Aflatoxin B1-exposed hepatocyte-derived extracellular vesicles: Initiating hepatic stellate cell-mediated liver fibrosis through a p53-Parkin-dependent mitophagy pathway.

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

The RNA m6A writer METTL3 in tumor microenvironment: emerging roles and therapeutic implications.

The tumor microenvironment (TME) is a heterogeneous ecosystem comprising cancer cells, immune cells, stromal cells, and various non-cellular components, all of which play critical roles in controlling tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), the core component of N 6-methyladenosine (m6A) writer, is frequently associated with abnormalities in the m6A epitranscriptome in different cancer types, impacting both cancer cells and the surrounding TME. While the impact of METTL3 on cancer cells has been extensively reviewed, its roles in TME and anti-cancer immunity have not been comprehensively summarized. This review aims to systematically summarize the functions of METTL3 in TME, particularly its effects on tumor-infiltrating immune cells. We also elaborate on the underlying m6A-dependent mechanism. Additionally, we discuss ongoing endeavors towards developing METTL3 inhibitors, as well as the potential of targeting METTL3 to bolster the efficacy of immunotherapy.

Hexarelin alleviates apoptosis on ischemic acute kidney injury via MDM2/p53 pathway.

INTRODUCTION: Hexarelin exhibits significant protection against organ injury in models of ischemia/reperfusion (I/R)-induced injury (IRI). Nevertheless, the impact of Hexarelin on acute kidney injury (AKI) and its underlying mechanism remains unclear. In this study, we investigated the therapeutic potential of Hexarelin in I/R-induced AKI and elucidated its molecular mechanisms. METHODS: We assessed the protective effects of Hexarelin through both in vivo and in vitro experiments. In the I/R-induced AKI model, rats were pretreated with Hexarelin at 100 µg/kg/d for 7 days before being sacrificed 24 h post-IRI. Subsequently, kidney function, histology, and apoptosis were assessed. In vitro, hypoxia/reoxygenation (H/R)-induced HK-2 cell model was used to investigate the impact of Hexarelin on apoptosis in HK-2 cells. Then, we employed molecular docking using a pharmmapper server and autodock software to identify potential target proteins of Hexarelin. RESULTS: In this study, rats subjected to I/R developed severe kidney injury characterized by tubular necrosis, tubular dilatation, increased serum creatinine levels, and cell apoptosis. However, pretreatment with Hexarelin exhibited a protective effect by mitigating post-ischemic kidney pathological changes, improving renal function, and inhibiting apoptosis. This was achieved through the downregulation of conventional apoptosis-related genes, such as Caspase-3, Bax and Bad, and the upregulation of the anti-apoptotic protein Bcl-2. Consistent with the in vivo results, Hexarelin also reduced cell apoptosis in post-H/R HK-2 cells. Furthermore, our analysis using GSEA confirmed the essential role of the apoptosis pathway in I/R-induced AKI. Molecular docking revealed a strong binding affinity between Hexarelin and MDM2, suggesting the potential mechanism of Hexarelin's anti-apoptosis effect at least partially through its interaction with MDM2, a well-known negative regulator of apoptosis-related protein that of p53. To validate these findings, we evaluated the relative expression of MDM2 and p53 in I/R-induced AKI with or without Hexarelin pre-administration and observed a significant suppression of MDM2 and p53 by Hexarelin in both in vivo and in vitro experiments. CONCLUSION: Collectively, Hexarelin was identified as a promising medication in protecting apoptosis against I/R-induced AKI.

Protein phosphatase 2A-B55ß mediated mitochondrial p-GPX4 dephosphorylation promoted sorafenib-induced ferroptosis in hepatocellular carcinoma via regulating p53 retrograde signaling.

Rationale: As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. Methods: To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells in vitro. Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55ß subunit (PP2A-B55ß) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55ß overexpression xenograft tumors in a nude mice model in vivo. Results: In the Sora-induced ferroptosis model of HCC in vitro, decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4Ser2) revealed that mitochondrial p-GPX4Ser2 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55ß, it was found that PP2A-B55ß directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55ß reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55ß enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Conclusion: Our data uncovered that the PP2A-B55ß/p-GPX4Ser2/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4Ser2 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55ß was an upstream signal modulator responsible for mitochondrial p-GPX4Ser2 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55ß activation.

