Regulation Of Protein Kinase Research Articles

Elucidating the molecular mechanism of angiogenic activity of sulfate glycosaminoglycan derived from fish swim bladder in human umbilical vein endothelial cells

Fish swim bladders are considered a traditional marine aquatic food that has medicinal and nutritional properties. Sulfate glycosaminoglycans (SBSG) obtained from fish swim bladders may have anti-angiogenic activity. However, the anti-angiogenic activity of SBSG and its mechanism has not been reported. Therefore, the present study investigated the effect of SBSG on angiogenesis and examined molecular pathways in human umbilical vein endothelial cells (HUVECs) using cell viability, chicken chorioallantoic membrane (CAM) assay, tube formation assay, and proteomic analyses. The results demonstrated that the SBSG significantly (P < 0.05) suppressed tube formation in the HUVECs while decreasing vascular density in CAM. Quantitative proteome analysis identified 1474 differentially expressed proteins (DEPs) involved in different molecular pathways. In the bladder cancer pathway, 10 proteins were significantly downregulated [matrix metalloproteinase-9 (MMP-9), epidermal growth factor receptor (EGFR), cyclin-dependent kinase 4 (CDK4), fibroblast growth factor receptor 3 (FGFR3), harvey rat sarcoma (H-Ras), rat sarcoma (Ras), mitogen-activated extracellular signal-regulated kinase (MEK), extracellular regulated protein kinases (ERK), receptor tyrosine-protein kinase erbB-2 (ERBB2), and E-Cadherin (E-cad)], whereas 2 proteins were significantly upregulated [thrombospondin-1 (TSP-1) and E-cad]. Notably, SBSG was effectively bound to the active sites of EGFR, MMP-9, and TSP-1, which led to reduced EGFR and MMP-9 protein expression (p < 0.05), and increased TSP-1 (P < 0.05), thereby inhibiting angiogenesis. These findings suggest that SBSG is a potential candidate for the nutraceuticals industry for angiogenesis management.

Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase

β-adrenergic signalling activates cAMP-dependent protein kinase (PKA), which regulates the activity of L-type voltage-gated calcium channels such as CaV1.2. Several PKA target sites in the C-terminal tail of CaV1.2 have been identified, and their phosphorylation has been suggested to increase currents in specific tissues or heterologous expression systems. However, augmentation of CaV1.2 currents in the heart is instead mediated by phosphorylation of Rad, a small GTPase that can inhibit CaV1.2. It is unclear how each of the proposed target sites in CaV1.2 and Rad rank towards their recognition by PKA, which could reveal a preferential phosphorylation. Here we used quantitative assays on three CaV1.2 and four Rad sites. Isothermal titration calorimetry (ITC) and enzyme kinetics show that there are two Tiers of targets, with CaV1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming Tier 1 substrates for PKA. These share a common feature with two Arginine residues at specific positions that can anchor the peptide into the substrate binding cleft of PKA. In contrast, PKA shows minimal activity for the other, Tier 2 substrates, characterized by low kcat values and undetectable binding via ITC. The existence of two Tiers suggests that PKA regulation of the CaV1.2 complex may occur in a graded fashion. We report crystal structures of the PKA catalytic subunit with and without a CaV1.2 and test the importance of several anchoring residues via mutagenesis. Different target sites utilize different anchors, highlighting the plasticity of PKAc to recognize substrates.

Dan-shen Yin attenuates myocardial fibrosis after myocardial infarction in rats: Molecular mechanism insights by integrated transcriptomics and network pharmacology analysis and experimental validation

