Search Results (61)

Background: Brain microvascular endothelial cells (BMECs) form the blood–brain barrier (BBB), a highly selective interface that restricts paracellular diffusion and regulates the transport of nutrients and drugs into the central nervous system via specialized transporters and receptors. Tight junction-associated protein 1 (Tjap1), also termed protein incorporated later into tight junctions (Pilt), has been localized to tight junctions (TJs) in epithelial cells and to the trans-Golgi network in fibroblasts; however, its expression, subcellular localization, and functional significance in BMECs are still unknown. Methods: We characterized Tjap1 subcellular localization in mouse and human BMEC cell lines as well as primary mouse BMECs by immunofluorescence with and without pharmacological Golgi disruption by treatment with Brefeldin A, Golgicide A or Pitstop 2. CRISPR/Cas9-mediated Tjap1 knockout cells were generated and examined with regard to their Golgi morphology using immunostaining. Tjap1 mRNA localization was examined by RNAscope in situ hybridization. Quantitative real-time PCR and Western blot was performed to assess the expression of BBB-associated efflux transporters, solute carrier transporters, and cellular receptors in control and Tjap1 knockout cells. Results: Tjap1 predominantly localized to the cis-Golgi compartment, co-localizing with Gm130 rather than Tgn38, and was absent from TJs in BMECs. Tjap1 knockout induced pronounced Golgi fragmentation BMECs. Importantly, Tjap1 knockout significantly downregulated mRNA-expression of Abcb1a, Abcb1b, Abcc4, Slc2a1, Slc7a1, Slc7a5 and Tfrc, while Abcg2 was upregulated. At the protein level, a decrease in the protein levels of Abcb1, Abcc4, Slc2a1, Slc7a1, and Tfrc was observed in Tjap1 knockout cEND cells. Conclusions: In BMECs, Tjap1 is a cis-Golgi-associated protein required for the structural integrity of the Golgi apparatus. Its deletion is associated with Golgi fragmentation and significant alterations in the mRNA and protein expression of drug transporters and receptors at the BBB. These findings identify Tjap1 as a candidate regulator of both Golgi architecture and the BBB transporter profile in vitro, with potential implications for modulating drug transport across the BBB. Full article

Aberrant protein glycosylation is a key driver of colorectal cancer (CRC) progression, contributing to tumour growth, metastasis, and immune evasion. In this study, computational approaches were employed to explore the potential of Brefeldin A as an inhibitor of two glycosylation-associated regulatory proteins: Protein Kinase C alpha (PrKCα) and Mitogen-Activated Protein Kinase 1 (MAPK1). Using computational docking and structural analyses, Brefeldin A was predicted to bind effectively to both targets, thereby inhibiting their enzymatic activities. Detailed investigations revealed that Brefeldin A interacts favourably within the active sites of MAPK1 and PrKCα, forming stable complexes by optimal binding interactions. Key residues contributing to binding stabilisation were identified in both MAPK1 and PrKCα. For MAPK1, residues such as Lys114 and Ser153 played a significant role in hydrogen bonding interactions, while for PrKCα, Gln105, Asn154, and Asp167 were notably involved. These interactions included both hydrogen bonds and hydrophobic contacts, which collectively contributed to the strength and specificity of ligand binding. The identification of these residues provides insight into the molecular mechanisms underlying the stabilisation of the Brefeldin A-kinase complexes. Binding affinity estimations showed that Brefeldin A bound to MAPK1 exhibited a binding energy of −22.18 ± 4.50 kcal/mol. In contrast, the Brefeldin A bound to PrKCα demonstrated a slightly stronger binding energy of −23.90 ± 5.36 kcal/mol. Collectively, these findings underscore Brefeldin A’s potential as a novel inhibitor targeting glycosylation-related proteins in CRC, offering a promising therapeutic strategy to impede CRC progression. This work not only proposes Brefeldin A as a promising therapeutic lead but also supports glycosylation inhibition as a valuable approach for CRC control, with broader implications for drug discovery in glycan-related oncogenic pathways. Full article