Aberrant RNA m6A modification in gastrointestinal malignancies: versatile regulators of cancer hallmarks and novel therapeutic opportunities.

Gastrointestinal (GI) cancer is one of the most common malignancies, and a leading cause of cancer-related death worldwide. However, molecular targeted therapies are still lacking, leading to poor treatment efficacies. As an important layer of epigenetic regulation, RNA N6-Methyladenosine (m6A) modification is recently linked to various biological hallmarks of cancer by orchestrating RNA metabolism, including RNA splicing, export, translation, and decay, which is partially involved in a novel biological process termed phase separation. Through these regulatory mechanisms, m6A dictates gene expression in a dynamic and reversible manner and may play oncogenic, tumor suppressive or context-dependent roles in GI tumorigenesis. Therefore, regulators and effectors of m6A, as well as their modified substrates, represent a novel class of molecular targets for cancer treatments. In this review, we comprehensively summarize recent advances in this field and highlight research findings that documented key roles of RNA m6A modification in governing hallmarks of GI cancers. From a historical perspective, milestone findings in m6A machinery are integrated with a timeline of developing m6A targeting compounds. These available chemical compounds, as well as other approaches that target core components of the RNA m6A pathway hold promises for clinical translational to treat human GI cancers. Further investigation on several outstanding issues, e.g. how oncogenic insults may disrupt m6A homeostasis, and how m6A modification impacts on the tumor microenvironment, may dissect novel mechanisms underlying human tumorigenesis and identifies next-generation anti-cancer therapeutics. In this review, we discuss advances in our understanding of m6A RNA modification since its discovery in the 1970s to the latest progress in defining its potential clinic relevance. We summarize the molecular basis and roles of m6A regulators in the hallmarks of GI cancer and discuss their context-dependent functions. Furthermore, the identification and characterization of inhibitors or activators of m6A regulators and their potential anti-cancer effects are discussed. With the rapid growth in this field there is significant potential for developing m6A targeted therapy in GI cancers.

Aflatoxin B1 exposure triggers hepatic lipotoxicity via p53 and perilipin 2 interaction-mediated mitochondria-lipid droplet contacts: An in vitro and in vivo assessment.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins widely found in food contaminants, and its target organ is the liver. It poses a major food security and public health threat worldwide. However, the lipotoxicity mechanism of AFB1 exposure-induced liver injury remains unclear and requires further elucidation. Herein, we investigated the potential hepatic lipotoxicity of AFB1 exposure using in vitro and in vivo models to assess the public health hazards of high dietary AFB1 exposure. We demonstrated that low-dose of AFB1 (1.25 µM for 48 h, about one-fifth of the IC50 in HepG2 and HepaRG cells, IC50 are 5.995 µM and 5.266 µM, respectively) exposure significantly induced hepatic lipotoxicity, including abnormal lipid droplets (LDs) growth, mitochondria-LDs contacts increase, lipophagy disruption, and lipid accumulation. Mechanistically, we showed that AFB1 exposure promoted the mitochondrial p53 (mito-p53) and LDs-associated protein perilipin 2 (PLIN2) interaction-mediated mitochondria-LDs contacts, resulting in lipid accumulation in hepatocytes. Mito-p53-targeted inhibition, knockdown of PLIN2, and rapamycin application efficiently promoted the lysosome-dependent lipophagy and alleviated the hepatic lipotoxicity and liver injury induced by AFB1 exposure. Overall, our study found that mito-p53 and PLIN2 interaction mediates three organelles-mitochondria, LDs, and lysosomal networks to regulate lipid homeostasis in AFB1 exposure-induced hepatotoxicity, revealing how this unique trio of organelles works together and provides a novel insight into the targeted intervention in inter-organelle lipid sensing and trafficking for alleviating hazardous materials-induced hepatic lipotoxicity.

Mesenchymal stem cells protect against sepsis-associated acute kidney injury by inducing Gal-9/Tim-3 to remodel immune homeostasis.

