Sweet sorghum (Sorghum bicolor Moench) seedling emergence and growth are significantly impeded by physical soil crusts (PSCs) in saline-alkaline soils. Abscisic acid (ABA) is a potent seed priming agent known for modulating plant physiological and metabolic responses under salinity stress. However, the influence of ABA priming on seedling emergence in PSCs remains unclear. This study conducted both pot and field experiment to examine the effects of ABA priming on enhancing seedling emergence under PSC conditions. ABA priming altered the balance of at least 24 endogenous phytohormones, including abscisic acid, jasmonic acid, gibberellins, ethylene, auxins, and cytokinins. Additionally, it reprogrammed starch and sucrose metabolism, resulting in the differential expression of genes encoding key enzymes such as AMY, BAM, and INV, which are crucial for converting complex sugars into readily available energy sources, thereby supporting seedling growth. Furthermore, 52 differentially expressed metabolites (DEMs) of flavonoids were identified in germinating seedlings, including 15 anthocyanins, 3 flavones, 7 flavonols, 6 isoflavones, 7 flavanones, and 14 other flavonoids. Genetic and metabolic co-expression network analysis, along with flavonoid biosynthesis pathway exploration, revealed that the biosynthesis of 17 key DEMs-including liquiritigenin, apigenin, kaempferide, syringetin, phloretin, formononetin, dihydrokaempferol, and xanthohumol-was regulated by 10 differentially expressed genes (DEGs) associated with flavonoid biosynthesis. These DEGs encoded 7 enzymes critical for this pathway, including chalcone synthase, shikimate O-hydroxycinnamoyltransferase, bifunctional dihydroflavonol 4-reductase, naringenin 7-O-methyltransferase, and anthocyanidin reductase. This regulation, along with reduced levels of superoxide anion (O2-) and malondialdehyde and increased antioxidant enzyme activities, suggested that flavonoids played a vital role in mitigating oxidative stress. These findings demonstrate that ABA priming can effectively enhance sweet sorghum seedling emergence in PSCs by accelerating emergence and boosting stress resistance.

Proteasome is an essential organelle responsible for maintaining cellular protein homeostasis, but its relationship with DNA methylation remains unknown. In this study, we assessed DNA methylation of colorectal cancer (CRC) cells following treatment with proteasome inhibitors, and investigated the underlying mechanism of DNA methylation changes and the biological effects on CRC cells. We established that inhibition of proteasome leads to significant alterations in DNA methylation profile in CRC by suppressing the synthesis of DNA methyltransferases (DNMTs). We found that treating CRC cells with proteasome inhibitors results in attenuated translation of DNMT1 and DNMT3B, mediated by the inactivation of AKT and mammalian target of rapamycin (mTOR), which is dependent on the accumulation of p300, an acetyltransferase that inhibits AKT through acetylation modification. Furthermore, we demonstrated that downregulation of DNMT1 and DNMT3B confers protection against proteasome inhibitor treatment, potentially through reprogramming the transcriptome of CRC cells, highlighting the significant role of DNMTs in response to disruptions in protein homeostasis. Interestingly, it appears that the proteasome inhibitor-induced downregulation of DNMT1 and DNMT3B is specific to CRC. Altogether, our findings reveal an epigenetic effect of proteasome on DNA methylation in CRC through its regulation of DNA methyltransferase synthesis.

Salt stress has devastating effects on agriculture, yet the key regulators modulating the transcriptional dynamics of salt-responsive genes remain largely elusive in plants. Here, we discover that salt stress substantially induces the kinase activity of Mediator cyclin-dependent kinase 8 (CDK8), which is essential for its positive role in regulating salt tolerance. CDK8 is identified to phosphorylate AT-hook motif nuclear-localized protein 10 (AHL10) at serine 314, leading to its degradation under salt stress. Consistently, AHL10 is found to negatively regulate salt tolerance. Transcriptome analysis further indicates that CDK8 regulates over 20% of salt-responsive genes, half of which are co-regulated by AHL10. Moreover, AHL10 is revealed to recruit SU(VAR)3-9 homologs (SUVH2/9) to AT-rich DNA sequences in the nuclear matrix-attachment regions (MARs) of salt-responsive gene promoters, facilitating H3K9me2 deposition and repressing salt-responsive genes. Our study thereby has identified the CDK8-AHL10-SUVH2/9 module as a key molecular switch controlling transcriptional dynamics in response to salt stress.

