Protein Kinase Family Research Articles

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.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases

Abstractc‐Jun N‐terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen‐activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3‐selective covalent inhibitor JC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine in the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160‐fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long‐lasting inhibition against cellular JNK3. In addition, its alkyne‐containing probe JC‐P1 could label JNK3 in SH‐SY5Y cell lysate and living cells, with good proteome‐wide selectivity. JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform‐selective and cell‐active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3‐targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Kinase function of TgTKL1 is essential for its role in Toxoplasma propagation and pathogenesis.

Toxoplasma gondii is a protozoan parasite that can cause life-threatening disease in humans. Hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. We have previously shown that a member of the TKL protein kinase family, TgTKL1, is a plant-like kinase that is required for effective Toxoplasma growth in vitro and essential for virulence in vivo. Herein, we show that the TgTKL1 is, indeed, a bona fide kinase, and loss of its kinase function in the Toxoplasma leads to similar defects seen in parasites with complete loss of TgTKL1. More specifically, the TgTKL1 kinase mutant exhibits defects in parasite growth, host-cell invasion, gene expression profile, and virulence in the animal model. Together, these findings suggest that TgTKL1 is a true kinase, and loss of its kinase activity leads to disruption of TgTKL1 function in Toxoplasma.

First De Novo genome assembly and characterization of Gaultheria prostrata

Gaultheria Kalm ex L. (Ericaceae), a type of evergreen shrub, known as a natural source of methyl salicylate, possesses rich germplasm resources, strong habitat adaptability, significant ornamental value, and noteworthy pharmacological activities. However, due to the paucity of whole genomic information, genetically deep research in these areas remains limited. Consequently, we intend to obtain genome data through high-throughput sequencing, gene annotation, flow cytometry, transcription factors prediction and genetic marker analysis for a representative species of this genus, with Gaultheria prostrata selected for our study. In this study, we preliminarily obtained the genome of G. prostrata through next-generation sequencing methods. Utilizing 47.94 Gb of high-quality sequence data (108.95× coverage), assembled into 114,436 scaffolds, with an N50 length of 33,667 bp. The genome size assembled by SOAPdenovo, approximately 417 Mb, corresponded closely to predictions by flow cytometry (440 Mb) and k-mer analysis (447 Mb). The genome integrity was evaluated using BUSCO with 91%. The heterozygosity ratio was 0.159%, the GC content was 38.85%, and the repetitive regions encompassed over 34.6% of the genome. A total of 26,497 protein-coding genes have been predicted and annotated across Nr, Swissprot, GO, KEGG, and Pfam databases. Among these, 14,377 and 2,387 genes received functional annotation in Nr and Swissprot, respectively; 21,895, 24,424, and 22,330 genes were similarly annotated in GO, KEGG, and Pfam. Moreover, A total of 279,785 SSRs were identified and 345,270 primers for these SSRs were designed. Within the various nucleotide types of SSRs, AG/CT and AAG/CTT constituted the predominant dinucleotide and trinucleotide repeat types in G. prostrata. In addition, 1,395 transcription factors (TFs) from 75 TF families, 462 transcription regulators (TRs) from 33 TR families and 840 protein kinase (PKs) from 118 PK families were identified in this genome. We also performed phylogenetic analyses of G. prostrata and related species, including estimation of divergence times and expansion and contraction analyses, followed by positive selection analyses of orthologous gene pairs of G. prostrata and its close relative Vaccinium corymbosum. These results provide a reference for in-depth study of genus Gaultheria, contributing to future functional and comparative genomics analyses and providing supporting data for the development of molecular markers.

Functions and mechanisms of CDPKs in plant responses to abiotic stress

Calcium-dependent protein kinases (CDPKs/CPKs) are members of the Ca2+-sensitive Ser/Thr protein kinase family and play a crucial role in plant growth and development and responses to abiotic stress. CDPKs are capable of rapidly sensing changes in intracellular Ca2+ signals and recognizing and phosphorylating specific substrates, thereby transmitting and amplifying Ca2+ signal cascades downstream. They are involved in plant responses to stress conditions such as drought, saline-alkali stress, and injuries and regulate plant growth and development, gene expression, ion channel activity, and stomatal movement. The autophosphorylation of CDPKs can affect their activities and substrate specificity. CDPKs have the ability to bind to and phosphorylate multiple substrates. In addition to participating in respiratory burst oxidase homolog (RBOH), mitogen-activated protein kinase (MAPK), and plant hormone signaling pathways, CDPKs can also bind to 14-3-3 proteins, which enables the regulation of plant responses to stress and promotes plant growth and development. This paper summarized the research findings on the discovery, structure, classification, and roles of CDPKs in plant responses to stress and proposed the future research directions, aiming to provide the genetic resources and a theoretical basis for improving the stress tolerance of crops.

