Protein Kinase Research Articles

Endoplasmic Reticulum Stress Nano-Orchestrators for Precisely Regulated Immunogenic Cell Death as Potent Cancer Vaccines.

Dying tumor cells regulated by immunogenic cell death (ICD) inducers are promising candidates for cancer vaccine development because of their comprehensive antigen spectrum. However, their limited immunogenicity and potential tumorigenicity hinder clinical translation. To address these challenges, a nano-orchestrator is developed that targets the endoplasmic reticulum (ER) stress, a critical pre-ICD event, to optimize the "precise dose" of ER stress. Using a clinical-range irradiation fluence (50‒200 J cm-2) with an 808 nm laser, the release of damage associated molecular patterns (DAMPs) and antigens are precisely regulated. A fluence of 150 J cm-2 (2 W cm-2 for 75 s) increases dendritic cell maturation and antitumor T cell proliferation, providing valuable clinical insights. The ER stress nano-orchestrator enhances both adjuvanticity and antigenicity via the protein kinase R-like endoplasmic reticulum kinase (PERK)-C/EBP homologousprotein (CHOP) pathway to regulate ICD-induced DAMPs and promote tumor cell apoptosis. These optimized ER stress phototherapeutic dying tumor cells can serve as prophylactic vaccines, achieving a remarkable 100% success rate against tumor rechallenge in vivo. Additionally, the nano-orchestrator shows the potential to develop in situ therapeutic tumor vaccines when combined with anti-PD-L1 treatment, providing important insights into enhancing the efficacy of immune checkpoint regulators by modulating endogenous immune responses.

Combination therapy of systemic and local metformin improves imiquimod-induced psoriasis-like lesions with type 2 diabetes: the role of AMPK/KGF/STAT3 axis.

Insulin resistance and a disturbed lipid profile are common associations with type 2 diabetes mellitus (T2DM) and different skin diseases, particularly psoriasis (PsO). We investigated potential therapeutic mechanisms of metformin in a murine animal model of psoriasiform lesions in T2DM. Forty-two rats were randomly divided into control, PsO, and type II DM (T2DM) groups. After confirmation of DM, the type II diabetic rats were allocated into T2DM+ PsO, T2DM+ PsO+ systemic metformin (S. met), T2DM+ PsO+ topical metformin (T. met)), and T2DM+ PsO + combined metformin (C. met). PsO was induced by topical imiquimod. Systemic administration of the cornerstone antidiabetic drug, metformin, was able to improve insulin resistance and lipid profile. At molecular levels, both topical and systemic metformin significantly increased AMP-activated protein kinase (AMPK), and lowered keratinocyte growth factor (KGF) / "Signal transducer and activator of transcription" (STAT)3 protein levels, and the IL-17RA and IL-17RC gene expression. Although its glucose-controlling effect was not optimum, T.met gel served anti-psoriatic and anti-inflammatory effects.

Deficiencies in corin and atrial natriuretic peptide-mediated signaling impair endochondral ossification in bone development

Corin is a protease that activates atrial natriuretic peptide (ANP), a hormone in cardiovascular homeostasis. Structurally, ANP is similar to C-type natriuretic peptide (CNP) crucial in bone development. Here, we examine the role of corin and ANP in chondrocyte differentiation and bone formation. We show that in Corin and Nppa (encoding ANP) knockout (KO) mice, chondrocyte differentiation is impaired, resulting in shortened limb long bones. In adult mice, Corin and Nppa deficiency impairs bone density and microarchitecture. Molecular studies in cartilages from newborn Corin and Nppa KO mice and in cultured chondrocytes indicate that corin and ANP act in chondrocytes via cGMP-dependent protein kinase G signaling to inhibit mitogen-activated protein kinase phosphorylation and stimulate glycogen synthase kinase-3β phosphorylation and β-catenin upregulation. These results indicate that corin and ANP signaling regulates chondrocyte differentiation in bone development and homeostasis, suggesting that enhancing ANP signaling may improve bone quality in patients with osteoporosis.

