Search Results (91)

Macropinocytosis is an actin-driven endocytic process mediating nonspecific uptake of extracellular fluid through membrane ruffling. Neutrophil macropinocytosis has been reported only in limited descriptive studies, and its signaling mechanisms have not been defined. Here, we provide a characterization of macropinocytosis in primary human neutrophils and investigate signaling pathways that contribute to the regulation of this process. Quantitative flow cytometry using a high-molecular-weight fluid-phase tracer showed that the diacylglycerol (DAG) mimetic 4β-phorbol 12-myristate 13-acetate (4β-PMA) induces macropinocytic activity in primary human neutrophils. Granulocyte macrophage-colony stimulating factor (GM-CSF) and hepatocyte growth factor (HGF) also promoted fluid-phase uptake. Inhibition of actin polymerization or macropinocytosis reduced tracer uptake, confirming dependence on actin-driven machinery. Scanning electron microscopy revealed dorsal membrane ruffling and cup-like structures after stimulation. Mechanistically, DAG-dependent activation of protein kinase C beta (PKCβ) acted upstream of NADPH oxidase 2 (NOX2)-derived superoxide anion production, driving membrane remodeling. Collectively, these findings define a DAG–PKCβ–NOX2–superoxide signaling axis, as a key regulator of macropinocytosis in primary human neutrophils and provide the first mechanistic framework for its redox-dependent regulation. Full article

Target of rapamycin (TOR) is a conserved protein kinase that integrates nutrient and energy signals to control growth and metabolism, yet its proteome-level impact in microalgae remains poorly understood. Here, we conducted quantitative proteomics analysis of the unicellular red alga Cyanidioschyzon merolae under rapamycin-induced TOR inactivation to characterize global changes in protein abundance. TOR inhibition triggered widespread metabolic remodeling, including coordinated shifts in carbon and nitrogen allocation, and pronounced changes in protein synthesis, photosynthesis, and energy metabolism. Specifically, proteins associated with ribosome biogenesis and ribosomal subunits declined broadly, indicating impaired translation, alongside pronounced reductions in photosynthetic components, including PSI/PSII subunits and chlorophyll biosynthesis enzymes. In contrast, triacylglycerol (TAG) biosynthesis and starch metabolism were enhanced, indicating a shift towards carbon storage. Notably, a diacylglycerol acyltransferase (DGAT; CMQ199C) and a UDP-glucose pyrophosphorylase (UGP; CMS159C) were strongly induced (2.02-fold and 3.48-fold, respectively), identifying them as candidate targets for enhancing TAG and starch accumulation. Proteins associated with nitrogen assimilation were also upregulated, supporting TOR-dependent regulation of nitrogen metabolism at the protein level. Together, these results indicate that TOR orchestrates proteome-level reprogramming in C. merolae, coordinating growth, energy production, and carbon storage across interconnected metabolic pathways. Full article