Impairment of neuronal tyrosine phosphatase STEP worsens post-ischemic inflammation and brain injury under hypertensive condition

Hypertension is associated with poor outcome and higher mortality in patients with ischemic stroke. The impairment of adaptive vascular mechanisms under hypertensive condition compromises collateral blood flow after arterial occlusion in patients with acute ischemic stroke resulting in hypoperfusion. The increased oxidative stress caused by hypoperfusion is thought to be a trigger for the rapid evolution of ischemic infarct volume under hypertensive condition. However, the cellular factors and pathways that contribute to the exacerbation of ischemic brain injury under hypertensive condition is not yet understood. The current study reveals that predisposition to hypertension leads to basal loss of function of the neuron-specific tyrosine phosphatase STEP, which plays a crucial role in neuroprotection against excitotoxic insult. The findings further show that a mild ischemic insult in hypertensive rats triggers an early onset and sustained activation of the neuronal extracellular signal regulated kinase (ERK MAPK), a member of the mitogen activated protein kinase family and a substrate of STEP. This leads to rapid increase in the activation of neuronal NF-κB, expression of neuronal cyclooxygenase-2 and subsequent biosynthesis of the pro-inflammatory mediator prostaglandin E2, resulting in rapid morphological transformation of microglia to the pro-inflammatory state and subsequent exacerbation of ischemic brain injury. Restoration of STEP signaling with intravenous administration of a STEP-derived peptide mimetic reduces the pro-inflammatory response in neurons, activation of microglia, and ischemic brain injury. The findings suggest that the basal loss of STEP function under hypertensive condition contributes to the exacerbation of ischemic brain injury by enhancing post-ischemic inflammatory response. The study not only presents a novel role of STEP in regulating neuroimmune communication but also highlights the therapeutic potential of a STEP-mimetic in mitigating ischemic brain damage under hypertensive condition.

Role of SIK1 in tumors: Emerging players and therapeutic potentials (Review).

Salt‑induced kinase 1 (SIK1) is a serine/threonine protein kinase that is a member of the AMP‑activated protein kinase family. SIK is catalytically activated through its phosphorylation by the upstream kinase LKB1. SIK1 has been reported to be associated with numerous types of cancer. The present review summarizes the structure, regulatory factors and inhibitors of SIK1, and also describes how SIK1 is a signal regulatory factor that fulfills connecting roles in various signal regulatory pathways. Furthermore, the anti‑inflammatory effects of SIK1 during the early stage of tumor occurrence and its different regulatory effects following tumor occurrence, are summarized, and through collating the tumor signal regulatory mechanisms in which SIK1 participates, it has been demonstrated that SIK1 acts as a necessary node in cancer signal transduction. In conclusion, SIK1 is discussed independent of the SIKs family, its research results and recent progress in oncology are summarized in detail with a focus on SIK1, and its potential as a therapeutic target is highlighted, underscoring the need for SIK1‑targeted regulatory strategies in future cancer therapy.

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

Comprehensive analysis of the expression and prognosis for cyclin-dependent protein kinase family in osteosarcoma

Background and Objective Cyclin-dependent protein kinases (CDKs) have been suggested as prospective therapeutic targets because they control processes vital to the survival and growth of cancer cells. However, research on the varied CDK expression profiles and prognostic factors in osteosarcoma is still lacking. Methods The osteosarcoma microRNA (GSE65071) and gene expression profiles were retrieved from the Gene Expression Omnibus (GEO) database (GSE42352). A substantial variation in prognosis was discovered in CDKs using the TARGET database. Cytoscape was used to construct the miRNAs-CDKs network, and functional and pathway enrichment analyses were completed. It was looked at how immune checkpoint genes, m6A-related genes, and CDKs interact. Results In patients with osteosarcoma compared to normal samples, CDK1-5, CDK18, CDK16, and CDK17 gene expression levels were considerably greater, whereas CDK7-9, CDK11B, CDK16, and CDK20 gene expression levels were significantly lower. Patients with osteosarcoma who had low CDK3 and 18 gene levels or high CDK6, 9 gene levels were predicted to have a favorable prognosis and a long-life expectancy. Immune checkpoint genes, m6A-related gene expression, and CDKs expression all showed some connection. Finally, a network of crucial CDKs and miRNAs was constructed. Conclusion According to our research, CDK3, 6, 9, and 18 have been identified as possible therapeutic targets for osteosarcoma, and CDKs may have a role in controlling m6A mutations in tumor cells as well as immune checkpoint regulation.