Application of Network Pharmacology, Molecular Docking, and In Vitro Experimental Evaluation to Decipher the Anti-Inflammatory Mechanisms of Cirsium japonicum

Cirsium japonicum, a traditional herb, exhibits significant anti-inflammatory activity. However, the main components and potential mechanisms of C. japonicum remain unclear. The aim of this study is to investigate the anti-inflammatory mechanism of Cirsium japonicum through network pharmacology and cellular experiments. The effective components of and potential targets for the anti-inflammatory activity of C. japonicum were identified using a traditional Chinese medicine systematic pharmacology database, the TCMSP analysis platform, and the GeneCards database. The drug–component–target–disease network diagram was constructed using Cytoscape 3.8.0 software, while the protein interaction network diagram was created using the STRING database and Cytoscape 3.8.0 software. Gene ontology (GO) enrichment and KEGG pathway enrichment analysis were carried out using the DAVID database. Molecular docking between key targets and active components was constructed with AutoDock 4.2.6 software to determine the best binding target. The results revealed that 14 active components of C. japonicum targeted 171 anti-inflammatory proteins. GO function enrichment analysis yielded 173 items, while KEGG pathway enrichment analysis identified 48 signaling pathways related to inflammation regulation. Molecular docking showed a strong affinity of sitosterol, stigmasterol, and other components with key targets such as peroxisome proliferator-activated receptor α recombinant protein (PPARA) and cyclooxygenase-2 (PTGS2). Vanillin, one active ingredient of C. japonicum, inhibited the release of lipopolysaccharide (LPS)-induced inflammatory factors in RAW264.7 cells. These findings suggest that C. japonicum may exert its anti-inflammatory effects by modulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signal pathway (PI3K-Akt) and apoptin signal pathway, highlighting the multi-component, multi-target, and multi-channel molecular mechanism underlying its anti-inflammatory properties. Finally, the anti-inflammatory effect of vanillin, an effective component of C. japonicum, was verified by cell experiments. This study provides a new understanding of the pharmacological mechanisms of C. japonicum in the treatment of inflammatory conditions.

Identification of HSPG2 as a bladder pro-tumor protein through NID1/AKT signaling

PurposeHeparan sulfate proteoglycans (HSPGs) are complex molecules found on the cell membrane and within the extracellular matrix, increasingly recognized for their role in tumor progression. This study aimed to investigate the involvement of Heparan sulfate proteoglycan 2 (HSPG2) in the progression of bladder cancer.MethodsWe identified HSPG2 as a promoter of bladder tumor progression using single-cell RNA sequencing and transcriptome analysis of sequencing data from seven patient samples obtained from the Gene Expression Omnibus (GEO) database (GSE135337). Transcript profiles of 28 normal tissues and 407 bladder tumor tissues were analyzed for HSPG2 expression and survival outcomes using the Sanger tools and cBioPortal databases. HSPG2-overexpressing T24 and Biu-87 cell lines were generated, and cell proliferation and migration were assessed using CCK-8 and Transwell assays. Western blotting and immunostaining were performed to evaluate the activation of Nidogen-1 (NID1)/protein kinase B (AKT) signaling. Mouse models with patient-derived tumor organoids (HSPG2high and HSPG2low) were established to assess anticancer effects.ResultsOur results demonstrated a marked upregulation of HSPG2 in malignant bladder tumors, which correlated significantly with poor patient prognosis. HSPG2 overexpression consistently enhanced bladder tumor cell proliferation and conferred chemotherapy resistance, as shown in both in vitro and in vivo experiments. Mechanistically, HSPG2 upregulated NID1 expression, leading to the activation of the AKT pro-survival signaling pathway and promoting sustained tumor growth in bladder cancer.ConclusionThis study highlights the critical pro-tumor role of HSPG2/NID1/AKT signaling in bladder cancer and suggests its potential as a therapeutic target in clinical treatment.

Alleviating vascular calcification with Bushen Huoxue formula in rats with chronic kidney disease by inhibiting the PTEN/PI3K/AKT signaling pathway through exosomal microRNA-32.

