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Poultry Science May 2024The proliferation and death of granulosa cells (GCs) in poultry play a decisive role in follicular fate and egg production. The follicular fluid (FF) contains a variety...
The proliferation and death of granulosa cells (GCs) in poultry play a decisive role in follicular fate and egg production. The follicular fluid (FF) contains a variety of nutrients and genetic substances to ensure the communication between follicular cells. Exosomes, as a new intercellular communication, could carry and transport the proteins, RNA, and lipids to react on GCs, which had been found in FF of various domestic animals. Whether exosomes of FF in poultry play a similar role is unclear. In this study, geese, a poultry with low egg production, were chosen, and the effect of FF exosomes on the proliferation and death of GCs was investigated. Firstly, there were not only a large number of healthy small yellow follicles (HSYFs) but also some atresia small yellow follicles (ASYFs) in the egg-laying stage. Also, the GC layers of ASYFs became loose interconnections, inward detachment, and diminished survival rate than that of HSYFs. Besides, compared to HSYFs, the contents of E2, P4, and the mRNA expression levels of ferroptosis-related genes GPX4, FPN1, and FTH1 were significantly decreased, while COX2, NCOA4, VDAC3 mRNA were significantly increased, and the structure of mitochondrial cristae disappeared and the outer membrane broke in the GC layers of ASYFs. Moreover, the ROS, MDA, and oxidation levels in the GC layers of ASYFs were significantly higher than those of HSYFs. All these hinted that ferroptosis might result in a large number of GCs death and involvement in follicle atresia. Secondly, FF exosomes were isolated from HSYFs and ASYFs, respectively, and identified by TEM, NTA, and detection of exosome marker proteins. Also, we found the exosomes were phagocytic by GCs by tracking CM-Dil. Moreover, the addition of ASYF-FF exosomes significantly elevated the MDA content, Fe levels, and the mitochondrial membrane potential (MMP) in GCs, thus significantly inhibiting the proliferation of GCs, which was restored by the ferroptosis inhibitor ferrostatin-1. Thirdly, the proteomic sequencing was performed between FF-derived exosomes of HSYFs and ASYFs. We obtained 1615 differentially expressed proteins, which were mainly enriched in the protein transport and ferroptosis pathways. Among them, HMOX1 was enriched in the ferroptosis pathway based on differential protein-protein interaction network analysis. Finally, the role of HMOX1 in regulating ferroptosis in GCs was further explored. The highly expressed HMOX1 was observed in the exosomes of ASYF-FF than that in HSYF-FF. Overexpression of HMOX1 increased ATG5, LC3II, and NCOA4 expression and reduced the expression of FTH1, GPX4, PCBP2, FPN1 in the ferroptosis pathway, also promoted intracellular Fe accumulation and MDA surge, which drove ferroptosis in GCs. The effects of HMOX1 on ferroptosis could be blocked by its inhibitor Znpp. Taken together, the important protein HMOX1 was identified in FF, which could be delivered to GCs via exosomes, triggering ferroptosis and thus determining the fate of follicles.
PubMed: 38943808
DOI: 10.1016/j.psj.2024.103912 -
Poultry Science Jun 2024Imidacloprid (IMI), an insecticide from the neonicotinoid group widely used in agriculture, has drawn attention due to its potential harmful effects on non-target...
Imidacloprid (IMI), an insecticide from the neonicotinoid group widely used in agriculture, has drawn attention due to its potential harmful effects on non-target species, including bird populations. In the present work, we investigated the effect of IMI on avian semen by in vitro exposure of rooster spermatozoa to this pesticide. The semen was collected twice a week. Samples collected on one day were pooled and incubated with the following IMI concentrations: 0 mM, 0.5 mM, 5 mM, 10 mM, and 50 mM at 36°C for 3 h. Comprehensive semen analysis was carried out after 1 h and 3 h of incubation, evaluating sperm motility parameters with the CASA system and using flow cytometry to assess membrane integrity, mitochondrial activity, acrosome integrity, chromatin structure, intracellular calcium level and apoptosis markers such as: early apoptosis and caspase activation and lipid peroxidation. The results of the first experiment suggest that low concentrations of IMI have a different effect on sperm motility compared to higher concentrations. In IMI samples, we also observed a lower percentage of cells with a high level of calcium ions compared to the control, and a lower level of lipid peroxidation. We concluded that IMI may act as a blocker of calcium channels, preventing the influx of these ions into the cell. To confirm this mechanism, we conducted a second experiment with calcium channel blockers: SNX 325, MRS-1845, and Nifedipine. The results of this experiment confirmed that the mechanism of action of IMI largely relies on the blockade of calcium channels in rooster sperm. Blocking the influx of calcium ions into the cell prevents the formation of Ca²⁺-dependent pores, thereby preventing an increase in cell membrane permeability, ultimately blocking early apoptosis and lipid peroxidation in chicken spermatozoa.
PubMed: 38943803
DOI: 10.1016/j.psj.2024.103959 -
Bioelectrochemistry (Amsterdam,... Jun 2024This study assessed the viability of an anion-exchange microbial fuel cell (MFC) for extracting electricity from palm oil mill effluent (POME), a major pollutant in...
