highly conserved among species, suggesting that FAM76B has important biological functions. However, the biological function of FAM76B is currently still unclear. To explore the biological function of FAM76B, we firstly used zebrafish as the experimental model to study the distribution and expression level of Fam76b. The results indicated that fam76b is highly expressed in hematopoiesis and immune systems of zebrafish by real-time quantitative PCR, in situ hybridization and Tg(fam76b: eGFP) transgenic zebrafish. Then, the fam76b gene was knocked out by CRISPR/Cas9 in zebrafish and fam76b rescue in fam76b-/- zebrafish was performed using the TOL2 transposable system. fam76b gene knockout zebrafish exhibit reduced thymus, excessive inflammatory response, and increased mortality. FAM76B was further found to be involved in regulating the development of hematopoiesis and immune system, and participate in the process of inflammatory response. Our findings in the study lay the groundwork for elucidating the function of the new molecule Fam76b and provide new insights into the development of zebrafish hematopoietic and immune system.

FAM76B regulates NF-κB-mediated inflammatory pathway by influencing the translocation of hnRNPA2B1.

FAM76B has been reported to be a nuclear speckle-localized protein with unknown function. In this study, FAM76B was first demonstrated to inhibit the NF-κB-mediated inflammatory pathway by affecting the translocation of hnRNPA2B1 in vitro. We further showed that FAM76B suppressed inflammation in vivo using a traumatic brain injury (TBI) mouse model. Lastly, FAM76B was shown to interact with hnRNPA2B1 in human tissues taken from patients with acute, organizing, and chronic TBI, and with different neurodegenerative diseases. The results suggested that FAM76B mediated neuroinflammation via influencing the translocation of hnRNPA2B1 in vivo during TBI repair and neurodegenerative diseases. In summary, we for the first time demonstrated the role of FAM76B in regulating inflammation and further showed that FAM76B could regulate the NF-κB-mediated inflammatory pathway by affecting hnRNPA2B1 translocation, which provides new information for studying the mechanism of inflammation regulation.

Comprehensive proteomic and phosphoproteomic reveal that Microcystin-LR contributed to the malignant progression of gastric cancer by estrogenic potency.

The widespread cyanotoxins in drinking water pose a threat to public health induced by Microcystins (MCs). MC-LR, a predominant toxic form of MCs, has been found to play critical roles in cancer progression. The role of MC-LR in hepatocarcinogenesis has attracted extensive attention. However, as a critical digestive organ, the precise mechanism of MC-LR-induced gastric cancer is still unclear. We found that 100 nM MC-LR promoted the proliferation, migration, invasion, and anti-apoptosis of SGC-7901 cells. Quantitative proteome and phosphoproteome analysis identified differential expression patterns and aberrant pathways of SGC-7901 cells exposed to MC-LR. The results indicated that 48,109 unique peptides from 6320 proteins and 1375 phosphoproteins with 3473 phosphorylation sites were detected after 24 h treatment of MC-LR. Proteome and phosphoproteome conjoint analysis indicated estrogen signaling pathway might play an essential step in MC-LR-treated molecular events. The mechanism underlying these changes may involve MC-LR excessively activating the estrogen signaling pathway by reducing Hsp90 phosphorylation, which results in nucleus translocation of activated ERα and Krt16 overexpression in gastric cells. In general, our results indicate multiple crucial signals triggered by MC-LR, among which MC-LR may promote the development of gastric cancer by exerting estrogenic potency.

Rutaecarpin reduces lipids by DGKθ-dependent activation of PPARα.