Novel treatment options are needed for the gastric pathogen Helicobacter pylori due to its increasing antibiotic resistance. The vitamin K analogue menadione has been extensively studied due to interest in its anti-bacterial and anti-cancer properties. Here, we investigated the effects of menadione on H. pylori growth, viability, antibiotic resistance, motility and gene expression using clinical isolates. The MIC of menadione was 313 µM for 11/13 isolates and 156 µM for 2/13 isolates. The minimum bactericidal concentrations were 1.25-2.5 mM, indicating that concentrations in the micromolar range were bacteriostatic rather than bactericidal. We were not able to experimentally evolve resistance to menadione in vitro. Sub-MIC menadione (16 µM for 24 h) did not significantly inhibit bacterial growth but significantly (P<0.05) changed the expression of 1291/1615 (79.9%) genes encoded by strain 322A. The expression of the virulence factor genes cagA and vacA was downregulated in the presence of sub-MIC menadione, while genes involved in stress responses were upregulated. Sub-MIC menadione significantly (P<0.0001) inhibited the motility of H. pylori, consistent with the predicted effects of the observed significant (P<0.05) downregulation of cheY, upregulation of rpoN and changes in the expression of flagellar assembly pathway genes seen in the transcriptomic analysis. Through in-depth interrogation of transcriptomic data, we concluded that sub-MIC menadione elicits a general stress response in H. pylori with survival in the stationary phase likely mediated by the upregulation of surE and rpoN. Sub-MIC menadione caused some modest increases in H. pylori susceptibility to antibiotics, but the effect was variable with strain and antibiotic type and did not reach statistical significance. Menadione (78 µM) was minimally cytotoxic to human gastric adenocarcinoma (AGS) cells after 4 h but caused a significant loss of cell viability after 24 h. Given its inhibitory effects on bacterial growth, motility and expression of virulence- and colonization-associated genes, menadione at low micromolar concentrations may have potential utility as a virulence-attenuating agent against H. pylori.

Diabetic kidney disease (DKD) is a prevalent complication associated with diabetes in which podocyte dysfunction significantly contributes to the development and progression of the condition. Ring finger protein 183 (RNF183) is an ER-localized, transmembrane ring finger protein with classical E3 ligase activity. However, whether RNF183 is involved in glomerular podocyte dysfunction, which is the mechanism of action of DKD, is still poorly understood. In this study, we first demonstrated that RNF183 expression in glomerular podocytes of patients with DKD decreased as the disease progressed. Additionally, our transcriptome sequencing analysis of kidney tissues from diabetic mice revealed a significant reduction in RNF183 expression within the kidney cortex. Similarly, the expression of RNF183 was significantly reduced both in the kidneys of diabetic mice and in human podocytes exposed to high glucose conditions. The downregulation of RNF183 resulted in a suppression of autophagic activity, an increase in apoptotic cell death, and reduced expression of cellular markers in HPC cells. We found that RNF183 was modified via N6-methyladenosine (m6A) RNA methylation. Meanwhile, treatment with meclofenamic acid 2 (MA2), an m6A demethylase inhibitor, resulted in the upregulation of RNF183 expression in HPC cells cultured in high glucose conditions. Furthermore, high glucose treatment decreased the transcription and protein levels in both the m6A writer methyltransferaselike3 (METTL3) and the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). IGF2BP2 assisted with METTL3, which is jointly involved in the transcription of RNF183. Furthermore, we confirmed that RNF183 directly ubiquitinates M2 pyruvate kinase (PKM2) through co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS) experiments. The level of PKM2 ubiquitination was increased following RNF183 overexpression, leading to enhanced PKM2 protein degradation and subsequently alleviating high glucose-induced podocyte damage. The results of this study indicated that RNF183 was regulated via m6A methylation modification and that RNF183 expression was reduced in HPC cells treated with high glucose, which resulted in decreased PKM2 ubiquitination levels and subsequently aggravated podocyte injury. The findings suggest that RNF183 may serve as a potential therapeutic target for diabetic kidney injury, offering new insights into its role in the progression of DKD.