Prediction of protein-ligand binding sites modulating activity of MAST protein kinases

Aim. Identification of the protein-ligand binding sites, that may be the target of compounds, affecting individual human protein kinases of the MAST family (MAST1, 2, 3, 4 and MASTL / GWL). Methods. Literature and database search. Comparison of protein and ligand structures. Protein structure modeling, structural superimposition, etc. Results. The structural alignment demonstrates significant similarity of catalytic domains in MAST1, 2, 3, 4 and MASTL (GWL). It justifies transferring of reference ligands from PDB structures to human MASTs, discovering potential sites of ligand binding. 13 sites of ligand-binding were specified based on refrence ligands, transferred from RCSB Protein Data Bank structures, and differences in sites amino acid composition of MAST family members were discovered. Сonclusions. Based on the differences in the amino acid composition of the studied pockets in MAST1, 2, 3, 4 and MASTL (GWL), the sites B, C, D, E, F, were selected for further study and virtual screening for new selective inhibitors of individual members of MAST protein kinase family.

RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1

One of the most critical axes for cell fate determination is how cells respond to excessive reactive oxygen species (ROS)-oxidative stress. Extensive lipid peroxidation commits cells to death via a distinct cell death paradigm termed ferroptosis. However, the molecular mechanism regulating cellular fates to distinct ROS remains incompletely understood. Through siRNA against human receptor-interacting protein kinase (RIPK) family members, we found that RIPK4 is crucial for oxidative stress and ferroptotic death. Upon ROS induction, RIPK4 is rapidly activated, and the kinase activity of RIPK4 is indispensable to induce cell death. Specific ablation of RIPK4 in kidney proximal tubules protects mice from acute kidney injury induced by cisplatin and renal ischemia/reperfusion. RNA sequencing revealed the dramatically decreased expression of acyl-CoA synthetase medium-chain (ACSM) family members induced by cisplatin treatment which is compromised in RIPK4-deficient mice. Among these ACSM family members, suppression of ACSM1 strongly augments oxidative stress and ferroptotic cell death with induced expression of ACS long-chain family member 4, an important component for ferroptosis execution. Our lipidome analysis revealed that overexpression of ACSM1 leads to the accumulation of monounsaturated fatty acids, attenuation of polyunsaturated fatty acid (PUFAs) production, and thereby cellular resistance to ferroptosis. Hence, knockdown of ACSM1 resensitizes RIPK4 KO cells to oxidative stress and ferroptotic death. In conclusion, RIPK4 is a key player involved in oxidative stress and ferroptotic death, which is potentially important for a broad spectrum of human pathologies. The link between the RIPK4-ASCM1 axis to PUFAs and ferroptosis reveals a unique mechanism to oxidative stress-induced necrosis and ferroptosis.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases.

c-Jun N-terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen-activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3-selective covalent inhibitorJC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160-fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long-lasting inhibition against cellular JNK3. In addition, its alkyne-containing probe JC-P1 could label JNK3 in SH-SY5Y cell lysate and living cells, with goodproteome-wide selectivity. Furthermore, JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform-selective and cell-active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3-targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Component-resolved diagnosis: enhancing precision diagnosis and clinical management of shellfish allergies

Shellfish, being one of the eight major food allergens, affects approximately 3% of the global population. The occurrence of shellfish allergy is not only related to the individual's immune system sensitivity but is also influenced by geographical environment, food availability, and dietary habits. Although crustaceans (such as shrimp, crab, and lobster) and mollusks (such as oysters, mussels, and squid) are collectively referred to as shellfish, they exhibit significant differences in biological evolution and the spectrum of allergenic molecules they contain, leading to various allergic reactions. Accurate identification of allergenic proteins is crucial for the diagnosis and management of shellfish allergies, with key allergenic protein families including tropomyosin, arginine kinase, and hemocyanin. Furthermore, due to the diversity of shellfish allergens and their cross-reactivity with dust mite and insect allergens, diagnosing and managing shellfish allergies is complex, especially concerning tropomyosin and arginine kinase protein families. Currently, there are no specific immunotherapy treatments for shellfish allergies, and clinical management primarily relies on avoiding allergens and using anti-allergy medications. This article thoroughly interprets the " Molecular Allergology User's Guide 2.0 (MAUG 2.0)" published by the European Academy of Allergy and Clinical Immunology (EAACI) and the latest research on shellfish allergies both domestically and internationally. It highlights the significant role of allergen component diagnostics in optimizing the diagnostic and treatment processes for shellfish allergies, effectively assisting clinicians in accurately identifying common allergens and cross-reactions, thereby providing patients with more personalized diagnosis and treatment plans.