OBJECTIVE: Lipid metabolic disorders pose a serious threat to human health, and currently no good treatments exist. In earlier studies by the authors, HepG2 cells with diacylglycerol kinase theta (DGKθ) knockout were found to cause significant lipid accumulation, suggesting that DGKθ may be a potential target for treating lipid metabolic disorders. METHODS: A high-throughput screening of natural products targeting the potential signaling pathway of lipid metabolism was carried out in the DGKθ-T2A-luciferase knock-in HepG2 cell. RNA-sequencing and bioinformatic approaches were used to analyze the potential pathway by which rutaecarpin decreases lipids. Western blot and quantitative polymerase chain reaction were performed to investigate the mechanisms of rutaecarpin's reduction in lipid levels. RESULTS: Rutaecarpin was found to significantly enhance DGKθ expression, and the potential mechanisms by which rutaecarpin accelerates lipid metabolism by targeting DGKθ was explored in vitro and in vivo. The results indicated that rutaecarpin could markedly reduce lipid accumulation in oleic acid-induced HepG2 cells and in high-fat diet-induced obese C57BL/6J mice by targeting the hepatocyte nuclear factor 1-beta (HNF1B)-DGKθ-peroxisome proliferator-activated receptor alpha (PPARα)-apolipoprotein C3 (APOC3) pathway. CONCLUSION: Rutaecarpin is effective in reducing lipid accumulation, and the development of a high-throughput screening platform based on a reporter knock-in cell line may facilitate the discovery of effective drugs for lipid metabolic disorders based on the DGKθ target.

Progranulin deficiency promotes persistent neuroinflammation and causes regional pathology in the hippocampus following traumatic brain injury.

Traumatic brain injury (TBI) can be progressive and can lead to the development of a long-term complication termed chronic traumatic encephalopathy. The mechanisms underlying the progressive changes are still unknown; however, studies have suggested that microglia-mediated neuroinflammation in response to TBI may play a fundamental role. This study aimed to determine whether progranulin (PGRN), a major modulator of microglial activity, plays a role in the progressive damage following TBI. PGRN-deficient and wild-type mice were subjected to controlled cortical impact and were observed neuropathologically after 3 days, 7 days, and 5 months. Compared to sham and wild-type mice, the PGRN-deficient mice showed overall stronger microgliosis and astrocytosis. The astrocytosis involved broader areas than the microgliosis and was more prominent in the basal ganglia, hippocampus, and internal capsule in PGRN-deficient mice. Ongoing neuronal death was uniquely observed in the hippocampal CA3 region of PGRN-deficient mice at 5 months after TBI, accompanying the regional chronic microgliosis and astrocytosis involving the CA3 commissural pathway. In addition, there was M1 microglial polarization in the pericontusional area with activated TLR4/MyD88/NF-κB signaling; however, the hippocampus showed only mild M1 polarization 7 days after TBI. Lastly, Morris water maze tests showed PGRN-deficient mice had poorer spatial learning and memory 5 months after TBI than wild-type or sham mice. The data indicated the PGRN deficiency caused TBI progression by promoting persistent microgliosis with microglial polarization and astrocytosis, as well as regional pathology in the hippocampus. The study suggests that PGRN should be evaluated as a potential therapy for TBI.

GSH-Responsive and Hypoxia-Activated Multifunctional Nanoparticles for Synergetically Enhanced Tumor Therapy.

The integration of reactive oxygen species (ROS)-based chemodynamic therapy (CDT) and photodynamic therapy (PDT) has attracted enormous attention for synergistic antitumor therapies. However, the strategy is severely hampered by tumor hypoxia and overproduced antioxidant glutathione (GSH) in the tumor microenvironment. Inspired by the concept of metal coordination-based nanomedicines, we proposed an effective strategy for synergistic cancer treatment in response to the special tumor microenvironmental properties. Herein, we present novel metal-coordinated multifunctional nanoparticles (NPs) by the Cu2+-triggered assembly of photosensitizer indocyanine green (ICG) and hypoxia-activated anticancer prodrug tirapazamine (TPZ) (Cu-ICG/TPZ NPs). After accumulating within tumor sites via the enhanced permeability and retention (EPR) effect, the Cu-ICG/TPZ NPs were capable of triggering a cascade of combinational therapeutic reactions, including hyperthermia, GSH elimination, and Cu+-mediated •OH generation and the subsequent hypoxia-triggered chemotherapeutic effect of TPZ, thus achieving synergistic tumor therapy. Both in vitro and in vivo evaluations suggested that the multifunctional Cu-ICG/TPZ NPs could realize satisfactory therapeutic efficacy with excellent biosafety. These results thus suggested the great potential of Cu-ICG/TPZ NPs to serve as a metallodrug nanoagent for synergetically enhanced tumor treatment.