Nephrin is an essential constituent of the slit diaphragm of the kidney filtering unit. Loss of nephrin expression leads to protein leakage into the urine, one of the hallmarks of kidney damage. Autoantibodies against nephrin have been reported in patients with minimal change disease and recurrent focal segmental glomerulosclerosis. Understanding the mechanism of nephrin loss may help improve or lead to the development of novel treatment strategies. In this study, we demonstrated the important function of miR-204-5p expression on the protection of nephrin from anti-nephrin antibodies present in nephrotoxic serum (NTS). In addition, we identified that aspartyl protease cathepsin D is one enzyme that may be involved in nephrin enzymatic degradation and that cathepsin D is a direct target of miR-204-5p gene regulation. The regulation of miR-204-5p expression was determined to be regulated by the long noncoding RNA Josd1-ps. In an NTS in vivo animal model, treatment with the pan aspartic protease inhibitor Pepstatin A ameliorated renal damage. Finally, we showed that the expression of miR-204-5p had a nephrin-protecting function in vitro. Developing a method of delivery of miR-204-5p specifically to podocytes in vivo may provide a novel method of nephroprotection against nephrin autoantibodies.

Ageing is a major risk factor for cognitive and physical decline, but its mechanisms remain poorly understood. This study aimed to detect early cognitive and physical changes, and to analyze the pathway involved by monitoring two groups of mice: a young and an adult group. The study has identified the types of molecules involved in the hippocampus. Adult mice (47 weeks) showed significantly reduced exploratory behavior compared to young mice (11 weeks), although spatial working memory showed no difference. In terms of physical function, grip strength was significantly reduced in adult mice. The Frailty Index (FI) further highlighted age-related changes in adult mice. To investigate the causes of cognitive decline, adult mice were categorized based on their declining cognitive function. Microarray analysis of their hippocampi revealed that the cholinergic receptor nicotinic α3 subunit (Chrna3) was significantly reduced in mice with cognitive decline compared to controls. Subsequent in vitro experiments showed that oxidative stress and cholinesterase inhibitors decreased Chrna3 expression, whereas nicotine and cytisine increased it. These results suggest that Chrna3 is a key factor in age-related cognitive decline. The development of therapeutic strategies targeting Chrna3 expression may offer promising avenues for preclinical and clinical research to mitigate cognitive ageing.

Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive- and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus, and SARS-CoV-2, not only in mammalian and insect cells but also in precision-cut lung slices and neuronal organoids. ULK1 activation inhibited enveloped viruses but not EV71. However, receptor presentation at the cytoplasmic membrane remained unaffected, indicating that receptor binding was unchanged, while later stages of endocytosis were targeted via two distinct pathways. Drug-induced activation of the AMPK pathway reduced early endocytic factor TXNIP by suppressing translation. In contrast, the amounts of Rab5 and the late endosomal marker Rab7 decreased due to translation inactivation and ULK1-dependent proteasome activation within minutes. Furthermore, activation of AMPK hindered the late replication steps of SARS-CoV-2 by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction in early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acid and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses.