Ethnopharmacological relevanceDan-shen Yin (DSY) originated from the “Shi Fang Ge Kuo” is a Chinese formula composed of three medicines: Salvia miltiorrhiza (Dan-shen), Santalum album L. (Tan-xiang) and Amomum villosum Lour. (Sha-ren). It has many years of clinical experience in the prevention of myocardial fibrosis (MF). However, the specific mechanism of DSY in prevent MF is not clear. Aim of the studyThis study aimed to assess the efficacy of DSY in the prevention of MF and reveal its underlying mechanism in a rat model of MF after myocardial infarction (MI) induced by ligation of the left anterior descending branch (LAD) of the coronary artery. Materials and methodsThe blood-entry components of DSY were analyzed by UHPLC-Q-TOF-MS/MS. LAD-ligated rats were used to assess the efficacy of DSY in the prevention of MF. Network pharmacology and transcriptomics analysis were used to predict possible target signaling pathways of DSY in MF. Echocardiography, immunohistochemistry and ELISA methods were used to evaluate the cardiac functions and biochemical changes of the rats. The mRNA expressions of target genes were measured by RT-qPCR. The proteins expressions, including Collagen I, Collagen III, α-smooth muscle actin (α-SMA), matrix metallopeptidase 2 (MMP 2), matrix metallopeptidase 9 (MMP 9), transforming growth factor-β (TGF-β), protein kinase B (AKT), phospho-AKT, extracellular regulated protein kinases (ERK), phospho-ERK, c-Jun N-terminal kinase (JNK), phospho-JNK, mothers against decapentaplegic protein (Smad3), and phospho-Smad3 were detected and quantified by Western Blot. ResultsUHPLC-Q-TOF-MS/MS analysis disclosed that 20 components within DSY could be absorbed into blood of rats. DSY improved myocardial injury in the myocardial tissue of LAD-ligated rats, as evidenced by the elevation of left ventricular ejection fraction and left ventricular fractional shortening, and the decrease of the serum CK-MB and LDH levels. Network pharmacology and transcriptomics predicted that DSY could interfere biological processes, such as extracellular matrix organization, focal adhesion and ECM-receptor interaction, and modulate TGF-β mediated signaling pathways, including PI3K/AKT, MAPK, and Smad3. Further study confirmed that DSY reduced MF, accompanied by reduced TGF-β, Collagen I, Collagen III, α-SMA, MMP 2 and MMP 9. Moreover, DSY repressed the phosphorylation of AKT, MAPKs and Smad3. In addition, DSY reduced inflammation and suppressed the mRNA expressions of IL-1β, IL-6, TNF-α, COX2 and iNOS in MF rats. ConclusionsOur study demonstrated that DSY prevented MF in vivo, the action of which was probably via reducing extracellular matrix organization, focal adhesion ECM-receptor interaction and inflammation by regulating TGF-β mediated PI3K/AKT, MAPK and Smad signaling pathways.

Expression Analysis of the Extensive Regulation of Mitogen-Activated Protein Kinase (MAPK) Family Genes in Buckwheat (Fagopyrum tataricum) During Organ Differentiation and Stress Response

The mitogen-activated protein kinase (MAPK) signaling cascade is a unique and relatively conserved signaling pathway in eukaryotes, transmitting extracellular signals into cells through successive phosphorylation and eliciting appropriate responses from the organism. While its mechanism in plant immune response has been partially elucidated in Arabidopsis, it has been rarely examined in Tartary buckwheat (Fagopyrum tataricum). Based on the conserved MAPK domain, we identified 16 MAPK family genes in Tartary buckwheat. The FtMAPKs have similar structures and motif compositions, indicating that this gene family is conserved yet functionally diverse. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, we observed significant expression variation in 15 genes across different organs, except for FtMAPK12. FtMAPK9 showed specific expression in vegetative organs, FtMAPK4 in reproductive organs, and FtMAPK1 and FtMAPK10 in leaves and flowers, respectively, indicating their regulatory roles in Tartary buckwheat development. Following drought and salt stress treatments, 12 and 14 FtMAPKs, respectively, showed significantly altered expression in leaves exhibiting notable biological oxidation. Among these, FtMAPK3, FtMAPK4, and FtMAPK8 demonstrated highly significant changes across both treatments. Transcriptome analysis confirmed these findings, suggesting that these three genes play pivotal roles in Tartary buckwheat’s response to abiotic stress and hold potential for molecular breeding improvements.

Super-Enhancer-Driven Syndecan-4 Regulates Intercellular Communication in Hypoxic Pulmonary Hypertension.