Esophageal Cancer-Derived Extracellular Vesicle miR-21-5p Contributes to EMT of ESCC Cells by Disorganizing Macrophage Polarization.

The disorganized polarization of tumor-associated macrophages (TAMs) exerts a critical effect on tumor progression. MicroRNAs (miRNAs) in extracellular vesicles (EVs) secreted from cancer cells may contribute to this process. However, the relationship between TAMs and EVs-miRNAs-mediated regulation in esophageal squamous cell carcinoma (ESCC) remains unclear. In the present study, immunoaffinity magnetic beads combined with antiepithelial cell adhesion molecules (EpCAM) were used to isolate and identify EVs-miR-21-5p from the plasma of ESCC patients. An in vitro coculture system was designed to evaluate the effect of esophageal cancer cells with miR-21-5p overexpression on macrophage polarization. We found that phorbol myristate acetate-induced THP-1 macrophages took up EVs-miR-21-5p from EC109 or EC9706 cells and were transformed into M2 macrophages. This, in turn, contributed to the excessive migration and invasion of esophageal cancer cells. The mechanism underlying these changes may involve activation of M2 macrophages by upregulated ESCC-derived EVs-miR-21-5p through the PTEN/AKT/STAT6 pathway. This may result in esophageal cancer cell epithelial-mesenchymal transition (EMT) via TGF-ß/Smad2 signaling. Our results indicate positive feedback between M2 macrophage polarization and EMT of esophageal cancer cells in the tumor microenvironment via shuttling of miR-21-5p in tumor-derived EVs.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying IL-24/endostatin enhances glioma therapy.

Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.

miR-34a-5p Attenuates EMT through targeting SMAD4 in silica-induced pulmonary fibrosis.

Silicosis is an incurable occupational disease, and its pathological feature is diffuse pulmonary fibrosis. Pulmonary epithelial-mesenchymal transition (EMT) is one of the important events in the pathogenesis of silicosis. Previous studies found that abnormal expression of various microRNAs (miRNAs) involved in the development of lung fibrosis. However, their roles in silicosis have not been elucidated. To research the biological effects of miR-34a in EMT process in silica-induced lung fibrosis, we established the silicosis model in mouse and miR-34a intervention in a cell model of TGF-ß1 stimulated lung epithelial cells (A549). The results showed that miR-34a expression was down-regulated in the fibrotic lung tissue after silica treatment, and it was similarly expressed in A549 cells stimulated by TGF-ß1. Meanwhile, silica-induced EMT process can increase expression of two mesenchymal markers, α-SMA and vimentin. Furthermore, overexpression miR-34a markedly inhibited EMT stimulated by TGF-ß1. Mechanistically, SMAD4 was identified as the target of miR-34a. SMAD4 levels decreased in mRNA and protein levels in A549 cells upon miR-34a overexpression. In addition, the knockdown of SMAD4 blocked the EMT process. Taken together, miR-34a regulated EMT, which might be partially realized by targeting SMAD4. Our data might provide new insight into treatment targets for silica-induced pulmonary fibrosis.

Transcriptome analysis reveals a protective role of liver X receptor alpha against silica particle-induced experimental silicosis.