OBJECTIVE: The present study investigated the specific mechanism by which mesenchymal stem cells (MSCs) protect against sepsis-associated acute kidney injury (SA-AKI). METHODS: Male C57BL/6 mice underwent cecal ligation and puncture surgery to induce sepsis and then received either normal IgG or MSCs (1 × 106 cells, intravenously) plus Gal-9 or soluble Tim-3 3 h after surgery. RESULTS: After cecal ligation and puncture surgery, the mice injected with Gal-9 or MSCs plus Gal-9 had a higher survival rate than the mice in the IgG treatment group. Treatment with MSCs plus Gal-9 decreased serum creatinine and blood urea nitrogen levels, improved tubular function recovery, reduced IL-17 and RORγt levels and induced IL-10 and FOXP3 expression. Additionally, the Th17/Treg cell balance was altered. However, when soluble Tim-3 was used to block the Gal-9/Tim-3 pathway, the septic mice developed kidney injury and exhibited increased mortality. Treatment with MSCs plus soluble Tim-3 blunted the therapeutic effect of MSCs, inhibited the induction of Tregs, and suppressed the inhibition of differentiation into Th17 cells. CONCLUSION: Treatment with MSCs significantly reversed the Th1/Th2 balance. Thus, the Gal-9/Tim-3 pathway may be an important mechanism of MSC-mediated protection against SA-AKI.

Mesenchymal Stem Cells Attenuate Sepsis-associated Acute Kidney Injury by Changing the Balance of Th17 cells/Tregs via Gal-9/Tim-3.

OBJECTIVE: The aim of the present study was to investigate the protective effect of MSCs on CLP-induced SA-AKI and determine the mechanisms of this effect. METHODS: The expression of Gal-9 and Tim-3 was assayed by qPCR and western blot. IL-10, IL-17, RORγt, and FOXP3 were assayed by qPCR and TNFα, INFγ, IL-4, and IL-6 were assayed by ELISA in renal samples after CLP with or without MSCs treatment. The expression of Gal-9 in MSCs was knocked down in vivo using RNA interference, and si-Gal-9-MSCs were injected in SA-AKI mice. The effect of MSCs on the differentiation of lymphocytes into Th17 cells and Tregs was evaluated in vitro by FAC in coculture of MSCs and CD4+ T cells and after blockade of the Gal-9/Tim-3 pathway. RESULTS: MSCs decreased serum creatinine and urea nitrogen levels and relieved tubular injury. Additionally, MSCs significantly improved the survival rate and markedly attenuated the infiltration of neutrophils and the levels of TNF-α, IFN-γ, IL-4, and IL-6 in the kidneys of septic mice (P < 0.05). Treatment with MSCs also reduced the proportion of Th17 cells and the levels of IL-17 and RORγt (P < 0.05). In contrast, MSCs increased the proportion of Tregs and the levels of IL-10 and FOXP3 related to these cells (P < 0.05). Furthermore, we determined whether Gal-9/Tim-3 and Th17 cells/Tregs are involved in the protective effects of MSCs in an SA-AKI model. The results of Western blot and real-time PCR indicated that MSCs inhibited the expression of Tim-3 and increased the expression of gal-9 (P < 0.05). Knockdown of gal-9 in MSCs using small interfering RNA blunted the therapeutic effect of MSCs, and blockade of the Gal-9/Tim-3 pathway using α-lactose or anti-Tim-3 inhibited the induction of Tregs and suppressed the inhibition of the differentiation to Th17 cells by MSCs after coculture of MSCs with CD4+ T cells (P > 0.05). CONCLUSION: Treatment with MSCs can protect against SA-AKI. The results suggested that the relieving effect of MSCs against SA-AKI may be partially mediated by the induction of Tregs and inhibition of Th17 cells via the Gal-9/Tim-3 pathway.

Madelung's Disease: Analysis of Clinical Characteristics, Fatty Mass Distribution, Comorbidities and Treatment of 54 Patients in China.