Unveiling pro-proliferation genes involved in crosstalk between pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs) are important to improving the therapeutic outcome of pulmonary hypertension (PH). Although growing studies have shown that super-enhancers (SEs) play a pivotal role in pathological and physiological processes, the SE-associated genes in PH and their impact on PASMC proliferation remain largely unexplored. We used serotype 5 adenovirus-associated virus to interfere with syndecan-4 and constructed an SU5416 combined with hypoxia-PH model. Chromatin immunoprecipitation sequencing analysis, chromatin immunoprecipitation quantitative polymerase chain reaction, and bioinformatics were used to confirm early growth response 1 was involved in regulating syndecan-4-associated SE in PASMCs. The effects of syndecan-4 and its underlying mechanisms were subsequently elucidated using Western blot, coimmunoprecipitation, and cell coculture assays. Herein, we identified a novel SE-associated gene, syndecan-4, in hypoxia-exposed PASMCs. Syndecan-4 was transcriptionally driven by early growth response 1 via an SE and was significantly overexpressed in hypoxic PASMCs and plasma from patients with PH. Mechanism studies revealed that syndecan-4 induces PASMC proliferation by interacting and regulating protein kinase C α ubiquitination. In addition, syndecan-4 was enriched in exosomes secreted from hypoxic PASMCs, which subsequently transported and led to pulmonary artery endothelial cell dysfunction. Syndecan-4 inhibition in hypoxia by serotype 5 adenovirus-associated virus treatment attenuated the pulmonary artery remodeling and development of PH invivo. Taken together, our results demonstrate that an SE-driven syndecan-4 modulates crosstalk of PASMCs and pulmonary artery endothelial cells and promotes vascular remodeling via the protein kinase C α and exosome pathway, thus providing potential targets for the early diagnosis and treatment of hypoxic PH.

Rosmarinic Acid Exhibits Antifungal and Antibiofilm Activities Against Candida albicans: Insights into Gene Expression and Morphological Changes.

Candida species, opportunistic pathogens that cause various infections, pose a significant threat due to their ability to form biofilms that resist antifungal treatments and immune responses. The increasing resistance of Candida spp. and the limited availability of effective treatments have prompted the research of natural compounds as alternative therapies. This study assessed the antifungal properties of RA against Candida species, focusing on its impact on C. albicans biofilms and the underlying mechanisms. The antifungal efficacy of RA was evaluated using the CLSI M27-A3 microdilution method on both fluconazole-susceptible and -resistant strains. Biofilm formation by C. albicans was assessed through a crystal violet assay, while its antibiofilm activity was analyzed using an MTT assay and field emission scanning electron microscopy (FESEM). Gene expression related to biofilm formation was studied using quantitative real-time PCR (qRT-PCR), and statistical analysis was performed with an ANOVA. Among the 28 Candida strains tested, RA exhibited minimum inhibitory concentration (MIC) values ranging from 160 to 1280 μg/mL. At a 640 μg/mL concentration, it significantly reduced the expression of genes associated with adhesion (ALS3, HWP1, and ECE1), hyphal development (UME6 and HGC1), and hyphal cAMP-dependent protein kinase regulators (CYR1, RAS1, and EFG1) in RAS1-cAMP-EFG1 pathway (p < 0.05). FESEM analysis revealed a reduction in hyphal networks and disruptions on the cell surface. Our study is the first to demonstrate the effects of RA on C. albicans adhesion, hyphae development, and biofilm formation through gene expression analysis with findings supported by FESEM. This approach distinguishes our study from previous studies on the effect of RA on Candida. However, the high MIC values of RA limit its antifungal potential. Therefore, more extensive research using innovative methods is required to increase the antifungal effect of RA.

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM.

MicroRNA-34a negatively regulates Netrin1 and mediates MEK/ERK pathway to regulate chemosensitivity of gastric cancer cells.