Tumors undergo metabolic reprogramming to meet the energetic, synthetic and redox demands essential for malignancy, often characterized by increased glycolysis and lactate production. However, the role of mitochondrial metabolism in tumor immunity remains unclear. The present study integrates spatial transcriptomics, bulk transcriptomics and proteomics, revealing a strong link between the metabolite succinyl-CoA and tumor immunity as well as the efficacy of anti-programmed cell death protein-1 (PD-1) therapy in patients with melanoma. Elevated succinyl-CoA levels, through α-ketoglutarate or succinate supplementation, enhanced T cell-mediated tumor elimination, both in vitro and in vivo. Mechanistically, succinylation of the ligand of PD-1 (PD-L1) at lysine 129 led to its degradation. Increased carnitine palmitoyltransferase 1A (CPT1A), identified as a succinyltransferase for PD-L1, boosted anti-tumor activity. Preclinically, bezafibrate, a hyperlipidemia drug, upregulated CPT1A and synergized with CTLA-4 monoclonal antibody to inhibit tumor growth. Clinically, higher PD-L1 and lower CPT1A levels in tumors correlated with better anti-PD-1 therapy responses, suggesting potential biomarkers for prediction of treatment efficacy.

Melatonin, also known as the pineal hormone, is secreted by the pineal gland and primarily regulates circadian rhythms. Additionally, it possesses immunomodulatory properties and anticancer effects. However, its specific mechanism in hepatocellular carcinoma (HCC) remains unclear, particularly regarding its effect on HCC-mediated immune escape through PD-L1 expression.In this study, in vitro experiments were conducted using Huh7 and HepG2 HCC cells. Melatonin treatment was applied to both cell types to observe changes in malignant phenotypes. Additionally, melatonin-pretreated Huh7 or HepG2 cells were co-cultured with T cells to simulate the tumor microenvironment. The results showed that melatonin inhibited cancer cell proliferation, migration, and invasion, as well as reduced PD-L1 expression in cancer cells, exhibiting similar anti-cancer effects in the co-culture system. In vivo experiments involved establishing ascitic HCC mouse models using H22 cells, followed by subcutaneous tumor models in Balb/c nude and Balb/c wild-type mice. Melatonin inhibited tumor growth and suppressed PD-L1 expression in cancer tissues in both subcutaneous tumor models, and it increased T lymphocyte activity in the spleen of Balb/c wild-type mice. Overall, the in vitro and in vivo experiments demonstrated that melatonin has dual anti-cancer effects in HCC: direct intrinsic anti-cancer activity and enhancement of anti-tumor immunity by reducing PD-L1 expression thereby inhibiting cancer immune escape. Furthermore, a decrease in the expression of the upstream molecule HIF-1α of PD-L1 and an increase in the expression levels of JNK, P38, and their phosphorylated forms were detected. Thus, the mechanism by which melatonin reduces PD-L1 may involve the downregulation of HIF-1α expression or the activation of the MAPK-JNK and MAPK-P38 pathways. This provides new insights and strategies for HCC treatment.

Exposure to di-n-butyl phthalate (DBP) during embryo development or lactation has been linked to reproductive toxicity. The ten-eleven translocation (TET) protein family plays a role in various pathological processes; however, its involvement in reproductive dysfunction in offspring mice exposed to DBP during gestation remains sparsely reported. In this study, SPF C57BL/6 pregnant mice were intragastrically administered DBP at doses of 0.5, 5, and 75 mg/kg body weight, or corn oil as a control, from gestational days 5-19. Following weaning, the offspring mice were maintained on a standard diet for 5 weeks. Additionally, mono-n-butyl phthalate (MBP)-induced TM3 cells were utilized to explore the underlying mechanisms in vitro. The results showed that in utero exposure to DBP resulted in diminished sperm quality, testicular damage, decreased reproductive hormone levels, and reduced expression of testosterone synthesis proteins in male offspring mice. Moreover, DBP exposure influenced the expression of steroidogenic acute regulatory protein (StAR) via the cAMP/PKA signaling pathway, associated with luteinizing hormone receptor (LHR)-mediated suppression of testosterone synthesis. Notably, DBP exposure led to decreased expression of TET methylcytosine dioxygenase 2 (TET2) in the progeny, and overexpression or silencing of TET2 affected the levels of proteins involved in the LHR-mediated testosterone synthesis pathway. Further investigations revealed that TET2 downregulation inhibits testosterone synthesis through the LHR-mediated LH/cAMP/PKA/StAR signaling pathway, ultimately impairing reproductive function in DBP-exposed offspring mice during gestation. This study provides a novel perspective for identifying molecular markers that may be more sensitive indicators of male reproductive damage from an epigenetic standpoint.