This experiment investigated the effects of dietary Artemisia ordosica Krasch. (AOK) supplementation on the n3-polyunsaturated fatty acid (n3-PUFA) profile of subcutaneous adipose tissue (SADT) in Arbas cashmere goats and explored the underlying transcriptional mechanisms. Forty healthy, weaned kids (120 ± 10 days of age; similar body weight) were randomly allocated to two groups (n = 20): a control group (CON, basal diet) and an AOK group (AOK, basal diet with 3% of the roughage replaced by AOK). The feeding trial spanned 104 days, consisting of a 14-day adaptation period and 90 days of data acquisition. Compared with the CON group, AOK significantly reduced the content of saturated fatty acids (SFAs) and n6-polyunsaturated fatty acids (n6-PUFAs)/n3-PUFAs (n6/n3). In contrast, the levels of n3-PUFAs in the SADT of cashmere goats increased markedly (p < 0.05). Compared with the CON group, AOK exhibited significantly higher activities of hormone-sensitive lipase (HSL) (p = 0.027), adenylyl cyclase 2 (ADCY2) (p = 0.010), adenylyl cyclase 5 (ADCY5) (p = 0.046), cluster of differentiation 36 (CD36) (p = 0.013), solute carrier family 27 member 4 (SLC27A4) (p = 0.021), and fatty acid binding protein 4 (FABP4) (p = 0.040), along with significantly lower activities of fatty acid synthase (FAS) (p = 0.002), lipoprotein lipase (LPL) (p = 0.048), and stearoyl-coa desaturase (SCD) (p = 0.026) in SADT. Compared with the CON group, the activities of superoxide dismutase (SOD) (p = 0.032), catalase (CAT) (p = 0.010), glutathione peroxidase (GSH-PX) (p = 0.029), and total antioxidant capacity (T-AOC) (p = 0.002) were significantly increased in the AOK group. Transcriptomic profiling revealed that AOK supplementation downregulated mRNA levels of ADCY2, ADCY5, LPL, FAS, SCD, stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), glycogen synthase 1 (GYS1), acyl-CoA oxidase 1 (ACOX1), acetyl-CoA carboxylase (ACC), diacylglycerol acyltransferase 1 (DGAT1), fatty acid desaturase 1 (FADS1), solute carrier family 27 member 2 (SLC27A2), erythroblastic leukemia viral oncogene homolog 4 (ERBB4), and carnitine palmitoyltransferase 1B (CPT1B) (p < 0.05). It also markedly induced acyl-CoA synthetase long-chain family member 4 (ACSL4) (p < 0.01) in SADT. Genes significantly enriched in the adenosine-monophosphate-activated protein kinase (AMPK) signaling pathway included LPL, SCD1, CPT1B, and GYS1 (p = 0.010). Genes significantly enriched in the phosphatidylinositol 3-kinase-akt (PI3K-Akt) signaling pathway included GYS1 and ERBB4 (p = 0.015). CPT1B, ADCY2, and GYS1 were identified as the genes significantly enriched in the insulin resistance signaling pathway (p = 0.048). LPL was the only gene significantly enriched in the cholesterol metabolism pathway (p = 0.049). Genes showing a tendency toward significant enrichment in the peroxisome-proliferator-activated receptor (PPAR) signaling pathway included ACSL4, CPT1B, SCD1, and LPL (p = 0.051). These interconnected cascades improve insulin sensitivity, stimulate triglyceride (TG) hydrolysis, and modulate n3-PUFA levels. Supplementation with AOK enhances n3-PUFA content by accelerating TG breakdown while simultaneously restraining FA oxidation in SADT. Consequently, AOK supplementation can be effectively used to enhance the nutritional value of cashmere goat meat through improved n3-PUFA deposition in SADT. Full article

Background/Objectives: Kidney proximal tubules reabsorb up to 70% of water and solutes from the glomerular ultrafiltrate, a Ca²⁺-modulated process essential for homeostasis. The plasma membrane Ca²⁺-ATPase (PMCA) in basolateral membranes (BLMs) plays a pivotal role in maintaining intracellular calcium homeostasis and regulating calcium reabsorption. Methods: Here, we investigated the regulatory influence of two key bioactive lipids, diacylglycerol (DG) and phosphatidic acid (PA), on PMCA activity from pig kidney, accompanied by lipidomic assays and transcriptomic data analyses. Results: Biochemical assays revealed dose- and time-dependent inhibition of PMCA by DG, fully reversed by Calphostin C, implicating PKC activation. Conversely, PA significantly stimulated PMCA activity, demonstrating an opposite regulatory effect. Our targeted lipidomics identified multiple DG species in HK-2 cells, suggesting substrate diversity. Analysis of transcriptomic data for hypoxic versus normoxic HK-2 cells revealed dramatic coordinated regulation of DG/PA metabolism genes, with upregulation of DG-producing enzymes (PLCB1, PLDs) and downregulation of DG-consuming kinases (DGKs), predicting enhanced DG accumulation under metabolic stress. ATP2B4 (PMCA4) upregulation indicated compensatory transcriptional responses. Conclusions: Our findings suggest that DG inhibits BLM-associated PMCA via classic and/or atypical PKC-dependent phosphorylation while PA exerts opposing stimulatory effects. Both transcriptional remodeling and post-translational modifications regulate this axis. These findings highlight the DG–Diacylglycerol Kinase–PA axis as a dynamic modulator of Ca²⁺ signaling in the kidney that responds to metabolic stress. Full article