To explore the mechanism of action of MicroRNAs-34a (miR-34a) and Eurite growth guiding factor 1 (Netrin1) in cisplatin resistance in gastric cancer (GC), providing new clues for overcoming tumor resistance and optimizing anti-tumor therapy for GC. The Cancer Genome Atlas (TCGA), Differentially Expressed MicroRNAs (miRNAs) in human cancers (dbDEMC), and Starbase online databases were used to analyze the correlation between miR-34a and Netrin-1 and prognosis in GC, and to predict and verify the targeted binding of miR-34a to Netrin-1. The experimental methods including Cell transfection, real-time polymerase chain reaction (RT-PCR), Cell-Counting-Kit-8 (CCK8) assay, flow cytometry, wound scratch assay, transwell assay, and western blotting were used to investigate the effects of miR-34a and Netrin1 on chemotherapy resistance and biological characteristics in cisplatin-resistant GC cells (HGC27/DDP), and to analyze the molecular mechanism of cisplatin resistance. miR-34a expression was downregulated in gastric cancer clinical samples and cisplatin-resistant cells, while Netrin1 was upregulated, and was related to overall survival (OS). Upregulation of miR-34a can significantly reduce the IC50 value of cisplatin(0.65 vs 1.6 ng/mL) and Multidrug Resistance 1 (MDR-1) protein level, inhibit the proliferation activity, reduce the expression levels of proliferating cell nuclear antigen (PCNA) and ki-67 protein, and induce the increase of apoptosis rate and the enhancement of cycle arrest. Upregulation of miR-34a can also significantly reduce the expression level of Matrix metalloproteinase 9 (MMP9) protein, promote the expression of E-cadherin protein, reduce the wound healing rate and invasion number to inhibit migration and invasion ability in drug-resistant gastric cancer cells. Moreover, overexpression of Netrin1 on the basis of upregulation of miR-34a can weaken the above changes caused by upregulation of miR-34a. In addition, upregulation of miR-34a can significantly inhibit the Mitogen-activated protein kinase kinase (MEK) / Extracellular regulated protein kinases (ERK) pathway, while overexpression of Netrin1 can activate the MEK/ERK pathway, and inhibition of MEK/ERK pathway can effectively counteract the protein expression of Netrin1, and reverse changes in the expression of cisplatin IC50 and MDR-1 proteins caused by co-upregulation of miR-34a/Netrin1 in HGC27/DDP, as well as changes in proliferation, apoptosis, migration and invasion. In addition, upregulation of miR-34a can significantly inhibit the MEK/ERK pathway, while overexpression of Netrin1 can activate the MEK/ERK pathway. If the MEK/ERK pathway was inhibited, it can effectively counteract the protein overexpression of Netrin1, and reverse the changes in the expression of cisplatin IC50 and MDR-1 proteins in HGC27/DDP induced by co-upregulation of miR-34a / Netrin1, as well as changes in proliferation, apoptosis, migration and invasion. miR-34a targets and negatively regulates Netrin1 to mediate the proliferation, apoptosis, apoptosis, migration, and invasion of drug-resistant gastric cancer cells via the MEK/ERK pathway, and change the chemosensitivity in GC cells. miR-34a/Netrin1/MEK/ERK axis may serve as a novel therapeutic target for chemoresistance in GC, it is of great significance for overcoming drug resistance and developing new therapeutic strategies for GC.

Regulation of AMPK and GAPDH by Transglutaminase 2 Plays a Pivotal Role in Microvascular Leakage in Diabetic Retinas.

Diabetic retinopathy is the most common microvascular complication caused by chronic hyperglycemia and is a leading cause of blindness; however, the underlying molecular mechanism has not been clearly elucidated. Thus, we investigated whether regulation of AMPactivated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by transglutaminase 2 (TGase2) is important for hyperglycemia-induced microvascular leakage in the diabetic retina. In HRECs and diabetic mouse retinas, we found that TGase2, activated by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels, played an essential role in hyperglycemia-induced vascular leakage. ROS generation and TGsae2 activation were involved in hyperglycemia-induced AMPK dephosphorylation, which resulted in VE-cadherin disassembly and increased fluorescein isothiocyanate-dextran extravasation. Furthermore, high glucose-induced TGase2 activation suppressed GAPDH activity, determined by an on-chip activity assay, through inhibition of AMPK, which induced VE-cadherin disassembly and endothelial permeability in HRECs. Overall, our findings suggest that inhibition of AMPK and GAPDH by TGase2 plays a pivotal role in hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