Purpose: Madelung's disease (MD) is a rare disease characterized by the deposition of unencapsulated fat masses on the face, neck, chest, back and other areas of patients. The aim of the study was to analyze the clinical characteristics, comorbidities and treatment of MD in Chinese populations. Patients and Methods: We retrospectively reviewed the medical records of 54 patients who were diagnosed with MD at the Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital from January 2005 to February 2021 and collected the subjects' demographic information, clinical indicators, location of fat deposits, treatment, complications and prognostic data. Results: Among 54 MD patients in the study, only 1 (1.85%) was female, and the subjects had an average age of 56.65 ± 7.93 years. More than 70% of patients had a history of long-term smoking or/and alcohol abuse. In our study, type I accounted for approximately 61.11% of cases according to Donhauser's classification, and almost all patients had neck fat deposition. MD patients often have multiple comorbidities across several systems, such as the endocrine, digestive, circulatory, urinary, and neurological systems. Among these, endocrine system diseases were the most common comorbidities in our study, accounting for 81.48%. Notably, up to 20.37% of cases were complicated with cancer, especially digestive system tumors. More than 70% of the patients received surgical treatment, and nearly 40% experienced postoperative recurrence. Conclusion: Considering that MD patients often have comorbidities of multiple systems and that a small number of cases are even complicated by cancer, we recommend that clinicians comprehensively assess a patient's condition and complications, advocate that patients quit consuming alcohol and smoking as soon as possible, establish healthy dietary and living habits, and formulate individualized and comprehensive diagnosis and treatment plans.

Intracellular antibody targeting HBx suppresses invasion and metastasis in hepatitis B virus-related hepatocarcinogenesis via protein phosphatase 2A-B56γ-mediated dephosphorylation of protein kinase B.

OBJECTIVES: Hepatitis B virus X (HBx) is closely associated with HBV-related hepatocarcinogenesis via the inactivation of tumour suppressors. Protein phosphatase 2A (PP2A) regulatory subunit B56 gamma (B56γ), as a tumour suppressor, plays a critical role in regulating cellular phosphorylation signals via dephosphorylation of signalling proteins. However, the underlying mechanism that B56γ involved in regulating HBx-associated hepatocarcinogenesis phenotypes and mediating anti-HBx antibody-mediated tumour suppression remains unknown. MATERIALS AND METHODS: We used bioinformatics analysis, paired HCC patient specimens, HBx transgenic (HBx-Tg) mice, xenograft nude mice, HBV stable replication in the HepG2.2.15 cells, and anti-HBx antibody intervention to systematically evaluate the biological function of protein kinase B (AKT) dephosphorylation through B56γ in HBx-associated hepatocarcinogenesis. RESULTS: Bioinformatics analysis revealed that AKT, matrix metalloproteinase 2 (MMP2), and MMP9 were markedly upregulated, while cell migration and viral carcinogenesis pathways were activated in HBV-infected liver tissues and HBV-associated HCC tissues. Our results demonstrated that HBx-expression promotes AKT phosphorylation (p-AKTThr308/Ser473 ), mediating the migration and invasion phenotypes in vivo and in vitro. Importantly, in clinical samples, HBx and B56γ were downregulated in HBV-associated HCC tumour tissues compared with peritumor tissues. Moreover, intervention with site-directed mutagenesis (AKTT308A , AKTS473A ) of p-AKTThr308/Ser473 mimics dephosphorylation, genetics-based B56γ overexpression, and intracellular anti-HBx antibody inhibited cell growth, migration, and invasion in HBx-expressing HCC cells. CONCLUSIONS: Our results demonstrated that B56γ inhibited HBV/HBx-dependent hepatocarcinogenesis by regulating the dephosphorylation of p-AKTThr308/Ser473 in HCC cells. The intracellular anti-HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV-related HCC.

Protein phosphatase 2A-B56γ-Drp1-Rab7 signaling axis regulates mitochondria-lysosome crosstalk to sensitize the anti-cancer therapy of hepatocellular carcinoma.

Mitochondria-lysosome crosstalk is an intercellular communication platform regulating mitochondrial quality control (MQC). Activated dynamin-related protein 1 (Drp1) with phosphorylation at serine 616 (p-Drp1Ser616) plays a critical role in mitophagy-dependent cell survival and anti-cancer therapy for hepatocellular carcinoma (HCC). However, the underlying mechanisms that p-Drp1Ser616 involved in regulating mitochondria-lysosome crosstalk and mediating anti-HCC therapy remain unknown. HCC cells and mouse xenograft models were conducted to evaluate the relationship between p-Drp1Ser616 and Ras-associated protein 7 (Rab7) and the underlying mechanism by protein phosphatase 2A (PP2A)-B56γ regulating mitophagy via dephosphorylation of p-Drp1Ser616 in HCC. Herein, we found that Drp1 was frequently upregulated and was associated with poor prognosis in HCC. Mitochondrial p-Drp1Ser616 was a novel inter-organelle tethering protein localized to mitochondrion and lysosome membrane contact sites (MCSs) via interaction with Rab7 to trigger an increase in the mitochondria-lysosome crosstalk, resulting in PINK1-Parkin-dependent mitophagy and anti-apoptosis in HCC cells under the treatment of chemotherapy drugs. Moreover, we demonstrate that B56γ-mediated direct dephosphorylation of p-Drp1Ser616 inhibited mitophagy and thus increased mitochondria-dependent apoptosis. Overall, our findings demonstrated that activation of B56γ sensitizes the anti-cancer effect of HCC chemoprevention via dephosphorylated regulation of p-Drp1Ser616 in inhibiting the interaction between p-Drp1Ser616 and Rab7, which may provide a novel mechanism underlying the theranostics for targeting intervention in HCC.