Silicosis, a severe and irreversible form of pulmonary fibrosis (PF) caused by long-term exposure to dust particles in production environments, is the biggest occupational health concern in China and most low-income countries. The transdifferentiation of pulmonary fibroblasts is the terminal event in silicosis, and specific transcription factors (TFs) play a crucial role in this condition. However, the relationship between TF-mediated regulation and silicosis remains unknown. We performed a transcriptomic analysis to elucidate this relationship, and our results revealed that two TFs, EGR2 and BHLHE40, were upregulated and five, i.e., TBX2, NR1H3 (LXRα), NR2F1, PPARG (PPARγ), and EPAS1, were downregulated in activated fibroblasts. Notably, PPARγ and LXRα expression was also decreased in an experimental mouse model of silicosis. The mechanism underlying these changes may involve TGF-ß1 secretion from silica-exposed alveolar macrophages, causing PPARγ and LXRα downregulation, which in turn would result in aberrant α-SMA transcription. Our results suggest that LXRα is a potential target for the prevention of silicosis and PF.

The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.

Regulation of Autophagy Orchestrates Pyroptotic Cell Death in Molybdenum Disulfide Quantum Dot-Induced Microglial Toxicity.

Molybdenum disulfide quantum dots (MoS2 QDs) represent an emerging class of two-dimensional (2D) atomically layered transition metal dichalcogenide nanostructures with few nanometers in lateral size, which show attractive potential as versatile platforms for theranostic applications in various neurological disorders. However, the potential impacts of MoS2 QDs on microglia remain unclear. In this report, we showed that exposure of microglia to MoS2 QDs triggered NLRP3 inflammasome activation as revealed by the cleavage of the inactive precursor of caspase-1 to its active form and the increased release of downstream pro-inflammatory cytokines, resulting in microglia cell death that occurred through caspase-1-dependent pyroptosis. We also found that MoS2 QDs activated autophagy, and suppression of autophagy by specific inhibitors potentiated MoS2 QD-induced pyroptosis. Additionally, MoS2 QDs stimulated mitochondria-derived reactive oxygen species (mtROS) generation in BV-2 cells. However, ROS scavengers could diminish the MoS2 QD-mediated NLRP3 inflammasome activation and pyroptotic cell death in microglia. Overall, our findings identified pyroptosis as a cellular response to MoS2 QD exposure in microglial cells, affording novel insights into the neurotoxicity of MoS2 QDs and facilitating the rational design and application of functional MoS2 QDs in neuroscience.

Molybdenum Disulfide Quantum Dots Attenuates Endothelial-to-Mesenchymal Transition by Activating TFEB-Mediated Lysosomal Biogenesis.

A defective lysosome-autophagy degradation pathway contributes to a variety of endothelial-to-mesenchymal transition (EndMT)-related cardiovascular diseases. Molybdenum disulfide quantum dots (MoS2 QDs) are nanoscale sizes in the planar dimensions and atomic structures of transition metal dichalcogenides (TMDs) materials with excellent physicochemical and biological properties, making them ideal for various biomedical applications. In this study, water-soluble MoS2 QDs with an average diameter of about 3.4 nm were synthesized by using a sulfuric acid-assisted ultrasonic method. The as-prepared MoS2 QDs exhibited low cytotoxicity of less than 100 µg/mL in both human umbilical vein endothelial cells and human coronary artery endothelial cells and showed novel biological properties to prevent EndMT and promote angiogenesis in vitro. We found that MoS2 QDs treatment-induced transcription factor (TFEB) mediated lysosomal biogenesis, which could cause autophagy activation. Importantly, using in vitro transforming growth factor (TGF)-ß-induced EndMT model, we demonstrated that the cardiovascular protective effect of MoS2 QDs against EndMT acted through triggering TFEB nucleus translocation and restoring an impairment of autophagic flux, whereas genetic suppression of TFEB impaired the protective action of MoS2 QDs against EndMT. Taken together, these results gain novel insights into the mechanisms by which MoS2 QDs regulate EndMT and facilitate the development of MoS2-based nanoagents for the treatment of EndMT-related cardiovascular diseases.

[Construction and application of PGRN and Rev-erbß double genes knockout HEK293 cell lines].

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbß, the PGRN gene in HEK293 (Rev-erbß-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbß and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbß-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbß on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbß enhanced the regulation of Rev-erbß on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbß-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbß on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbß in transcriptional regulation remains to be further studied.