Vascular calcification (VC) significantly raises cardiovascular mortality in chronic kidney disease (CKD) patients. VC is characterized by the phenotypic transformation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells, mediated by exosomes derived from calcified VSMCs and the exosomal microRNAs (miRNA) which may trigger some signals to recipient VSMCs. Bushen Huoxue (BSHX) formula has demonstrated its clinical efficacy in CKD and its protective role in CKD-VC rats has also been observed. However, little is known about its underlying mechanism. To establish a VC model, aortic VSMCs from rats were induced to osteogenic differentiation by high-level phosphate (HP) in vitro. The expression of exosome and calcification makers were analyzed by western blot, including CD9, CD63, α-SMA, BMP-2, and Runx2, respectively. Differential expression of exosomal miRNAs in normal and HP-induced VSMCs were identified by using whole miRNA microarray technology. GO and KEGG analyses were performed to determine the significant enrichment of functions and signaling pathways in the target genes. In vivo, the CKD-VC rat model was established by administering adenine gavage combined with a high phosphorus diet. The rats were divided into normal control, model, low-dose BSHX, medium-dose BSHX, high-dose BSHX groups, and sevelamer groups. The blood biochemical parameters were measured. Renal histopathology and aortic calcification were observed. Western blot detected the levels of the calcification markers. Quantitative real-time PCR (qPCR) assay detected exosomal microRNA-32 (miR-32) mRNA expression in the aorta, the most differentially expressed exosomal miRNA previously identified. Phosphatase and tensin homolog located on chromosome ten (PTEN)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway components were also tested by western blot. Exosomal miRNA-32 and PI3K/AKT signaling pathways were highly differentially expressed between normal and HP-induced VSMCs. In vivo, BSHX improved blood biochemical parameters, renal histopathology, and aortic calcification in CKD-VC rats. BSHX increased the expression level of α-SMA and decreased the level of BMP-2 and Runx2. BSHX also lowered the expression level of exosomal miR-32 mRNA, enhanced PTEN expression, therefore, reduced p-PI3K and p-AKT levels in the aorta. BSHX alleviated VC in CKD rats by downregulating exosomal miR-32 expression in the aorta, thereby promoting PTEN expression and inhibiting the PI3K/AKT signaling pathway.

Integration of metabolomics and transcriptomics reveals the toxicological mechanism of deltamethrin exposure in Chinese mitten crab Eriocheir sinensis

This study investigated the toxicological mechanism of deltamethrin on Chinese mitten crab Eriocheir sinensis juveniles in fresh water. We first conducted an acute toxicity test, followed by laboratory methods to detect changes in immune-related indices in terms of antioxidant enzyme markers, lipid metabolism-related genes, and autophagy-related and apoptosis genes. The acute toxicity (96-h LC50) of deltamethrin to E. sinensis was 7.195 μg/L. After 48 h of exposure, serum showed elevated immune-related indices (P < 0.05) for alkaline phosphatase (AKP), acid phosphatase (ACP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), complement components C3 and C4, and the key pro-inflammatory cytokines interleukin-6, interleukin-1β, and tumor necrosis factor alpha (TNF-α). In hepatopancreas at 48 h, indicators related to the antioxidant system, namely superoxide dismutase (SOD) and glutathione (GSH), were significantly elevated, whereas nitric oxide and total antioxidant capacity (T-AOC) were decreased (P < 0.05). In contrast, lipid metabolism indices for triglyceride (TG), total cholesterol (TC), and malondialdehyde (MDA) were increased (P < 0.05). Transcriptomics and metabolomics revealed that exposure to deltamethrin disrupted the lipid metabolic process in the hepatopancreas mainly by altering fatty acid synthesis, amino acid metabolism, immune signaling, and autophagy activation, while the exposure increased the content of phospholipids and cholesterol but decreased the levels of amino acids and palmitoleic acid. Quantitative genetics revealed significantly aberrantly expressed (P < 0.05) lipid metabolism-related genes, including acc1, fasn, scd1, and pnpla2, all key genes involved in lipid accumulation. Deltamethrin exposure also significantly altered (P < 0.05) gene expression levels for Toll-like receptor (tlr), myeloid differentiation factor 88 (myd88), crustin1, anti-lipopolysaccharide factor isoform 3 (alf3), tumor necrosis factor alpha (tnf-α), and NF-κB transcription factor relish. Furthermore, deltamethrin activated the toll-like receptor/major myeloid differentiation response gene 88/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR/MyD88/NF-kB) signaling pathway, which activates a nonspecific immune response in E. sinensis. Additionally, carnitine palmitoyltransferase 1 A (cpt1a), cytochrome c (cyt-c), adenosine 5′-monophosphate (amp)-activated protein kinase (ampk), the autophagosomal protein microtubule-associated protein 1 light chain 3c (lc3c), and the autophagy-related proteins beclin1, atg5, atg12 were significantly induced (P < 0.05) in the adenosine monophosphate-activated protein kinase/rapamycin (AMPK/mTOR) signaling pathway. These changes resulted in excess free radicals, causing oxidative stress in the mitochondrial membrane, promoting mitochondrial autophagy. The results confirm that deltamethrin exposure can induce hepatopancreatic injury by promoting mitochondrial autophagy, activating an immune response, and inhibiting lipid metabolism. Overall, this study provides multi-level information to reveal the toxic effects of deltamethrin on E. sinensis.