Toscana virus (TOSV), a negative-sense RNA phlebovirus transmitted by Phlebotomus sandflies and endemic in Mediterranean regions, is an emerging pathogen capable of causing diseases ranging from mild febrile illness to severe central nervous system involvement. With no licensed vaccines or specific antiviral therapies available, the identification of novel therapeutic approaches is urgently needed. Microbial secondary metabolites have recently attracted attention for their broad-spectrum antiviral activities. Among them, monensin and brefeldin A have shown antiviral efficacy against a variety of viruses, often by disrupting viral protein trafficking and inducing Golgi-associated stress responses. However, their potential activity in the context of TOSV infection has not yet been explored. This study aimed to evaluate the in vitro antiviral activity of monensin and brefeldin A against TOSV and to gain mechanistic insights into their effects at the cellular level. Vero cells were infected with TOSV and treated with monensin (1.5–25 µM) or brefeldin A (10.9–175 nM) at different time points (4, 6, 12, 24 h). Cytotoxicity was assessed using MTT and hemolysis assays. Antiviral activity was measured via plaque reduction assays and quantitative real-time PCR targeting the viral L gene. Western blot analysis was performed to assess TFE3 expression, a transcription factor associated with the Golgi stress response. Monensin exhibited rapid antiviral activity, achieving IC₅₀ values of 2.7 µM and 2.5 µM at 4 and 6 h post-treatment, respectively, with dose-dependent suppression of viral L gene expression. Brefeldin A displayed a delayed effect, with maximal inhibition after 12 h (IC₅₀ = 66.9 nM). Monensin treatment induced a concentration-dependent upregulation of TFE3, while brefeldin A caused only a modest increase, suggesting differential activation of the Golgi stress response during TOSV infection. These findings support the potential of microbial metabolites as therapeutic candidates for emerging arboviral infections in the Mediterranean region. Full article

Although single-cell analyses have advanced our understanding of pancreatic ductal adenocarcinoma (PDAC), most studies to date have focused on primary and metastatic tumors. Here, we map cell composition, phenotypic plasticity, and microenvironmental remodeling from human normal pancreas through preneoplastic lesions to PDAC, with the preneoplastic phase recognized as a critical window for carcinogenesis. We pinpoint genes that are persistently dysregulated throughout malignant transformation and are associated with a poor prognosis. Focusing on ductal and acinar cells as the principal origins of PDAC, we delineate malignant preneoplastic cell clusters that exhibit strong carcinogenic potential. Immune profiling reveals marked expansion and functional reprogramming of macrophages during disease progression. Integrative analysis with human PDAC bulk transcriptomic cohorts identifies candidate compounds, such as Brefeldin A, with potential for intervention in preneoplastic disease. Together, our study elucidates dynamic molecular and cellular mechanisms underlying PDAC carcinogenesis and provides actionable insights for early intervention and targeted therapy. Full article