Hyaluronic acid methacrylate/Pluronic F127 hydrogel enhanced with spermidine-modified mesoporous polydopamine nanoparticles for efficient synergistic periodontitis treatment

Bacterial infection, reactive oxygen species (ROS) accumulation, and persistent inflammation pose significant challenges in the treatment of periodontitis. However, the current single-modal strategy makes achieving the best treatment effect difficult. Herein, we developed a double-network hydrogel composed of Pluronic F127 (PF-127) and hyaluronic acid methacrylate (HAMA) loaded with spermidine-modified mesoporous polydopamine nanoparticles (M@S NPs). The PF-127/HAMA/M@S (PH/M@S) hydrogel was injectable and exhibited thermosensitivity and photocrosslinking capabilities, which enable it to adapt to the irregular shape of periodontal pockets. In vitro, the PH/M@S displayed multiple therapeutic effects, such as photothermal antibacterial activity, a high ROS scavenging capacity, and anti-inflammatory effects, which are beneficial for the multimodal treatment of periodontitis. The underlying anti-inflammatory mechanism of this hydrogel involves suppression of the extracellular regulated protein kinase 1/2 and nuclear factor kappa-B signalling pathways. Furthermore, in lipopolysaccharide-stimulated macrophage conditioned media, the PH/M@S effectively restored the osteogenic differentiation potential. In a rat model of periodontitis, the PH/M@S effectively reduced the bacterial load, relieved local inflammation and inhibited alveolar bone resorption. Collectively, these findings highlight the versatile functions of the PH/M@S, including photothermal antibacterial activity, ROS scavenging, and anti-inflammatory effects, indicating that this hydrogel is a promising multifunctional filling material for the treatment of periodontitis.

Integrating network pharmacology, molecular docking and non-targeted serum metabolomics to illustrate pharmacodynamic ingredients and pharmacologic mechanism of Haizao Yuhu Decoction in treating hyperthyroidism.

To explore the pharmacodynamic ingredients and pharmacologic mechanism of Haizao Yuhu Decoction (HYD) in treating hyperthyroidism via an analysis integrating network pharmacology, molecular docking, and non-targeted serum metabolomics. Therapeutic targets of hyperthyroidism were searched through multi-array analyses in the Gene Expression Omnibus (GEO) database. Hub genes were subjected to the construction of a protein-protein interaction (PPI) network, and GO and KEGG enrichment analyses. Targets of active pharmaceutical ingredients (APIs) in HYD and those of hyperthyroidism were intersected to yield hub genes, followed by validations via molecular docking and non-targeted serum metabolomics. 112 hub genes were identified by intersecting APIs of HYD and therapeutic targets of hyperthyroidism. Using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) in both negative and positive ion polarity modes, 279 compounds of HYD absorbed in the plasma were fingerprinted. Through summarizing data yielded from network pharmacology and non-targeted serum metabolomics, 214 common targets were identified from compounds of HYD absorbed in the plasma and therapeutic targets of hyperthyroidism, including PTPN11, PIK3CD, EGFR, HRAS, PIK3CA, AKT1, SRC, PIK3CB, and PIK3R1. They were mainly enriched in the biological processes of positive regulation of gene expression, positive regulation of MAPK cascade, signal transduction, protein phosphorylation, negative regulation of apoptotic process, positive regulation of protein kinase B signaling and positive regulation of MAP kinase activity; and molecular functions of identical protein binding, protein serine/threonine/tyrosine kinase activity, protein kinase activity, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding and protein binding. A total of 185 signaling pathways enriched in the 214 common targets were associated with cell proliferation and angiogenesis. HYD exerts a pharmacological effect on hyperthyroidism via inhibiting pathological angiogenesis in the thyroid and rebalancing immunity.

Will the antioxidant supplements impact the mitigation of oxidative stress in Parkinson's disease?