DNA-PK inhibition shows differential radiosensitization in orthotopic GBM PDX models based on DDR pathway deficits.

Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi's) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi's is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study we used GBM patient derived xenografts (PDXs) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300nM and above, with significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knock-down of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. While the mechanism underpinning this discordant effect in vitro vs. in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wildtype GBM lines significantly delayed growth and decreased NHEJ efficiency (but not Homologous Recombination), after peposertib exposure.

"Osteo-Organogenesis Niche" Hyaluronic Acid Engineered Materials Directing Re-Osteo-Organogenesis via Manipulating Macrophage CD44-MAPK/ERK-ETV1/5-MRC1 Axis.

The strategy of re-organogenesis provides an optimal framework for restoring complex organ structures and functions in adult damage. While the focus has often been on restoring organogenesis stem cells, there is limited investigations of reverting the environmental niche to support this approach. The guiding principle of "Nature selects the fittest to survive" drives the intricate dynamic changes in cellular events within the niche environment, especially through immune surveillance. The extracellular matrix (ECM) serves as the "self-associated molecular patterns" of the niche, containing extensive data on cell-niche reaction data and acting as the active tuner of immune surveillance. In this study, hyaluronic acid (HA) is identified as a unique component of the ECM in cranial osteo-organogenesis. Mechanistically, HA activates the Cluster of Differentiation 44 (CD44)-Mitogen-Activated Protein Kinase (MAPK)/Extracellular Signal-Regulated Kinase (ERK)-Ets Variant 1/5 (ETV1/5)- Mannose Receptor C-Type 1 (MRC1) axis in macrophages, establishing a distinct immune surveillance during osteo-organogenesis. Furthermore, HA is utilized as a novel engineered material for an "Osteo-organogenesis niche", restoring immune surveillance and synergistically regulating stem cells to achieve re-osteo-organogenesis in cranial defects of rats. Taken together, the study unveils a previously unknown strategy for leveraging re-organogenesis by utilizing "organogenesis niche" ECM engineered materials to manipulate immune surveillance, thereby comprehensively regulating stem cells and other tissue cells effectively for re-organogenesis.

Expert consensus on BRAF inhibitors in the treatment of malignant solid tumors (2024 edition)

The global aging population is leading to an increasing incidence of cancer, exacerbating the global burden of cancer. By 2040, the total number of cancer patients worldwide is projected to reach 28 million. With advancements in tumor molecular biology research and the widespread application of next-generation sequencing technology, precision treatment for cancer has made significant progress in clinical settings. Selective targeting of the Braf gene has emerged as one of the early successful cases of precision medicine for tumors. Braf gene mutations can result in unrestricted activation of downstream kinases in the mitogen-activated protein kinase (MAPK) cell signaling pathway, promoting rapid proliferation of tumor cells. BRAF inhibitors, either as monotherapy or in combination with MEK inhibitor, have been approved for various cancers, including melanoma, non-small cell lung cancer, thyroid cancer, colorectal cancer and glioma, among others. Clinical studies related to BRAF inhibitors are continuously exploring new applications. However, there is currently no consensus on the use of BRAF inhibitors in the diagnosis and treatment of multiple solid cancers in China. This article integrates clinical evidence of Braf mutations in multiple solid cancers to establish an expert consensus on the diagnosis and treatment of malignant solid cancers with Braf gene mutations. The goal is to promote and guide the standardized application of BRAF inhibitors.