Background/Objectives: Mesothelioma is a highly aggressive tumor with a poor prognosis that typically develops after a long latency period following asbestos exposure. Although immunotherapy combined with chemotherapy is increasingly used, the efficacy of standard treatments remains limited. This study aimed to explore ferroptosis induction as a potential therapeutic strategy for mesothelioma. Methods: We first screened microbial culture extracts collected from soil and marine environments to identify compounds with selective cytotoxicity against mesothelioma cells. Gene expression profiling was performed to investigate the mechanism of cell death induced by the identified compound. To assess intrinsic ferroptosis susceptibility, patient-derived mesothelioma cell lines and immortalized mesothelial cell lines were treated with RSL3, a GPX4 inhibitor. Results: Screening identified brefeldin A as a compound that selectively induces cell death in mesothelioma cells. Gene expression profiling revealed transcriptional changes consistent with ferroptosis induction. Treatment with RSL3 demonstrated marked variability in ferroptosis sensitivity across cell lines; the subgroup showing high sensitivity to RSL3 did not exhibit significant genetic alterations in NF2 or BAP1, but contained a significantly higher proportion of epithelioid tumors in histological classification. Conclusions: Our findings highlight ferroptosis induction as a promising antitumor mechanism in mesothelioma, particularly in the epithelioid subtype. While GPX4 inhibitors such as RSL3 are effective in vitro, further studies are needed to overcome pharmacological limitations and define molecular determinants of ferroptosis susceptibility, which may inform future personalized therapeutic strategies. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of targetable receptors, leading to limited treatment options and a critical need for novel therapeutic strategies. This study aimed to evaluate the potential of azelastine, a clinically approved H1-antihistamine, for drug repositioning against TNBC and to elucidate its underlying HRH1-independent mechanism of action. Cell viability assays (CCK-8) were performed on TNBC cell lines (MDA-MB-231 and BT-549) following treatment with azelastine and its major metabolite, desmethyl azelastine. After observing ambiguous clinical associations between HRH1 expression and patient prognosis, HRH1 dependency was assessed through histamine stimulation and HRH1 knockdown (siRNA). Subsequently, the role of ADP-ribosylation factor 1 (ARF1), found to be overexpressed in TNBC and linked to poor prognosis, was investigated using ARF1 knockdown (siRNA), co-treatment with the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) inhibitor golgicide A (GCA), and co-treatment with the Drp1 inhibitor M-divi 1. Azelastine and desmethyl azelastine potently reduced MDA-MB-231 cell viability in a dose- and time-dependent manner, achieving cell survivals of 61.3 ± 6.1% (30 µM) and 34.9 ± 3.7% (50 µM) for azelastine, and 52.4 ± 12.5% (30 µM) for desmethyl azelastine, respectively, after 72 h, with an IC₅₀ of 35.93 µM determined for azelastine in MDA-MB-231 cells. Additionally, azelastine significantly reduced the viability of BT-549 cells. Bioinformatic analysis of clinical datasets revealed HRH1 downregulation in tumors and, functionally, neither histamine stimulation nor HRH1 knockdown mediated azelastine cytotoxicity in cell culture. Importantly, ARF1 expression was significantly upregulated in TNBC and associated with poor prognosis. Co-treatment with GCA, preventing ARF1 activation, restored viability to near-control levels, supporting dependence on the GBF1–ARF1 activation axis of azelastine, whereas the Dynamic-related protein 1 (Drp1) inhibitor M-divi 1 not only partially rescued CCK-8-based cell viability but also normalized azelastine-induced loss of MitoTracker™ Red CMXRos signal and partially preserved (4′,6-diamidino-2-phenylindole) DAPI-based cell density, indicating Drp1-dependent mitochondrial dysfunction. Furthermore, azelastine selectively reduced p-ERK phosphorylation in the cell signaling pathway. Azelastine exerts potent anticancer effects in TNBC cells via an HRH1-independent, ARF1-dependent mechanism that attenuates the Extracellular signal-regulated kinase (ERK)–Drp1 axis, and induces Drp1-dependent mitochondrial dysfunction, independent of its canonical HRH1 receptor function. This ARF1-dependent mechanism provides strong scientific rationale for the drug repositioning of azelastine as an effective therapeutic agent for ARF1-driven TNBC. Full article

Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies. Full article

Chlorella, recognized for its high lipid and protein content, is increasingly studied for its potential in the food and bio industries. To enhance its production and understand the underlying mechanisms of lipid accumulation, this study investigated the role of endoplasmic reticulum (ER) stress in modulating lipid metabolism in Chlorella sorokiniana UTEX 2714, using six putative ER stress inducers: 2-deoxy-D-glucose (2-DG), dithiothreitol (DTT), tunicamycin (TM), thapsigargin (TG), brefeldin A (BFA), and monensin (Mon). The results showed that 2-DG, DTT, TM, BFA, and Mon significantly inhibited cell growth in C. sorokiniana. Treatment with 2-DG, DTT, TM, BFA, or Mon resulted in substantial increases in the triacylglycerol (TAG) to total fatty acid (tFA) ratio, with fold changes of 14.8, 7.9, 6.2, 10.1, and 8.9, respectively. Among the tFAs, cells treated with these compounds exhibited higher levels of saturated fatty acids and lower levels of polyunsaturated fatty acids (PUFAs). In contrast, the fatty acid composition of TAGs showed the opposite trend, with relative enrichment in PUFAs. This study enhances our understanding of Chlorella lipid metabolism, providing valuable insights for optimizing lipid production, particularly TAGs enriched with PUFA content, for applications in functional foods, nutraceuticals, and sustainable bioresources. Full article