Wheat is one of the world’s most important crops, cultivated across diverse ecogeographic zones on more than ~245 million hectares annually. Classified by vernalization requirement into spring, facultative, or winter types, the latter typically achieves higher yields due to its extended growing season, reaching ~18 t ha⁻¹ and 9–10 t ha⁻¹ as a national average for Western European countries such as Germany, France, and England, compared with the global average of barely above 3 t ha⁻¹. Despite this potential, winter wheat is largely confined to regions with relatively mild winters, while vast temperate zones with extremely cold winters rely on spring wheat. Breeding has traditionally targeted the vernalization–C-repeat Binding Factor (VRN–CBF) pathway, which confers tolerance to moderately severe winters but is insufficient for extreme cold, implying the need for additional layers of adaptive mechanisms. Using multiple genotypic datasets, we identified genomic regions underlying low-temperature tolerance. Genome- and chromosome-wide scans revealed strong differentiation on chromosome 5A (526–703 Mb), overlapping the VRN–CBF loci. SNP-level FST analysis between spring and winter cultivars highlighted the VRN-A1 (586–588 Mb) region and a locus spanning 549 and 559 Mb on chromosome 6A. Further comparisons between winter accessions adapted to extreme cold (≤−12 °C) and mild winters (>0 °C) revealed a differentiated region on chromosome 3B (561–564 Mb) harbouring two key genes conferring CBF-independent cold tolerance, TRAESCS3B02G351100 and TRAESCS3B02G354000, encoding diacylglycerol kinase1 (DGK1) and peroxidase 56 (PRX56), respectively. These findings underscore alternative pathways in shaping cold adaptation, highlighting the need to broaden breeding strategies for extreme environments. We further detected a pronounced haplotype divergence between Chinese and U.S. winter cultivars reflecting distinct breeding trajectories; notably, China, where ~90% of wheat production is of the winter type, achieves national yields >5 t ha⁻¹, compared with ~3 t ha⁻¹ in the United States, where over 70% of production is winter wheat. This contrast suggests that the haplotypes enriched in Chinese winter cultivars could represent valuable resources for enhancing winter wheat performance in other regions with comparable environments. Full article

Cardiovascular disease is the leading cause of mortality in insulin-resistant individuals, with metabolic cardiomyopathy preceding overt heart failure in a substantial proportion of patients with diabetes. Skeletal muscle accounts for approximately 40% of body mass and nearly 80% of insulin-stimulated glucose disposal, positioning it as a major determinant of systemic lipid flux. Dysregulation of lipid droplet dynamics, lipolysis, and fatty acid trafficking in skeletal muscle alters circulating lipid availability and promotes ectopic lipid deposition and mitochondrial stress in the myocardium. Intramyocellular lipid handling is governed by coordinated actions of lipid droplets, perilipin proteins (PLIN2 and PLIN3), adipose triglyceride lipase (ATGL), and diacylglycerol acyltransferases (DGAT1/2), which together regulate the rate and composition of fatty acid release into the circulation. Impaired coupling between intramyocellular lipid droplet turnover and mitochondrial oxidation in insulin-resistant muscle increases circulating free fatty acids, reducing cardiac oxidative capacity. In response, the myocardium undergoes mitochondrial lipid remodeling, including alterations in cardiolipin composition that impair cristae structure and electron transport chain efficiency. Excess lipid exposure activates apoptosis signal-regulating kinase-1 (ASK-1), promoting cardiomyocyte apoptosis and inflammatory signaling, while peroxisome proliferator-activated receptor gamma (PPARγ) modulates lipid uptake, storage, and mitochondrial oxidation in a context-dependent manner. This review integrates skeletal muscle–cardiac lipid crosstalk with ASK-1 and PPARγ signaling to define mechanisms linking peripheral insulin resistance to early myocardial dysfunction and to identify targets for intervention before irreversible cardiac remodeling develops. Full article