The tumor suppressor p53 can trigger tumor resistance to chemotherapy by facilitating DNA damage repair and maintaining genomic integrity. Here, we report that a p53-induced circular RNA circASCC3 promotes chemotherapeutic resistance by resolving R-loops. Our results reveal that p53 directly activates the transcription of ASCC3, the host gene of circASCC3. In addition, the RNA-binding protein SFPQ is identified to inhibit the formation of circASCC3 by associating with its flanking regions. Importantly, p53 facilitates the formation of circASCC3 by repressing the expression of SFPQ. CircASCC3 has a marginal effect on the survival and growth of cancer cells under normal growing conditions but surprisingly boosts their survival and growth in response to DNA damage stress. Mechanistic analysis reveals that circASCC3 binds to the DEAD-box RNA helicase DDX5 to inhibit its proteasomal degradation. This results in the prevention of R-loop accumulation due to DNA damage, thereby conferring tumor resistance to chemotherapy. Together, our study uncovers that p53 activates circASCC3 to promote R-loop resolution, which maintains genomic stability and potentially contributes to chemoresistance.

Esophageal cancer is one of the most common malignant tumors of the digestive tract, and miR-224 can promote the hypoxia tolerance of esophageal cancer cells. The expression of miR-224 and HIF-1α in esophageal cancer cells under hypoxic induction and their relationship with ROS was studied using RT-qPCR and Western Blot assays; cell viability and apoptosis under hypoxia, as well as the effects of miR-224 on cell proliferation and drug resistance, were investigated using CCK8, Annexin V-FITC/PI, H2DCFDA staining and Western Blot assays. Under hypoxic induction, miR-224 and HIF-1α expressions were upregulated, with the upregulation of miR-224 being related to ROS accumulation, while HIF-1α upregulation was not affected by ROS. Furthermore, the upregulation of miR-224 facilitated the survival of esophageal cancer cells under hypoxic conditions and reduced their chemosensitivity to CDDP. This effect was also validated in vitro, as miR-224 overexpression promoted the malignant behaviors in ESCC cells. Under hypoxic induction, ROS accumulation can lead to the upregulation of miR-224. MiR-224 facilitates the survival of esophageal cancer cells under hypoxic conditions and induces chemotherapeutic drug resistance.

Fetal alcohol spectrum disorders (FASD) describe ethanol-induced developmental defects including craniofacial malformations. While ethanol-sensitive genetic mutations contribute to facial malformations, the impacted cellular mechanisms remain unknown. Signaling via bone morphogenetic protein (Bmp) is a key regulatory step of epithelial morphogenesis driving facial development, providing a possible ethanol-sensitive mechanism. We found that zebrafish carrying mutants for Bmp signaling components are ethanol-sensitive and affect anterior pharyngeal endoderm shape and gene expression, indicating that ethanol-induced malformations of the anterior pharyngeal endoderm cause facial malformations. By integrating FASD patient data, we provide the first evidence that variants of the human Bmp receptor gene BMPR1B associate with ethanol-related differences in jaw volume. Our results show that ethanol exposure disrupts proper morphogenesis of, and tissue interactions between, facial epithelia that mirror overall viscerocranial shape changes and are predictive for Bmp-ethanol associations in human jaw development. Our data provide a mechanistic paradigm linking ethanol to disrupted epithelial cell behaviors that underlie facial defects in FASD.

Changweiqing (CWQ) is a Chinese herbal formula for the treatment of the gastrointestinal tract diseases, but its role in the treatment of colorectal cancer (CRC) has not been clarified. This study aimed to explore the molecular mechanism of CWQ in CRC treatment through bioinformatics analysis and network pharmacology.