Transcriptional Modulation of Plant Defense Genes by a Bipartite Begomovirus Promotes the Performance of Its Whitefly Vector

The majority of plant viruses rely on insect vectors for inter-plant transmission. Amid virus transmission, vector-borne viruses such as begomoviruses may significantly modulate host plants in various ways and, in turn, plant palatability to insect vectors. While many case studies on monopartite begomoviruses are available, bipartite begomoviruses are understudied. More importantly, detailed elucidation of the molecular mechanisms involved is limited. Here, we report the mechanisms by which an emerging bipartite begomovirus, the Sri Lankan cassava mosaic virus (SLCMV), modulates plant defenses against whitefly. SLCMV infection of tobacco (Nicotiana tabacum) plants significantly downregulated defenses against whitefly, as whitefly survival and fecundity increased significantly on virus-infected plants when compared to the controls. We then profiled SLCMV-induced transcriptomic changes in plants and identified a repertoire of differentially expressed genes (DEGs). GO enrichment analysis of DEGs demonstrated that the term defense response was significantly enriched. Functional analysis of DEGs associated with defense response revealed that four downregulated DEGs, including putative late blight resistance protein homolog R1B-17 (R1B-17), polygalacturonase inhibitor-like (PGI), serine/threonine protein kinase CDL1-like (CDL1), and Systemin B, directly contributed to plant defenses against whitefly. Taken together, our findings elucidate the role of novel plant factors involved in the modulation of plant defenses against whitefly by a bipartite begomovirus and shed new light on insect vector–virus–host plant tripartite interactions.

Stromal reprogramming overcomes resistance to RAS-MAPK inhibition to improve pancreas cancer responses to cytotoxic and immune therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is often resistant to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have both been implicated as drivers of resistance to therapy. Mitogen-activated protein kinase (MAPK) inhibition has not yet shown clinical efficacy, likely because of rapid acquisition of tumor-intrinsic resistance. However, the unique PDAC TME may also be a driver of resistance. We found that long-term focal adhesion kinase (FAK) inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in PDAC cells in mouse models and tissues from patients with PDAC. Concomitant inhibition of both FAK (with VS-4718) and rapidly accelerated fibrosarcoma and MAPK kinase (RAF-MEK) (with avutometinib) induced tumor growth inhibition and increased survival across multiple PDAC mouse models. In the TME, cancer-associated fibroblasts (CAFs) impaired the down-regulation of MYC by RAF-MEK inhibition in PDAC cells, resulting in resistance. By contrast, FAK inhibition reprogramed CAFs to suppress the production of FGF1, which can drive resistance to RAF-MEK inhibition. The addition of chemotherapy to combined FAK and RAF-MEK inhibition led to tumor regression, a decrease in liver metastasis, and improved survival in KRAS-driven PDAC mouse models. Combination of FAK and RAF-MEK inhibition alone improved antitumor immunity and priming of T cell responses in response to chemotherapy. These findings provided the rationale for an ongoing clinical trial evaluating the efficacy of avutometinib and defactinib in combination with gemcitabine and nab-paclitaxel in patients with PDAC and may suggest further paths for combined stromal and tumor-targeting therapies.

A Spatiotemporally Controlled and Mitochondria-Targeted Prodrug of Hydrogen Sulfide Enables Mild Mitochondrial Uncoupling for the Prevention of Lipid Deposition.

Mild mitochondrial uncoupling offers therapeutic benefits for various diseases like obesity by regulating cellular energy metabolism. However, effective chemical intervention tools for inducing mild mitochondria-targeted uncoupling are limited. Herein, we have developed a mitochondria-targeted H2S prodrug M1 with a unique property of on-demand photoactivated generation of H2S accompanied by self-reporting fluorescence for real-time tracking. Upon photoirradiation, M1 decomposes in mitochondria to generate H2S and a turn-on fluorescent coumarin derivative for the visualization and quantification of H2S. M1 is confirmed to induce reactive oxygen species (ROS)-dependent mild mitochondrial uncoupling, activating mitochondria-associated adenosine monophosphate-activated protein kinase (AMPK) to suppress palmitic acid (PA)-induced lipid deposition in hepatocytes. The uncoupling functions induced by M1 are strictly controlled in mitochondria, representing a fresh strategy to prevent lipid deposition and improve metabolic syndrome by increasing cellular energy expenditure.