Lipid signaling plays a crucial role in how plants perceive and respond to environmental challenges. Among the various lipid mediators, phosphatidic acid (PA) serves as a key metabolic intermediate and second messenger that links membrane dynamics with stress signaling. It is produced rapidly through the coordinated actions of phospholipase C, phospholipase D and diacylglycerol kinase, and its transient accumulation enables plants to adjust defense and acclimation responses with remarkable precision. Recent studies have shown that PA participates in immune signaling, osmotic regulation, and redox control, functioning at the intersection of membrane remodeling and intracellular signal transduction. Through interactions with hormone signaling, calcium fluxes, and reactive oxygen species production, PA integrates multiple stress-responsive pathways, thereby helping to maintain physiological homeostasis under adverse conditions. This review summarizes current understanding of the biosynthetic regulation and signaling roles of PA, and discusses emerging perspectives that highlight its central role in plant immunity and stress adaptation. Full article

Soil salinization poses a major threat to plant growth and ecosystem sustainability. Zoysia japonica, a salt-tolerant turfgrass, shows promise for saline–alkali soil remediation, yet its metabolic adaptation mechanisms remain poorly understood. Here, we applied non-targeted liquid chromatography/mass spectrometry (LC/MS) metabolomics to compare the responses of salt-tolerant (accession 68) and salt-sensitive (accession 9) genotypes of Z. japonica under salt stress. The sensitive genotype exhibited stronger metabolic disruption, with 843 differentially accumulated metabolites (largely down-regulated), compared with 595 in the tolerant genotype (predominantly up-regulated). We identified a coordinated tolerance mechanism primarily centered on lipid remodeling and energy maintenance. The tolerant genotype enhanced membrane stability through the accumulation of saturated glycerophospholipids and an increased phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratio, while maintaining phosphatidic acid (PA) homeostasis which may facilitate SOS-dependent Na⁺ efflux. It also mitigated oxidative damage by stabilizing diacylglycerol (DAG), thereby potentially limiting protein kinase C (PKC) overactivation. Furthermore, sustained cardiolipin and riboflavin metabolism supported mitochondrial energy production in the tolerant genotype. Together, these findings provide new insights into the early metabolic basis of salt tolerance in Z. japonica, suggesting a potential crucial role for PA-mediated regulation of SOS-dependent sodium sequestration during the initial phase of stress, and implying potential targets for breeding stress-resilient turfgrasses. Full article

Transient receptor potential canonical 6 (TRPC6) channels are promising drug targets for kidney, lung, and neurological diseases, making a detailed understanding of their regulation crucial to developing novel channel modulators with more precise modes of action. TRPC6 channels are commonly accepted as calcium-permeable, receptor-operated cation channels activated by diacylglycerol (DAG) downstream of phospholipase C (PLC) signaling. DAG, the endogenous activator of TRPC channels, also activates protein kinase C (PKC), which can phosphorylate TRPC6 and potentially modify its function. This study examined whether five putative PKC phosphorylation sites located in the C-terminus of TRPC6 affect channel gating. Using whole-cell patch-clamp recordings and utilizing photopharmacology with photoswitchable TRPC6 activators (OptoBI-1 and OptoDArG), we analyzed the activation, inactivation, and deactivation kinetics. Pharmacological modulation of PKC activity and strategic mutation of the phosphorylation sites—either to prevent or mimic phosphorylation—altered the current kinetics as well as the normalized slope conductances that were used to quantify differences in the curve progression of current–voltage relations, even when maximally induced current density amplitudes were unchanged. Our findings reveal activator-specific differences in TRPC6 current kinetics associated with C-terminal amino acid exchanges and PKC-dependent signaling, suggesting that phosphorylation-related mechanisms may fine-tune channel activity. Full article