Extracellular signal-regulated kinase is activated in podocytes from patients with diabetic nephropathy.

In the past few decades, the global prevalence of diabetes has provided us with a warning about future chronic complications. Diabetic nephropathy (DN) is the main cause of end-stage kidney disease. Podocytes in the glomerulus play a critical role in regulating glomerular permeability, and podocyte injury is one of the main causes of DN. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. In human patients with DN, phosphorylated ERK (pERK), the active form of ERK, is increased in the glomeruli. However, information on the expression of pERK, specifically in podocytes in DN, is limited. Meanwhile, high glucose induces ERK activation in immortalized podocyte cell lines, suggesting the involvement of podocytic ERK in DN. We performed an immunohistochemical study using Wilms' tumor-1 (WT-1) as a podocyte-specific marker to investigate whether podocytic pERK levels are increased in patients with DN. In the glomeruli of the DN group, we observed remarkable co-staining for WT-1 and pERK. In contrast, the glomeruli of the control group contained only a few pERK-positive podocytes. Statistical analyses revealed that, relative to healthy controls, patients with DN showed significantly increased pERK expression levels in cells that were positive for WT-1 (DN: 51.3 ± 13.1% vs. control: 7.3 ± 1.6%, p = 0.0158, t-test, n = 4 for each group). This suggests that ERK activation in podocytes is involved in the pathogenesis of DN.

Genome-wide association study of lignin trait in elite spring wheat against spot blotch disease

Spot blotch disease in wheat is one of major yield-limiting factor in warm humid conditions of South Asia. Developing the disease-resistant variety through molecular methods is an economically and environmentally eco-friendly approach. Diseased wheat leaves associated with lignin content increased by two or three times at 72 h compared with control. The genomic region associated with lignin content was investigated in a set of 289 diverse spring wheat genotypes. The genome - wide association studies panel employed a 90K Illumina SNP array and phenotyped in four environments. The GWAS analysis showed a total of 86 marker-trait associations on 1A, 1B, 2A, 2B, 2D, 3A, 3D, 4A, 5A, 5B, 6A, 6D, 7A, 7B, 7D were linked lignin trait. Insilco analysis revealed that significant SNPs were located on import putative candidate genes thirteen in the Transmembrane domain of the protein-kinase family and were involved in the host-pathogen interaction. The identified significant MTAs for further validation and useful for the wheat breeding programs to develop the spot blotch disease resistance.

Targeting MELK in tumor cells and tumor microenvironment: from function and mechanism to therapeutic application.

Maternal embryonic leucine zipper kinase (MELK), a member of the adenosine monophosphate-activated protein kinase (AMPK) protein family, has been reported to be involved in the regulation of many cellular events. The aberrant expression of MELK is associated with tumorigenesis and malignant progression of various tumors. Moreover, MELK plays an essential role in the regulation of tumor microenvironment (TME), which affects the function of immune cells and the responsiveness to immunotherapy. Currently, small molecule inhibitors targeting MELK have been developed and evaluated in clinical trials. A comprehensive understanding of MELK may provide clues and confidence for subsequent basic research and scientific transformation. In this review, we provide a comprehensive overview of the structural features, molecular biological functions, and critical roles of MELK in tumors and TME, as well as the targeted agents under development for the treatment of tumors and discuss the perspective for MELK-targeted therapies for tumors.