Fusarium neocosmosporiellum is the main pathogen of peanut pod rot in China. To investigate the type of F. neocosmosporiellum toxin and its pathogenic mechanism, a macrolide, brefeldin A, was isolated. The structure of the compound was identified by 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization–mass spectrometry (HR-ESI-MS). At the same time, the content of the compound in healthy and diseased peanut capsules was detected, and its plant toxicity to radish, mung bean, rice, and peanut seed radicle elongation and pathogenicity to peanut pod rot were evaluated. The results showed that brefeldin A at 50 μg/mL could significantly inhibit the radicle elongation of rice seeds. Brefeldin A was detected only in pods with peanut rot. Injecting 2 mg/mL brefeldin A solution into peanut pods caused the severe decay of peanut pods at the R3R4 stage, which is consistent with the symptoms of peanut rot. Full article

Stress on the Endoplasmic reticulum (ER) can severely disrupt cellular function by impairing protein folding and post-translational modifications, thereby leading to the accumulation of poor-quality proteins. However, research on its impact on photosynthesis remains limited. In this study, we investigated the impact of ER stress on the photosynthetic efficiency of Chlamydomonas reinhardtii using pharmacological inducers, tunicamycin (TM) and brefeldin A (BFA), which specifically target the ER. Our measurements of photosynthetic parameters showed that these ER stress-inducing compounds caused a significant decline in photosynthetic efficiency. A proteomic analysis confirmed that TM and BFA effectively induce ER stress, as evidenced by the upregulation of ER stress-related proteins. Furthermore, we observed a widespread downregulation of photosynthesis-related proteins, which is consistent with the results obtained from our measurements of photosynthetic parameters. These findings suggest that the stress on ER has a profound impact on chloroplast function, disrupting photosynthetic processes. This study highlights the critical interdependence between the ER and chloroplasts, and it underscores the broader implications of ER stress on the cellular metabolism and energy efficiency of photosynthetic organisms. Full article

Background/Objectives: Axonal Charcot–Marie–Tooth disease type 2 (CMT2) accounts for 24% of Hereditary Motor/Sensory Peripheral Neuropathies. CMT2 type GG, due to four distinct heterozygous mutations in the Golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1) gene (OMIM 606483), was described in seven cases from four unrelated families with autosomal dominant inheritance. It is characterized by slowly progressive distal muscle weakness and atrophy, primarily affecting the lower limbs. Here, we present two siblings sharing a novel GBF1 variant. Methods: Patient II.1 (male, 61 years at onset) presented lower limb hypoesthesia and walking difficulty; the examination revealed a postural tremor, a positive Romberg test, and muscle atrophy in the lower limbs and hands. Patient II.2 (his sister, 59 years at onset) had lower limb dysesthesias, hand paresthesia, and lower-limb stiffness. They underwent clinical evaluations, blood tests, and electroneurography. Their father represents a potentially affected individual, although a genetic analysis was not conducted. Results: All tests for peripheral neuropathies were unremarkable, including metabolic and autoimmune screening. Both showed a mixed demyelinating–axonal sensory–motor neuropathy. Genetic analysis revealed a new heterozygous GBF1 variant of uncertain significance. Conclusions: Based on autosomal dominant inheritance, as well as clinical and physiological features, a possible novel CMT2GG was diagnosed. Further research, including functional assays and in vitro studies, is necessary to confirm this variant’s causal link. Full article

Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While Arabidopsis and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of SiAAP9, both of which were induced by NO₃⁻ and showed similar expression patterns. The overexpression of SiAAP9L and SiAAP9S in Arabidopsis inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, SiAAP9-OX transgenic Arabidopsis showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of SiAAP9 suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9’s sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of SiAAP9 have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding. Full article