Acute myeloid leukemia (AML) is a heterogeneous disease with an unmet need for novel therapeutic drugs. Previous studies have reported the upregulation of diacylglycerol kinases (DGKs) in AML. This study investigated the effects of ritanserin, a DGKα-specific inhibitor, and DGKζ-IN4 or BAY 2965501, DGKζ-selective inhibitors, on a panel of AML cell lines. Ritanserin induced apoptotic cell death across all tested models, whereas DGKζ inhibitors triggered both apoptosis and necrosis to variable extents, with HL-60 cells being the most responsive to both compounds. Drug sensitivity did not correlate with DGKα or DGKζ expression levels, indicating that additional factors may influence cellular susceptibility. THP-1 proteomic profiling revealed that ritanserin broadly downregulated proteins involved in antigen presentation, cell cycle and metabolism, while BAY 2965501 affected a smaller and distinct but functionally similar protein subset, implying different mechanisms of action. Gene silencing confirmed AML cell line-specific dependence on DGK isoforms: HEL cells were sensitive to DGKα knockdown, HL-60 to DGKζ silencing, whereas K562 and THP-1 were resistant to both. These findings indicate that DGKs targeting can effectively reduce AML cell viability. However, AML heterogeneity and the limited selectivity of current inhibitors underscore the need for predictive biomarkers and combinatorial strategies to translate DGK inhibition into effective therapy. Full article

Ceramide 1-phosphate (C1P) is a key regulator of cell proliferation and survival in both normal and transformed cells. Major pathways implicated in the mitogenic actions of C1P include activation of the mitogen-activated protein kinases (MAPKs) ERK1-2 and JNK, as well as stimulation of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, the product of retinoblastoma, or the sphingomyelin synthase (SMS)/diacylglycerol (DAG)/protein kinase C-alpha (PKC-α) pathway. C1P-stimulated cell proliferation can also be mediated through enhanced secretion of vascular endothelial growth factor (VEGF) in macrophages or by releasing lysophosphatidic acid (LPA) in myoblasts. Also, the production of low levels of reactive oxygen species (ROS) can mediate the stimulation of cell growth by C1P, particularly in macrophages. Upregulation of the PI3K/Akt/mTOR pathway is also involved in the inhibition of cell death by C1P, which can also contribute to cell survival by blocking the activity of the ceramide-generating enzymes acid sphingomyelinase (ASMase) and serine palmitoyl transferase (SPT). Moreover, C1P-promoted cell survival involves upregulation of inducible nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Using photosensitive C1P analogues, it could be concluded that promotion of cell growth and inhibition of cell death were elicited by intracellularly generated C1P in a receptor-independent manner. The aim of the present review is to evaluate in detail the implication of the CerK/C1P axis in controlling cell proliferation and survival in mammalian cells, as well as to discuss and update on the molecular mechanisms by which C1P can accomplish these actions. Full article

A new class of spiro[1,2,4]triazolo[1,5-c]quinazoline derivatives is presented as promising modulators of diacylglycerol kinase α (DGK-α), a target implicated in cancer, neurological disorders, and immune dysfunction. Through structure-based computational design using the CB-Dock2 platform with human DGK-α (PDB ID: 6IIE), 40 novel compounds were systematically evaluated along with established inhibitors (ritanserin, R59022, R59949, BMS502, and (5Z,2E)-CU-3) across five distinct binding pockets. Several compounds demonstrated binding profiles at the level of or surpassing the reference compounds. The physicochemical analysis revealed balanced drug-like properties with favorable molecular weights (252–412 g/mol) and appropriate three-dimensionality. The toxicological assessment indicated reassuring safety profiles with predicted LD₅₀ values of 1000–2000 mg/kg and minimal hepatotoxicity, carcinogenicity, and mutagenicity potential. Notably, compound 33 (adamantyl-substituted) emerged as exceptionally promising, exhibiting strong binding affinity, moderate solubility, and selective CYP inhibition patterns that minimize drug–drug interaction risks. Detailed molecular interaction mapping identified critical binding determinants, including strategic hydrogen bonding with TRP151, GLU166, and ARG126. The multidimensional evaluation identified compounds 13, 18, 33, and 40 as particularly promising candidates that balance potent target engagement with favorable pharmaceutical profiles, establishing this scaffold as a valuable platform for developing next-generation therapeutics targeting DGK-α -mediated signaling pathways. Full article