NpCIPK6–NpSnRK1 module facilitates inter-subgeneric hybridization barriers in water lily (nymphaea) by reducing abscisic acid content

Abstract Pre-fertilization hybridization barriers are the main causes of inter-subgeneric hybridization challenges in water lily. However, the mechanism underlying low compatibility between pollen and stigma of water lily remains unclear. This study demonstrates that CBL-interacting protein kinase 6 (CIPK6) responded to the signaling exchange between incompatible pollen and stigma through interactions with SNF1-related kinase 1 (SnRK1) and promotes the accumulation of SnRK1 protein. Activated SnRK1 interacted with 9-cis-epoxycarotenoid dioxygenase 2 (NCED2) to promote its degradation, thereby inhibiting abscisic acid (ABA) synthesis. A decrease in ABA content in the stigma impaired the ABA-mediated removal of reactive oxygen species (ROS), ultimately resulting in the rejection of the incompatible pollen by the stigma. Our results highlight the essential role of the NpCIPK6–NpSnRK1–NpNCED2 module in conferring inter-subgeneric hybridization barriers in water lily by interfering with ABA synthesis and promoting ROS accumulation. This study offers valuable mechanistic insights into cellular signaling and reproductive barriers in water lily as well as across other biological contexts.

Alkaline Mineral Complex Water Attenuates Transportation-Induced Hepatic Lipid Metabolism Dysregulation by AMPKα-SREBP-1c/PPARα Pathways

Transportation, an unavoidable process in livestock farming, causes metabolic disorders in the body, which then lead to endocrine disruption, being immunocompromised, and growth suppression. Lipid metabolism dysregulation is a critical phenotype induced by transportation. The liver is a vital organ in lipid metabolism, with a role in both lipid synthesis and lipolysis. However, the specific mechanisms by which transportation affects hepatic lipid metabolism remain unclear. This study employed rats as a model to investigate the effects of transportation on hepatic lipid metabolism. Rats subjected to transportation showed altered serum lipid profiles, including decreased serum triglyceride (TG), low-density lipoprotein cholesterol (VLDL-C), and non-esterified fatty acid (NEFA) immediately after transportation (IAT) and serum total cholesterol (TC) on day 3, and increasing serum TG, TC, and low-density lipoprotein cholesterol (LDL-C) on day 10. Meanwhile, fatty droplets in the liver were also reduced at IAT and increased on days 3 and 10. Notably, transportation also affected hepatic-lipid-metabolism-related enzyme activities and signaling pathways, such as increased AMP-activated protein kinase alpha (AMPKα) phosphorylation and modulations in key proteins and genes related to lipid metabolism, decreased hepatic acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) activities at IAT, and increased carnitine palmitoyl transferase 1 alpha (CPT-1α) at IAT and ACC and CPT-1α activities on days 3 and 10. Supplementation with alkaline mineral complex water (AMC) before and after transportation mitigated the adverse effects on hepatic lipid metabolism by modulating the AMPKα-SREBP-1c/PPARα pathway, enhancing lipid synthesis, and reducing the oxidative catabolism of fatty acids. AMC inhibited the transportation-induced activation of AMPKα and restored the balance of lipid-metabolism-related enzymes and pathways. These findings highlight AMC’s potential as a therapeutic intervention to alleviate transportation-induced lipid metabolism disorders, offering significant implications for improving animal welfare and reducing economic losses in livestock farming.

Virtual Screening, Molecular Dynamics, and Mechanism Study of Homeodomain-Interacting Protein Kinase 2 Inhibitor in Renal Fibroblasts

Virtual Screening, Molecular Dynamics, and Mechanism Study of Homeodomain-Interacting Protein Kinase 2 Inhibitor in Renal Fibroblasts

Activation of platelet-derived growth factor receptors regulate connective tissue growth factor protein levels via the AKT pathway in malignant mesothelioma cells.