Targeting Mitochondrial COX-2 Enhances Chemosensitivity via Drp1-Dependent Remodeling of Mitochondrial Dynamics in Hepatocellular Carcinoma.

Mitochondria are highly dynamic organelles and undergo constant fission and fusion, which are both essential for the maintenance of cell physiological functions. Dysregulation of dynamin-related protein 1 (Drp1)-dependent mitochondrial dynamics is associated with tumorigenesis and the chemotherapeutic response in hepatocellular carcinoma (HCC). The enzyme cyclooxygenase-2 (COX-2) is overexpressed in most cancer types and correlates with a poor prognosis. However, the roles played by the translocation of mitochondrial COX-2 (mito-COX-2) and the interaction between mito-COX-2 and Drp1 in chemotherapeutic responses remain to be elucidated in the context of HCC. Bioinformatics analysis, paired HCC patient specimens, xenograft nude mice, immunofluorescence, transmission electron microscopy, molecular docking, CRISPR/Cas9 gene editing, proximity ligation assay, cytoplasmic and mitochondrial fractions, mitochondrial immunoprecipitation assay, and flow cytometry analysis were performed to evaluate the underlying mechanism of how mito-COX-2 and p-Drp1Ser616 interaction regulates the chemotherapeutic response via mitochondrial dynamics in vitro and in vivo. We found that COX-2 and Drp1 were frequently upregulated and confer a poor prognosis in HCC. We also found that the proportion of mito-COX-2 and p-Drp1Ser616 was increased in HCC cell lines. In vitro, we demonstrated that the enhanced mitochondrial translocation of COX-2 promotes its interaction with p-Drp1Ser616 via PTEN-induced putative kinase 1 (PINK1)-mediated Drp1 phosphorylation activation. This increase was associated with higher colony formation, cell proliferation, and mitochondrial fission. These findings were confirmed by knocking down COX-2 in HCC cells using CRISPR/Cas9 technology. Furthermore, inhibition of Drp1 using pharmacologic inhibitors (Mdivi-1) or RNA interference (siDNM1L) decreased mito-COX-2/p-Drp1Ser616 interaction-mediated mitochondrial fission, and increased apoptosis in HCC cells treated with platinum drugs. Moreover, inhibiting mito-COX-2 acetylation with the natural phytochemical resveratrol resulted in reducing cell proliferation and mitochondrial fission, occurring through upregulation of mitochondrial deacetylase sirtuin 3 (SIRT3), which, in turn, increased the chemosensitivity of HCC to platinum drugs in vitro and in vivo. Our results suggest that targeting interventions to PINK1-mediated mito-COX-2/p-Drp1Ser616-dependent mitochondrial dynamics increases the chemosensitivity of HCC and might help us to understand how to use the SIRT3-modulated mito-COX-2/p-Drp1Ser616 signaling axis to develop an effective clinical intervention in hepatocarcinogenesis.

Biotoxicity Evaluation of a Cationic Star Polymer on a Predatory Ladybird and Cooperative Pest Control by Polymer-Delivered Pesticides and Ladybird.