Laboratory evolution is an effective means of understanding microbial adaptation to the environment. We previously isolated four thermoadapted Zymomonas mobilis mutants, which showed a 2 °C rise in the critical high temperature (CHT), by performing multiple-parallel adaptation experiments. In the present study, the individual mutations in these mutants were intensively analyzed. Two mutations in each adapted mutant were found to primarily contribute to the increase in the upper temperature limit. RNA sequencing (RNA-seq) analysis revealed that the two mutations led to the upregulation of 79–185 genes and the downregulation of 242–311 genes. The findings from transcriptomic and physiological experiments suggest two common and primary mechanisms for thermal resistance: a decrease in the activity of diacylglycerol kinase, which may change the structure of lipopolysaccharide (LPS) probably to strengthen the membrane structure, and an increase in the expression of genes for GroEL/GroES or cell wall hydrolase to repair the protein or membrane damage that occurs at such critical temperatures. Additionally, transporters including efflux pumps may contribute to intracellular homeostasis by expelling toxic compounds such as ethanol and acetate or by maintaining the K⁺ concentration. The results of this study on four independently thermoadapted mutants led to the conclusion that the mutants have almost the same thermal adaptation strategies and thus their molecular diversity is limited. Full article

Phallus impudicus is a fungus used as a medicine and nutrient-rich food. However, the shelf life of mature Phallus impudicus is only a few hours. Therefore, research on its preservation technology is essential for improving its economic value. This study investigated the effects of hexanal concentrations (25–100 μL/L) and treatment time (4–8 h) on the inhibition of peach-shaped Phallus impudicus (CK) maturation and found that the maturation rate was 25% under optimal conditions of 25 μL/L hexanal treatment for 6 h. Quantitative transcriptomic and lipidomic analyses were conducted among CK, mature Phallus impudicus (M-P), and hexanal-treated peach-shaped Phallus impudicus (H-P-P). In total, 2933 and 2746 differentially expressed genes (DEGs) and 156 and 111 differentially abundant lipids (DALs) were identified in CK vs. H-P-P and M-P vs. H-P-P, respectively. Functional analysis demonstrated that hexanal treatment inhibited phospholipase D gene expression and reduced phosphatidic acid abundance, thereby inhibiting the activation of the phosphatidylinositol signaling system and the signal amplification of the cell wall integrity mitogen-activated protein kinase pathway. These blocked signal transductions inhibited the gene expression of most β-glucanases, chitinases and chitin synthases, further affecting cell wall reconstruction. Moreover, hexanal treatment enhanced membrane stability by reducing the monogalactosyl diglyceride/digalactosyl diacylglycerol ratio and increasing the phosphatidylcholine/phosphatidylethanolamine ratio. This study contributed to the development of hexanal treatment as a postharvest preservation technology for Phallus impudicus. Full article

Sphingosine kinases (SPHKs) are essential enzymes that catalyze the phosphorylation of sphingosine to produce sphingosine-1-phosphate (S1P), which plays pivotal roles in inflammation and immune regulation. In this study, genome-wide association analysis (GWAS) identified the Ydsphk1 gene as closely associated with the resistance of yellow drum (Nibea albiflora) to Vibrio harveyi. Structural prediction showed that YDSPHK1 contains a typical diacylglycerol kinase catalytic (DAGKc) domain (154–291 aa). By constructing and transfecting Ydsphk1 expression plasmids into yellow drum kidney cells, we found that YDSPHK1 is localized in the cytoplasm. Subsequent RNA-Seq analysis of an overexpression plasmid identified 25 differentially expressed genes (DEGs), including 13 upregulated and 12 downregulated. Notably, nsun5 and hsp90aa1 were significantly upregulated, while Nfkbia and hmox1 were downregulated. Promoter analysis indicated that the core regulatory regions of Ydsphk1 are located between −1931~−1679 bp and −419~+92 bp, with two predicted TFAP2A binding sites in the −419~+92 bp region. Further studies demonstrated that varying concentrations of TFAP2A significantly reduced Ydsphk1 promoter activity. These findings underscore the pivotal role of Ydsphk1 in regulating immune responses in yellow drum, particularly through its impact on key immune-related genes and pathways such as NF-κB signaling and ferroptosis. The identification of Ydsphk1 as a mediator of immune regulation provides valuable insights into the molecular mechanisms of immune defense and highlights its potential as a target for enhancing pathogen resistance in aquaculture practices. This study lays a strong foundation for future research aimed at developing innovative strategies for disease management in aquaculture species. Full article