Parkinson's disease is a frequent neurodegenerative condition marked by both non-motor and motor symptoms. It is brought on by the selective depletion of dopamine neurons from the substantia nigra region of the midbrain. Numerous variables, including lifestyle, environment, age, smoking, and underlying medical disorders, affect the occurrence of Parkinson's disease. The free radicals created by oxidative stress have an impact on the morphology and function of neuronal cells and are a factor in many neurodegenerative disorders, including Parkinson’s and Alzheimer’s, disorder. Although the pathophysiological mechanism causing neuronal degeneration is still unknown, oxidative stress plays a critical role in the pathogenesis of idiopathic Parkinson's disease. It also has an association with numerous proteins, including α-synuclein, amyloid, DJ-1 protein and several signalling pathways like extracellular regulated protein kinases. Reactive oxygen species also contribute to complex I's mitochondrial respiratory activity. The naturally occurring antioxidants are the phenols, anthocyanins, carotenoids, flavonoids, vitamins, and lignans. These antioxidants retain the reactive oxygen species generation and plays a role in several biological effects. As a result, natural plant antioxidants may have an impact on Parkinson's disease and offer an alternative therapy that minimises oxidative stress and slows down the evolution of the disease.

The Ca2+-Regulated Protein Kinase CIPK1 Modulates Plant Response to Nitrate Deficiency in Arabidopsis.

Nitrogen is an essential macroelement for plant growth and productivity. Calcium (Ca2+) acts as a critical second messenger in numerous adaptations and developmental processes in plants. The Calcineurin B-like protein (CBL)-interacting protein kinase (CIPK) signaling pathway has been demonstrated to be involved in multiple intracellular ion homeostasis of plants in response to stresses. However, whether CIPKs are involved in nitrate deficiency stress remains largely unknown. In this study, we screened Arabidopsis thaliana T-DNA insertion mutants of the CIPK family under nitrate deficiency conditions by a reverse genetic strategy. We found that the cipk1 mutant showed a shorter primary root and had a lower fresh weight and total N content compared with wildtype (WT) plants under nitrate deficiency. The CIPK1 complementation lines completely rescued the sensitive phenotype. Additionally, CIPK1 mutation caused nitrogen-starvation marker genes to be decreased under nitrate deficiency. We further found that CIPK1 interacted with teosintebranched 1/cycloidea/proliferating cell factor 1-20 (TCP20) in a yeast two-hybrid system. Collectively, our results reveal a novel role of CIPK1 in response to nitrate deficiency in Arabidopsis.

MAPK signaling pathway enhances tolerance of Mytilus galloprovincialis to co-exposure of sulfamethoxazole and polyethylene microplastics

Microplastics (MPs) and antibiotics often coexist in complex marine environments, yet their combined detrimental effects on marine organisms remain underexplored. This study evaluated the effects of polyethylene microplastics (PE, 200 μg/L) and sulfamethoxazole (SMX, 50 μg/L), both individually and in combination, on Mytilus galloprovincialis. The exposure lasted 6 days, followed by a 6-day recovery period. Bioaccumulation, DNA damage, pollutants transport/metabolism related responses and responding alterations of mitogen-activated protein kinase (MAPK) signaling pathway were detected in gills and digestive glands. Bioaccumulation of SMX/PE in mussels occurred in a tissue-specific manner, co-exposure altered SMX contents in investigated tissues. Co-exposure did not induce extra DNA damage, elevated DNA damage was alleviated during the recovery period in all treated groups. The exposure of SMX/PE exerted different alterations in pollutants transport/metabolism related responses, characterized by multixenobiotic resistance and relative expression of key genes (cytochrome P450 monooxygenase, glutathione S-transferase, ATP-binding cassette transporters). Key molecules (p38 MAPK, c-jun N-terminal kinase, extracellular regulated protein kinase, nuclear factor-κB and tumor protein p53) in MAPK signaling pathway were activated at transcriptional and translational levels after SMX/PE and co-exposure. Co-regulation between MAPK members and pollutants transport/metabolism related factors was revealed, suggesting MAPK signaling pathway served as a regulating hub in exposed mussels to conquer SMX/PE stress. Overall, this study provides new insights on SMX/PE induced health risks in marine mussels and potential mechanism through MAPK cascades regulation.