Although employing nanocarriers for gene/drug delivery shows great potential in agricultural fields, the biotoxicity of nanocarriers is a major concern for large-scale applications. Herein, we synthesized a cationic star polymer (SPc) as a pesticide nanocarrier/adjuvant to evaluate its safety against a widely used predatory ladybird (Harmonia axyridis). The application of SPc at extremely high concentrations nearly did not influence the hatching of ladybird eggs but it led to the death of ladybird larvae at lethal concentration 50 (LC50) values of 43.96 and 19.85 mg/mL through the soaking and feeding methods, respectively. The oral feeding of SPc downregulated many membrane protein genes and lysosome genes significantly, and the cell membrane and nucleus in gut tissues were remarkably damaged by SPc application, revealing that the lethal mechanism might be SPc-mediated membrane damage. Furthermore, the oral feeding of SPc increased the relative abundance of Serratia bacteria in ladybird guts to result in bacterial infection. Coapplication of ladybird and SPc-loaded thiamethoxam/matrine achieved desired control efficacies of more than 80% against green peach aphids, revealing that the coapplication could overcome the slow-acting property of ladybirds. To our knowledge, this is the first attempt to investigate the polymer-mediated lethal mechanism toward natural enemies and explore the possibility of coapplying SPc-loaded pesticides and natural enemies for pest management.

Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling.

Glioblastoma (GBM) is a prevalent and highly lethal form of glioma, with rapid tumor progression and frequent recurrence. Excessive outgrowth of pericytes in GBM governs the ecology of the perivascular niche, but their function in mediating chemoresistance has not been fully explored. Herein, we uncovered that pericytes potentiate DNA damage repair (DDR) in GBM cells residing in the perivascular niche, which induces temozolomide (TMZ) chemoresistance. We found that increased pericyte proportion correlates with accelerated tumor recurrence and worse prognosis. Genetic depletion of pericytes in GBM xenografts enhances TMZ-induced cytotoxicity and prolongs survival of tumor-bearing mice. Mechanistically, C-C motif chemokine ligand 5 (CCL5) secreted by pericytes activates C-C motif chemokine receptor 5 (CCR5) on GBM cells to enable DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-mediated DDR upon TMZ treatment. Disrupting CCL5-CCR5 paracrine signaling through the brain-penetrable CCR5 antagonist maraviroc (MVC) potently inhibits pericyte-promoted DDR and effectively improves the chemotherapeutic efficacy of TMZ. GBM patient-derived xenografts with high CCL5 expression benefit from combined treatment with TMZ and MVC. Our study reveals the role of pericytes as an extrinsic stimulator potentiating DDR signaling in GBM cells and suggests that targeting CCL5-CCR5 signaling could be an effective therapeutic strategy to improve chemotherapeutic efficacy against GBM.

Serum response factor, a novel early diagnostic biomarker of acute kidney injury.

OBJECTIVE: Studies have shown that serum response factor (SRF) is increased in chronic kidney injury, such as diabetic nephropathy, hyperuricemic nephropathy and renal cell carcinoma. The objective is to explore the early diagnostic value of SRF in acute kidney injury (AKI). METHODS: AKI-related microarray data were analyzed, and the expression and location of SRF were investigated in the early phase of AKI. RESULTS: Bioinformatics results demonstrated that SRF was dramatically elevated 2-4 h after ischemia/reperfusion (I/R) in mouse renal tissue. In I/R rats, SRF was mostly expressed and located in renal tubular epithelial cells (TECs). SRF started to increase at 1 h, peaked at 3-9 h and started to decrease at 12 h after I/R. The areas under the ROC curve of renal SRF mRNA, renal SRF protein, urinary SRF, serum SRF and serum creatinine (Scr) were 87.9%, 83.0%, 81.3%, 78.8%, 68.8%, respectively. CONCLUSION: SRF is remarkably upregulated in early (before 24 h) AKI and can replace Scr as a potential new early diagnostic biomarker of AKI.

SHARPIN stabilizes ß-catenin through a linear ubiquitination-independent manner to support gastric tumorigenesis.