Several Pleurocordyceps species have been reported as hyperparasitic fungi. A new species, Pleurocordyceps fusiformispora, and a known species, Perennicordyceps elaphomyceticola, are described here based on morphology and phylogenetic evidence from six genes (ITS, SSU, LSU, TET1-α, RPB1, and RPB2). Pl. fusiformispora differed from the other Pleurocordyceps species by producing flaky colonies, ovoid or elliptic α-conidia, and fusiform or long fusiform β-conidia. Both full genomes of Pe. elaphomyceticola and Pl. fusiformispora were sequenced, annotated, and compared. The antiSMASH and local BLAST analyses revealed significant differences in the number and types of putative secondary metabolite biosynthetic gene clusters, i.e., NPPS, PKS, and hybrid PKS–NRPS domains, between the two species. In addition, the putative BGCs of six compounds, namely ε-poly lysine, 4-epi-15-epi-brefeldin A, Monorden D/monocillin IV/monocillin VII/pochonin M/monocillin V/monocillin II, Tolypyridone, Piperazine, and Triticone DABFC, were excavated in the present study. This study motivates the use of heterologous expression and gene knockout methods to discover novel biologically active SMs from Polycephalomycetaceae. Full article

The aim of this study was to verify whether small molecules can improve the efficiency of precision gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoprotein (RNP) in porcine cells. CRISPR associated 9 (Cas9) protein, small guide RNA (sgRNA), phosphorothioate-modified single-stranded oligonucleotides (ssODN), and different small molecules were used to generate precise nucleotide substitutions at the insulin (INS) gene by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by polymerase chain reaction (PCR) for the target site. All samples were sequenced and analyzed, and the efficiencies of different small molecules at the target site were compared. The results showed that the optimal concentrations of the small molecules, including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A, for in vitro-cultured PFFs’ viability were determined. Compared with the control group, the single small molecules including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A increased the efficiency of HDR-mediated precise gene editing from 1.71-fold to 2.28-fold, respectively. There are no benefits in using the combination of two small molecules, since none of the combinations improved the precise gene editing efficiency compared to single small molecules. In conclusion, these results suggested that a single small molecule can increase the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells. Full article

Citrus target spot, caused by Pseudofabraea citricarpa, was formerly considered a cold-tolerant fungal disease. However, it has now spread from high-latitude regions to warmer low-latitude regions. Here, we conducted physiological observations on two different strains of the fungus collected from distinct regions, and evaluated their pathogenicity. Interestingly, the CQWZ collected from a low-latitude orchard, exhibited higher temperature tolerance and pathogenicity when compared to the SXCG collected from a high-latitude orchard. To further understand the evolution of temperature tolerance and virulence in these pathogens during the spread process, as well as the mechanisms underlying these differences, we performed genomic comparative analysis. The genome size of CQWZ was determined to be 44,004,669 bp, while the genome size of SXCG was determined to be 45,377,339 bp. Through genomic collinearity analysis, we identified two breakpoints and rearrangements during the evolutionary process of these two strains. Moreover, gene annotation results revealed that the CQWZ possessed 376 annotated genes in the “Xenobiotics biodegradation and metabolism” pathway, which is 79 genes more than the SXCG. The main factor contributing to this difference was the presence of salicylate hydroxylase. We also observed variations in the oxidative stress pathways and core pathogenic genes. The CQWZ exhibited the presence of a heat shock protein (HSP SSB), a catalase (CAT2), and 13 core pathogenic genes, including a LysM effector, in comparison to the SXCG. Furthermore, there were significant disparities in the gene clusters responsible for the production of seven metabolites, such as Fumonisin and Brefeldin. Finally, we identified the regulatory relationship, with the HOG pathway at its core, that potentially contributes to the differences in thermotolerance and virulence. As the global climate continues to warm, crop pathogens are increasingly expanding to new territories. Our findings will enhance understanding of the evolution mechanisms of pathogens under climate change. Full article