Unveiling the Role of Sik1 in Osteoblast Differentiation: Implications for Osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative disease characterized by subchondral osteosclerosis, mainly due to osteoblast activity. This research investigates the function of Sik1, a member of the AMP-activated protein kinase family, in OA. Proteomic analysis was conducted on clinical samples from 30 OA patients, revealing a negative correlation between Sik1 expression and OA. In vitro experiments utilized BMSCs to examine the effect of Sik1 on osteogenic differentiation. BMSCs were cultured and induced toward osteogenesis with specific media. Sik1 overexpression was achieved through lentiviral transfection, followed by analysis of osteogenesis-associated proteins using Western blotting, RT-qPCR, and alkaline phosphate staining. In vivo experiments involved destabilizing the medial meniscus in mice to establish an OA model, assessing the therapeutic potential of Sik1. The CT scans and histological staining were used to analyze subchondral bone alterations and cartilage damage. The findings show that Sik1 downregulation correlates with advanced OA and heightened osteogenic differentiation in BMSCs. Sik1 overexpression inhibits osteogenesis-related markers invitro and reduces cartilage damage and subchondral osteosclerosis invivo. Mechanistically, Sik1 modulates osteogenesis and subchondral bone changes through Runx2 activity regulation. The research emphasizes Sik1 as a promising target for treating OA, suggesting its involvement in controlling bone formation and changes in the subchondral osteosclerosis.

Protective Effects of 7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid (diHEP-DPA) against Blue Light-Induced Retinal Damages in A2E-Laden ARPE-19 Cells.

The purpose of this study was to investigate the protective effects of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA) in retinal pigment epithelial (RPE) cell damage. ARPE-19 cells, a human RPE cell line, were cultured with diHEP-DPA and Bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E), followed by exposure to BL. Cell viability and cell death rates were determined. Western blotting was performed to determine changes in apoptotic factors, mitogen-activated protein kinase (MAPK) family proteins, inflammatory proteins, and oxidative and carbonyl stresses. The levels of pro-inflammatory cytokines in the culture medium supernatants were also measured. Exposure to A2E and BL increased the ARPE-19 cell death rate, which was alleviated by diHEP-DPA in a concentration-dependent manner. A2E and BL treatments induced apoptosis in ARPE-19 cells, which was also alleviated by diHEP-DPA. Analysis of the relationship with MAPK proteins revealed that the expression of p-JNK and p-P38 increased after A2E and BL treatments and decreased with exposure to diHEP-DPA in a concentration-dependent manner. DiHEP-DPA also affected the inflammatory response by suppressing the expression of inflammatory proteins and the production of pro-inflammatory cytokines. Furthermore, it was shown that diHEP-DPA regulated the proteins related to oxidative and carbonyl stresses. Taken together, our results provide evidence that diHEP-DPA can inhibit cell damage caused by A2E and BL exposure at the cellular level by controlling various pathways involved in apoptosis and inflammatory responses.

The combination of a microbial and a non-microbial biostimulant increases yield in lettuce (Lactuca sativa) under salt stress conditions by up-regulating cytokinin biosynthesis.

Salinization poses a significant challenge in agriculture, exacerbated by anthropogenic global warming. Biostimulants, derived from living microorganisms or natural extracts, have emerged as valuable tools for conventional and organic agriculture. However, our understanding of the molecular mechanisms underlying the effects of biostimulants is very limited, especially in crops under real cultivation conditions. In this study, we adopted an integrative approach to investigate the effectiveness of the combined application of plant growth-promoting bacterium (Bacillus megaterium strain BM08) and a non-microbial biostimulant under control conditions (normal watering) and salt stress. After confirming the yield increase under both conditions, we investigated the molecular mechanisms underlying the observed effect by measuring a number of physiological parameters (i.e., lipid peroxidation, antioxidants, chlorophylls, total phenolics and phytohormone content), as well as RNA sequencing and primary metabolite analyses. Our findings reveal that the combined effect of the microbial and non-microbial biostimulants led to a decrease in the antioxidant response and an up-regulation of genes involved in cytokinin biosynthesis under salt stress conditions. This, in turn, resulted in a higher concentration of the bioactive cytokinin, isopentenyladenosine, in roots and leaves and an increase in γ-aminobutyric acid, a non-proteic amino acid related to abiotic stress responses. In addition, we observed a decrease in malic acid, along with an abscisic acid (ABA)-independent up-regulation of SR-kinases, a family of protein kinases associated with abiotic stress responses. Furthermore, we observed that the single application of the non-microbial biostimulant triggers an ABA-dependent response under salt stress; however, when combined with the microbial biostimulant, it potentiated the mechanisms triggered by the BM08 bacterial strain. This comprehensive investigation shows that the combination of two biostimulants is able to elicit a cytokinin-dependent response that may explain the observed yield increase under salt stress conditions.