BACKGROUND: Aberrant activation of Wnt/ß-catenin signaling by dysregulated post-translational protein modifications, especially ubiquitination is causally linked to cancer development and progression. Although Lys48-linked ubiquitination is known to regulate Wnt/ß-catenin signaling, it remains largely obscure how other types of ubiquitination, such as linear ubiquitination governs its signaling activity. METHODS: The expression and regulatory mechanism of linear ubiquitin chain assembly complex (LUBAC) on Wnt/ß-catenin signaling was examined by immunoprecipitation, western blot and immunohistochemical staining. The ubiquitination status of ß-catenin was detected by ubiquitination assay. The impacts of SHARPIN, a core component of LUBAC on malignant behaviors of gastric cancer cells were determined by various functional assays in vitro and in vivo. RESULTS: Unlike a canonical role in promoting linear ubiquitination, SHARPIN specifically interacts with ß-catenin to maintain its protein stability. Mechanistically, SHARPIN competes with the E3 ubiquitin ligase ß-Trcp1 for ß-catenin binding, thereby decreasing ß-catenin ubiquitination levels to abolish its proteasomal degradation. Importantly, SHARPIN is required for invasiveness and malignant growth of gastric cancer cells in vitro and in vivo, a function that is largely dependent on its binding partner ß-catenin. In line with these findings, elevated expression of SHARPIN in gastric cancer tissues is associated with disease malignancy and correlates with ß-catenin expression levels. CONCLUSIONS: Our findings reveal a novel molecular link connecting linear ubiquitination machinery and Wnt/ß-catenin signaling via SHARPIN-mediated stabilization of ß-catenin. Targeting the linear ubiquitination-independent function of SHARPIN could be exploited to inhibit the hyperactive ß-catenin signaling in a subset of human gastric cancers.

MicroRNA-101 inhibits cadmium-induced angiogenesis by targeting cyclooxygenase-2 in primary human umbilical vein endothelial cells.

Exposure to toxic metal contaminants, such as cadmium compounds (Cd2+), has been shown to induce adverse effects on various organs and tissues. In particular, blood vessels are severely impacted by Cd2+ exposure, which may lead to cardiovascular diseases (CVDs). According to previous studies, CVDs are associated with increased cyclooxygenase 2 (COX-2) levels. However, the mechanisms by which CdCl2-induced COX-2 overexpression leads to cardiovascular dysfunction remain unclear. Herein, we show that the relative gene expressions of VEGF and PTGS2 (COX-2 encoding gene) are positively correlated in CVDs patients. Moreover, we demonstrate that the in vitro administration of CdCl2 induces cytotoxicity and endoplasmic reticulum (ER) stress in primary human umbilical vein endothelial cells (HUVECs). The induction of ER stress and the overexpression of COX-2 in CdCl2-treated cells alters the protein level of vascular endothelial growth factor (VEGF), resulting in abnormal angiogenesis and increased cytotoxicity. At the pre-transcription level, the inhibition of ER stress by siGRP78 (a key mediator of ER stress) can restore normal angiogenesis in the CdCl2-exposed cells. Meanwhile, at the transcription level, the adverse effects of CdCl2 exposure may be reversed via genetic modification with siRNA (siPTGS2) or by using phytochemical inhibitors (parthenolide, PN) of COX-2. Finally, at the post-transcription level, COX-2 expression may be restricted by the binding of microRNA-101 (miR-101) to the 3'-UTR of PTGS2 mRNA. The use of mimic miR-101 (mi101) to induce the expression of miR-101 eventually leads to reduced COX-2 protein levels, relieved ER stress, and less abnormal angiogenesis and cytotoxicity of CdCl2-exposed primary HUVECs. Overall, our results suggest that CdCl2-induced abnormal angiogenesis is mediated by miR-101/COX-2/VEGF-axis-dependent ER stress, and that cardiovascular dysfunction may be controlled by manipulating COX-2 at the pre-transcription, transcription, and post-transcription levels.

Mitochondrial redox-driven mitofusin 2 S-glutathionylation promotes neuronal necroptosis via disrupting ER-mitochondria crosstalk in cadmium-induced neurotoxicity.

Reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress and mitochondrial dysfunction are known to affect the structural and functional damage in the neural system. Cadmium (Cd) is an environmental contaminant that is widely found in numerous environmental matrices and exhibits potential neurotoxic risk. However, it remains unclear how mitochondrial redox status induces, and whether Cd destabilizes, the ER-mitochondria crosstalk to have a toxic effect on the nervous system. Herein, in our present study, bioinformatics analysis revealed an important role of protein interaction and mitochondrial machinery in brain samples from Alzheimer's disease (AD) patients. Furthermore, we established a neurotoxicity model in vivo and in vitro induced by cadmium chloride (CdCl2). We demonstrated that CdCl2 exposure disrupts the balance in mitochondrial redox represented by enhanced mitochondrial ROS (mitoROS) levels, which enhance mitofusin 2 (Mfn2) S-glutathionylation and interrupt the mitochondria-associated ER membranes (MAMs) for crosstalk between the ER and mitochondria to induce neuronal necroptosis. Mechanistically, it was shown that CdCl2 exposure significantly enhances the mitochondria-associated degradation (MAD) of Mfn2 via S-glutathionylation, which inhibits Mfn2 localization to the MAMs and subsequently leads to the formation of the RIPK1-RIPK3-p-MLKL complex (a key component of the necrosome) at MAMs, to promote neuronal necroptosis. Furthermore, the glutaredoxin 1 (Grx1) catalyzed and Mfn2 overexpression restored S-glu-Mfn2, MAMs perturbation, necrosome formation, and necroptosis in neurons induced by CdCl2 exposure in vitro. Moreover, the intervention with antioxidants to reduce mitochondrial redox, such as N-acetyl-l-cysteine (NAC) and mitochondria-targeted antioxidant Mito-TEMPO, reduced the S-glutathionylation of Mfn2 involved in the antagonism of CdCl2-induced necroptosis and neurotoxicity in vivo and in vitro. Taken together, our results are the first time to demonstrate that S-glutathionylation of Mfn2 promotes neuronal necroptosis via disruption of ER-mitochondria crosstalk in CdCl2-induced neurotoxicity, providing the novel mechanistic insight into how hazardous chemical-induced adverse effects in various organs and tissues could be interpreted by intraorganellar pathways under the control of MAMs components in neurons.

A visual risk assessment tool for acute kidney injury after intracranial aneurysm clipping surgery.

OBJECTIVE: The aim of the study was to establish a predictive postoperative nomogram for acute kidney injury (AKI) after intracranial aneurysm clipping surgery, in order to early identify patients with high postoperative AKI risk. METHODS: This is a retrospective study, which included patients who underwent intracranial aneurysm clipping surgery. Multivariate logistic regression was employed to select confound factors that associated with AKI, then incorporated into the nomogram. The predictive accuracy of the model was assessed by concordance index (C-Index). RESULTS: A total of 365 patients after intracranial aneurysm clipping surgery were enrolled in the study eventually, of which 68 (18.63%) suffered postoperative AKI, and the incidence of stage 1, stage 2 and stage 3 were 92.65% (63/68), 5.88% (4/68), and 1.47% (1/68), respectively. Univariate logistic regression revealed that high density lipoprotein (HDL), prothrombin time (PT), estimated glomerular filtration rate (eGFR), size of aneurysm ≥10 mm, and aneurysm ruptured before surgery were associated with AKI after surgery, while multivariate logistic regression showed same results as the size of aneurysm ≥10 mm and aneurysm ruptured were independent AKI risk factors. In addition, the nomogram demonstrated a good accuracy in estimating intracranial aneurysm clipping associated AKI, as a C-Index and a bootstrap-corrected one of 0.772 and 0.737, respectively. Moreover, calibration plots showed consistency with the actual presence of AKI. CONCLUSION: The novel nomogram model can serve as a promising predictive tool to improve the identification of AKI among those who underwent intracranial aneurysm clipping surgery.

Suppressing endoplasmic reticulum stress-related autophagy attenuates retinal light injury.

Excessive light exposure is a principal environmental factor, which can cause damage to photoreceptors and retinal pigment epithelium (RPE) cells and may accelerate the progression of age-related macular degeneration (AMD). In this study, oxidative stress, endoplasmic reticulum (ER) stress and autophagy caused by light exposure were evaluated in vitro and in vivo. Light exposure caused severe photo-oxidative stress and ER stress in photoreceptors (661W cells) and RPE cells (ARPE-19 cells). Suppressing either oxidative stress or ER stress was protective against light damage in 661W and ARPE-19 cells and N-acetyl-L-cysteine treatment markedly inhibited the activation of ER stress caused by light exposure. Moreover, suppressing autophagy with 3-methyladenine significantly attenuated light-induced cell death. Additionally, inhibiting ER stress either by knocking down PERK signals or with GSK2606414 treatment remarkably suppressed prolonged autophagy and protected the cells against light injury. In vivo experiments verified neuroprotection via inhibiting ER stress-related autophagy in light-damaged retinas of mice. In conclusion, the above results suggest that light-induced photo-oxidative stress may trigger subsequent activation of ER stress and prolonged autophagy in photoreceptors and RPE cells. Suppressing ER stress may abrogate over-activated autophagy and protect the retina against light injury.