Diazinon is a commonly used organophosphate (OP) insecticide especially in developing countries for the control of insect pests, however, exposure to its toxic impact especially in humans and other non-target species remains an important public health concern. The study aimed to investigate the effect of epigallocatechin -3- gallate (EGCG), abundant in green tea plants on neurobehavioural, biochemical, and pathological changes in the brain of male Wistar rats following exposure to diazinon toxicity. Sixty adult male Wistar rats were acclimatized for seven days and subsequently randomly assigned into six treatment groups as follows: Group I: Control group (0.2 mL distilled water); Group II: Diazinon at 3 mg/kg (1% LD50); Group III: Diazinon (3 mg/kg) + EGCG (50 mg/kg, ~ 2% of LD50); Group IV: Diazinon (3 mg/kg) + EGCG (100 mg/kg, ~ 5% of LD50); Group V: EGCG (50 mg/kg) and Group VI: EGCG (100 mg/kg). All treatments were administered orally once daily for 14 days. Neurobehavioural studies, biomarkers of oxidative stress, histology, immunohistochemistry, and quantitative polymerase chain reaction (RT qPCR) were performed. Diazinon alone impaired recognition memory, increased oxidative stress markers and altered antioxidant defense in the brain. It upregulated TNF-α and IL-6 genes and repressed GPx 4 gene expressions. It was also associated with increased GFAP, Tau, and α-SN immunoreactivity. Microscopic examination revealed loss of Purkinje and hippocampal cells in brain. Co-treatment with EGCG however improved cognition, lowered oxidative stress markers, improved antioxidant status and suppressed TNF-α and IL-6. In conclusion, findings from this study demonstrated that EGCG offered protection against diazinon-induced neurotoxicity. Hence, natural sources of epigallocatechin -3- gallate such as fruits and vegetables could offer immense benefits by protecting against oxidative stress and inflammation in neurodegenerative disease conditions.Clinical trial number Not applicable.

Aloe vera is a popular medicinal plant in the cosmetic, pharmaceutical, and food industries. Acemannan (ACE), a β-(1,4)-acetylated mannan, is one of the bioactive compounds isolated from the A. vera gel. The pharmacological effects of ACE have been reported regarding digestive disease protection, antimicrobia, and prebiotic activity. Here, we used human HaCaT cells as a model to uncover the potential biological functions of ACE in keratinocytes. ACE increased cell growth in a concentration-dependent manner, and a higher incorporation of BrdU was detected in ACE-treated cells than in vehicle-treated cells, indicating ACE promotes cell proliferation. Furthermore, ACE concentration-dependently promoted cell migration in the wound scratch model. ACE regulated cell differentiation by transiently decreasing p63α expression, but increasing the expression of involucrin, loricrin, and transglutaminase 1 (TGase 1). These effects were non-additive to those induced by phorbol myristate acetate (PMA), but additive to epidermal growth factor (EGF), which are complete and incomplete differentiation agents of keratinocytes, respectively. Moreover, ACE activated EGF receptor (EGFR), protein kinase C (PKC), and protein kinase B (AKT/PKB). PKC inhibitor Ro320432 enhanced cell growth and migration, while EGFR inhibitor osimertinib blocked both responses. In summary, ACE is a potential therapeutic agent in wound healing. ACE activates PKC, leading to keratinocyte differentiation and activates EGFR, contributing to keratinocyte proliferation and migration.