Cytoarchitecture of Breast Cancer Cells under Diabetic Conditions: Role of Regulatory Kinases-Rho Kinase and Focal Adhesion Kinase.

Diabetes greatly reduces the survival rates in breast cancer patients due to chemoresistance and metastasis. Reorganization of the cytoskeleton is crucial to cell migration and metastasis. Regulatory cytoskeletal protein kinases such as the Rho kinase (ROCK) and focal adhesion kinase (FAK) play a key role in cell mobility and have been shown to be affected in cancer. It is hypothesized that diabetes/high-glucose conditions alter the cytoskeletal structure and, thus, the elasticity of breast cancer cells through the ROCK and FAK pathway, which can cause rapid metastasis of cancer. The aim of the study was to investigate the role of potential mediators that affect the morphology of cancer cells in diabetes, thus leading to aggressive cancer. Breast cancer cells (MDA-MB-231 and MCF-7) were treated with 5 mM glucose (low glucose) or 25 mM glucose (high glucose) in the presence of Rho kinase inhibitor (Y-27632, 10 mM) or FAK inhibitor (10 mM). Cell morphology and elasticity were monitored using atomic force microscopy (AFM), and actin staining was performed by fluorescence microscopy. For comparative study, normal mammary breast epithelial cells (MCF-10A) were used. It was observed that high-glucose treatments modified the cytoskeleton of the cells, as observed through AFM and fluorescence microscopy, and significantly reduced the elasticity of the cells. Blocking the ROCK or FAK pathway diminished the high-glucose effects. These changes were more evident in the breast cancer cells as compared to the normal cells. This study improves the knowledge on the cytoarchitecture properties of diabetic breast cancer cells and provides potential pathways that can be targeted to prevent such effects.

Exploring the mechanism of action of Bidens pilosa L. in combating hepatic fibrosis through network pharmacology and molecular docking: An observational study.

Based on network pharmacology and molecular docking methods, to explore the possible targets and mechanisms of Bidens pilosa L. in treatment of liver fibrosis. The TCMSP, GeneCard, OMIM, TTD and DrugBank databases were used to obtain the targets of Bidens pilosa L and liver fibrosis, than the intersection targets were screened out by Venny 2.1.0, the protein-protein interaction (PPI) network and the core targets were obtained by the STRING database. Use Cytoscape3.7.2 software to draw the "traditional Chinese medicine-component-target-disease" network. The DAVID database platform was explored to analyze the biological process and pathway, and predict the anti-liver fibrosis mechanism of Bidens pilosa L. AutoDock and PyMol were used to verify the molecular docking between the active ingredients of Bidens pilosa L. and the core targets. Six active components of Bidens pilosa L. and 106 intersection targets were screened. PIK3R1, HSP90AA1, SRC, TP53, AKT1, RELA and other core targets were screened by PPI network analysis. The results of GO and KEGG enrichment analysis showed that the anti-liver fibrosis of Bidens pilosa L mainly involved in the regulation and negative regulation of apoptosis process, positive regulation of protein kinase B signal transduction, positive regulation of cell migration and other biological processes. Pathways acting on cancer, fluid shear stress and atherosclerosis, lipids and atherosclerosis, PI3K-AKT signaling pathway, MAPK signaling pathway and other signaling pathways. Molecular docking showed that the active components of Bidens pilosa L. displayed good binding activity with core target proteins, and the average binding energy was -7.47 kcal/mol. The possible mechanism of the active components against liver fibrosis is to regulate the PI3K-AKT, MAPK, and other signaling pathways by acting on core targets such as PIK3R1, HSP90AA1, SRC, TP53, AKT1, RELA, and induce the apoptosis of activated HSC cells to reverse and improve liver fibrosis.

MTORC1 and mTORC2 Co-Protect against Cadmium-Induced Renal Tubular Epithelial Cell Apoptosis and Acute Kidney Injury by Regulating Protein Kinase B.