The incidence of malignant mesothelioma (MM), a disease linked to refractory asbestos exposure, continues to increase globally, and remains largely resistant to various treatments. Our previous studies have identified a strong correlation between connective tissue growth factor (CTGF) protein expression and MM malignancy, underscoring the importance of understanding CTGF regulation in MM cells. In this study, we demonstrate for the first time that stimulation with platelet-derived growth factor receptor (PDGFR) ligand, PDGF-BB, increases CTGF protein expression levels without affecting CTGF mRNA levels. Inhibition of PDGFR resulted in a reduction of CTGF protein expression, indicating that PDGFR activation is essential in regulating CTGF protein expression in MM cells. PDGF-BB also activated the protein kinase B (AKT) pathway, and inhibition of AKT phosphorylation abolished the PDGFR-induced CTGF protein expression, suggesting that PDGFR acts upstream of CTGF via the AKT pathway. This reinforces the role of CTGF protein as a key regulator of MM malignancy. Additionally, PDGFR activation led to the phosphorylation of mTOR and 4E-BP1, critical regulators of protein synthesis downstream of AKT, suggesting that PDGFR controls CTGF protein expression through the regulation of CTGF mRNA translation.

T lymphocyte-dependent IL-10 down-regulates a cytokine storm driven by Toxoplasma gondii GRA24.

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb-/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4+ T cells and most predominantly CD8+ T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma, the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb-/- and IL-10-/- mice. Furthermore, resistance against OMP was restored in Tcrb-/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4+ and CD8+ T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii. Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma.

OsPP2C49, a Negative Regulatory Factor in the Abscisic Acid Signaling Pathway, Positively Regulates Grain Yield in Rice

Clade A type 2C protein phosphatases (PP2Cs) are crucial components of the abscisic acid (ABA) signaling pathway. Research on clade A PP2Cs has focused more on their roles related to ABA signaling and stress responses than on the molecular mechanisms mediating their effects on plant growth and grain yield. Rice (Oryza sativa L.) is an important food crop worldwide. We previously determined that OsPP2C49, which encodes a rice clade A PP2C family member, negatively controls rice responses to drought, salt, and high-temperature stresses. In this study, we investigated the regulatory effects of OsPP2C49 on ABA responses and rice grain yield. By analyzing potential interactions with core ABA components, including pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory component of the ABA receptor (RCAR) and stress-activated protein kinases (SAPKs), we confirmed that OsPP2C49 is involved in the ABA signaling pathway. OsPP2C49 overexpression led to decreased ABA sensitivity and increased rice grain yield; the opposite phenotypes were observed in the ospp2c49 knockout mutants. Therefore, OsPP2C49 negatively regulates ABA responses, but positively modulates rice grain yield. Furthermore, we found that OsPP2C49 can interact with and dephosphorylate five OsSAPKs in vitro. Unlike OsPP2C49, these OsSAPKs positively modulate ABA responsiveness, but negatively affect rice yield. These findings indicate that OsPP2C49 may partially regulate ABA responses and rice grain production by dephosphorylating OsSAPKs. This study preliminarily explored the molecular basis of the regulatory effects of OsPP2C49 on rice plant growth and grain yield.

4-Anilinoquinazoline Derivatives as the First Potent NOD1-RIPK2 Signaling Pathway Inhibitors at the Nanomolar Range.

Inflammation is a defense mechanism that restores tissue damage and eliminates pathogens. Among the pattern recognition receptors that recognize danger or pathogenic signals, nucleotide oligomerization domains 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses, and inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 versus NOD2 pathway inhibitors at the nanomolar range based on a 4-anilinoquinazoline scaffold. We demonstrated that NOD1 inhibition occurs through the inhibition of receptor interacting protein kinase 2 (RIPK2), which is involved in its downstream signaling pathways. Compound 37 demonstrates no cytotoxicity, a selectivity for RIPK2 over epithelial and vascular endothelial growth factor receptors (EGFR/VEGFR), and a capacity to reduce pro-inflammatory cytokine IL-8 secretion. The structure of the RIPK2-compound 37 complex was resolved by crystallography. The 4-anilinoquinazoline scaffold offers novel perspectives to design NOD1-RIPK2 signaling inhibitors.