Synovitis represents the initial pathological change in osteoarthritis and contributes to its progression. Resolvin D1 (RV-D1) is a novel and endogenous docosahexaenoic acid-derived lipid mediator, which regulates the duration and magnitude of inflammation by downregulating pro-inflammatory genes and mediators. However, the effects of RV-D1 on synovitis remain unknown. The aim of the present study was to investigate the anti-inflammatory effects of RV-D1 in human fibroblast-like synoviocytes (HFLSs) and the underlying mechanisms. The expression of the HFLS formyl peptide receptor 2 (ALX/FPR) was examined via immunocytochemical analysis. HFLSs were treated with 1 ng/mL recombinant human interleukin-1β (IL-1β) and RV-D1. The gene expression of interleukin-1β (IL1B), matrix metalloproteinase 3 (MMP3), and MMP13 was examined using real-time reverse transcription-polymerase chain reaction after treatment with IL-1β and RV-D1. The effect of RV-D1 on apoptosis was examined based on fluorescence intensity. Phosphorylation of p-38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, nuclear factor kappa B (NF-κB), and AKT was analyzed via western blotting. ALX/FPR staining was observed on the cell surface. RV-D1 significantly suppressed the IL-1β-induced increase in gene and protein expression of IL-1β, MMP-3, and MMP-13. Pretreatment with 100 nM RV-D1 significantly increased the fluorescence intensity compared to that in the non-treatment group. Furthermore, pretreatment with RV-D1 significantly suppressed the phosphorylation of p-38, NF-κB, and AKT. Whereas WRW4, an antagonist of ALX/ FPR2, treatment weakened the effect of RV-D1, resulting in p-38, NF-κB, and AKT phosphorylation and the protein expression of MMP-13 at levels comparable to those in the IL-1β without RV-D1. In conclusion, RV-D1 suppressed IL-1β and MMP expression by inhibiting the phosphorylation of p-38, NF-κB, and AKT in inflammation in HFLSs. RV-D1 can be used to develop treatments for osteoarthritis and other inflammatory disorders.

Recently, we presented cell-permeable CaaX peptides as versatile tools to study intracellular prenylation of proteins. These peptides consist of a cell-penetrating peptide (CPP) and a C-terminal CaaX motif derived from Ras proteins and demonstrated high cellular accumulation and the ability to influence Ras signaling in cancerous cells. Here, we aimed to gain a deeper insight into how such cell-permeable CaaX peptides, particularly the KRas4B-derived CaaX-1 peptide, interact with farnesyltransferase (FTase) and likely influence further intracellular processes. We show that CaaX-1 is farnesylated by FTase ex cellulo and that an intact CaaX motif is required for modification. A competition experiment revealed a slower farnesylation of CaaX-1 by FTase compared to a CaaX motif-containing control peptide. CaaX-1 inhibited farnesylation of this control peptide at considerably lower concentrations; thus, a higher affinity for FTase is hypothesized. Notably, AlphaFold3 not only predicted interactions between CaaX-1 and FTase but also suggested interactions between the peptide and geranylgeranyltransferase type I. This finding encourages further investigation, as cross-prenylation is a well-known drawback of FTase inhibitors. Our results are further evidence for the usefulness of CaaX peptides as tools to study and manipulate the prenylation of proteins. They offer real potential for the development of novel inhibitors targeting the prenylation pathway.

Ovarian cancer therapy remains a challenge for human health, partly due to chemotherapy resistance. Understanding the molecular mechanisms underlying this resistance is crucial. Therefore, to identify genes involved in cisplatin resistance in ovarian cancer, RNA-seq analysis of A2780cp (cisplatin-resistant) and A2780 (cisplatin-sensitive) cell lines was performed, revealing 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3) as a differentially expressed candidate gene. First, MTT analysis confirmed the drug resistance of A2780cp and the sensitivity of A2780 cell lines. Subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting analyses revealed elevated AGPAT3 and mTOR expression in A2780cp cells compared with A2780 cells. Additionally, western blotting showed increased p-mTOR (phospho-mTOR)/mTOR and p-S6K (phospho-S6K)/S6K ratios in A2780cp cells. The overexpression of AGPAT3 in A2780 cells led to increased p-mTOR/mTOR and p-S6K/S6K ratios and increased IC50 values, as shown by RT-qPCR, western blotting, and MTT analysis. Conversely, shRNA-mediated downregulation of AGPAT3 resulted in reduced p-mTOR/mTOR and p-S6K/S6K ratios. At the cellular level, AGPAT3 overexpression in A2780 cells increased survival rates, decreased apoptosis, and caused G2/M cell cycle arrest under cisplatin treatment, as detected by apoptosis assay, and cell cycle flow cytometry analysis. Overall, we conclude that AGPAT3 is involved in cisplatin resistance in A2780cp cells and propose that targeting this gene or its enzymatic product could help overcome drug resistance.