The potential threat of cadmium (Cd)-induced acute kidney injury (AKI) is increasing. In this study, our primary goal was to investigate the individual roles played by mTOR complexes, specifically mTORC1 and mTORC2, in Cd-induced apoptosis in mouse kidney cells. We constructed a mouse model with specific deletion of Raptor/Rictor renal cells. Inhibitors and activators of mTORC1 or mTORC2 were also applied. The effects of protein kinase B (AKT) activation and autophagy were studied. Both mTORC1 and mTORC2 were found to mediate the antiapoptotic mechanism of renal cells by regulating the AKT activity. Inhibition of mTORC1 or mTORC2 exacerbated Cd-induced kidney cell apoptosis, suggesting that both proteins exert antiapoptotic effects under Cd exposure. We further found that the AKT activation plays a key role in mTORC1/TORC2-mediated antiapoptosis, protecting Cd-exposed kidney cells from apoptosis. We also found that mTOR activators inhibited excessive autophagy, alleviated apoptosis, and promoted cell survival. These findings provide new insights into the regulatory mechanisms of mTOR in renal diseases and provide a theoretical basis for the development of novel therapeutic strategies to treat renal injury.

CTGF regulated by ATF6 inhibits vascular endothelial inflammation and reduces hepatic ischemia–reperfusion injury

Vascular endothelial inflammation is crucial in hepatic ischemia–reperfusion injury (IRI). Our previous research has shown that connective tissue growth factor (CTGF), secreted by endothelial cells, protects against acute liver injury, but its upstream mechanism is unclear. We aimed to clarify the protective role of CTGF in endothelial cell inflammation during IRI and reveal the regulation between endoplasmic reticulum stress-induced activating transcription factor 6 (ATF6) and CTGF. Hypoxia/reoxygenation in endothelial cells, hepatic IRI in mice and clinical specimens were used to examine the relationships between CTGF and inflammatory factors and determine how ATF6 regulates CTGF and reduces damage. We found that activating ATF6 promoted CTGF expression and reduced liver damage in hepatic IRI. In vitro, activated ATF6 upregulated CTGF and downregulated inflammation, while ATF6 inhibition had the opposite effect. Dual-luciferase assays and chromatin immunoprecipitation confirmed that activated ATF6 binds to the CTGF promoter, enhancing its expression. Activated ATF6 increases CTGF and reduces extracellular regulated protein kinase 1/2 (ERK1/2) phosphorylation, decreasing inflammatory factors. Conversely, inhibiting ATF6 decreases CTGF and increases the phosphorylation of ERK1/2, increasing inflammatory factor levels. ERK1/2 inhibition reverses this effect. Clinical samples have shown that CTGF increases after IRI, inversely correlating with inflammatory cytokines. Therefore, ATF6 activation during liver IRI enhances CTGF expression and reduces endothelial inflammation via ERK1/2 inhibition, providing a novel target for diagnosing and treating liver IRI.

GNA15 facilitates the malignant development of thyroid carcinoma cells via the BTK-mediated MAPK signaling pathway.

G protein subunit alpha 15 (GNA15) is recognized as an oncogene for some cancers, however, its role in thyroid carcinoma (TC) is elusive and is investigated in this study. Concretely, bioinformatics was employed to analyze the GNA15 expression profile in TC. The effect of GNA15 on TC cell functions was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and Transwell assays. Expressions of extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were determined using Western blot. The involvement of Bruton tyrosine kinase (BTK) in the mechanism of GNA15 was investigated by BTK knockdown and rescue assay. GNA15 presented an overexpression pattern in TC samples, which facilitated the viability, proliferation, migration, and invasion of TC cells; GNA15 silencing led to converse results. Ratios of p-ERK/ERK, p-JNK/JNK, and p-p38/p38 were upregulated by GNA15 overexpression. The BTK deficiency weakened the aforementioned behaviors of TC cells and blocked the MAPK signaling pathway, however, these effects were counteracted by GNA15 overexpression. Collectively, GNA15 contributes to the malignant development of TC cells by binding to BTK and thus activating the MAPK signaling pathway.