This study assessed the viability of an anion-exchange microbial fuel cell (MFC) for extracting electricity from palm oil mill effluent (POME), a major pollutant in palm-oil producing regions due to increasing demand. The MFC incorporated a tubular membrane electrode assembly (MEA) with an air core, featuring a carbon-painted carbon-cloth cathode, an anion exchange membrane (AEM), and a nonwoven graphite fabric (NWGF) anode. An additional carbon brush (CB) anode was placed adjacent to the tubular MEA. The MFC operated under semi-batch conditions with POME replacement every 7 days. Results showed superior performance of the AEM, with the highest power density (P) observed in POME-treated MFCs. Current and power density increased with CB addition; the best chemical oxygen demand (COD) removal efficiency reached 73 %, decreasing from 1249 to 332 mg/L with three CBs. The P was 0.18 W/m(-|-) with 1000 mg/L COD and three CBs, dropping to 0.0031 W/m(-|-) without CB and at 410 mg/L COD. Anode resistance, calculated using organic matter supplementation, COD, and anode surface area, decreased with increased COD or surface area, improving electricity production. AEM and CB compatibility synergistically enhanced MFC performance, offering potential for POME wastewater treatment and energy recovery.
PubMed: 38943780
DOI: 10.1016/j.bioelechem.2024.108770 -
Current Opinion in Structural Biology Jun 2024The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a... (Review)
Review
The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs).
PubMed: 38943706
DOI: 10.1016/j.sbi.2024.102869 -
STAR Protocols Jun 2024Brown adipose tissue (BAT) is mitochondria rich, enabling high oxidative metabolism for non-shivering thermogenesis. The release of large/small extracellular vesicles...
Brown adipose tissue (BAT) is mitochondria rich, enabling high oxidative metabolism for non-shivering thermogenesis. The release of large/small extracellular vesicles (EVs) containing mitochondria or mitochondrial fragments, termed mito-EVs, may support mitochondrial quality control or intercellular communication. We present a protocol to isolate and characterize mito-EVs. We detail steps for BAT processing, cell debris removal, differential centrifugation (dC), and mito-EV analysis by flow cytometry and immunoblotting assays. For complete details on the use and execution of this protocol, please refer to Rosina et al..
PubMed: 38943650
DOI: 10.1016/j.xpro.2024.103161 -
Cell Reports Jun 2024Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor...
Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor (PAFR). However, drug development targeting the PAFR has been limited, partly due to an incomplete understanding of its activation mechanism. Here, we present a 2.9-Å structure of the PAF-bound PAFR-G complex. Structural and mutagenesis analyses unveil a specific binding mode of PAF, with the choline head forming cation-π interactions within PAFR hydrophobic pocket, while the alkyl tail penetrates deeply into an aromatic cleft between TM4 and TM5. Binding of PAF modulates conformational changes in key motifs of PAFR, triggering the outward movement of TM6, TM7, and helix 8 for G protein coupling. Molecular dynamics simulation suggests a membrane-side pathway for PAF entry into PAFR via the TM4-TM5 cavity. By providing molecular insights into PAFR signaling, this work contributes a foundation for developing therapeutic interventions targeting PAF signal axis.
PubMed: 38943642
DOI: 10.1016/j.celrep.2024.114422 -
Journal of the American Chemical Society Jun 2024Ascorbic acid (AA) has been attracting great attention with its emerging potential in T cell-dependent antitumor immunity. However, premature blood clearance and...
Ascorbic acid (AA) has been attracting great attention with its emerging potential in T cell-dependent antitumor immunity. However, premature blood clearance and immunologically "cold" tumors severely compromise its immunotherapeutic outcomes. As such, the reversal of the immunosuppressive tumor microenvironment (TME) has been the premise for improving the effectiveness of AA-based immunotherapy, which hinges upon advanced AA delivery and amplified immune-activating strategies. Herein, a novel coli ( ) outer membrane vesicle (OMV)-red blood cell (RBC) hybrid membrane (ERm)-camouflaged immunomodulatory nanoturret is meticulously designed based on gating of an AA-immobilized metal-organic framework (MOF) onto bortezomib (BTZ)-loaded magnesium-doped mesoporous silica (MMS) nanovehicles, which can realize immune landscape remodeling by chemotherapy-assisted ascorbate-mediated immunotherapy (CAMIT). Once reaching the acidic TME, the acidity-sensitive MOF gatekeeper and MMS core within the nanoturret undergo stepwise degradation, allowing for tumor-selective sequential release of AA and BTZ. The released BTZ can evoke robust immunogenic cell death (ICD), synergistically promote dendritic cell (DC) maturation in combination with OMV, and ultimately increase T cell tumor infiltration together with Mg. The army of T cells is further activated by AA, exhibiting remarkable antitumor and antimetastasis performance. Moreover, the CD8-deficient mice model discloses the T cell-dependent immune mechanism of the AA-based CAMIT strategy. In addition to providing a multifunctional biomimetic hybrid nanovehicle, this study is also anticipated to establish a new immunomodulatory fortification strategy based on the multicomponent-driven nanoturret for highly efficient T cell-activation-enhanced synergistic AA immunotherapy.
PubMed: 38943624
DOI: 10.1021/jacs.4c04840 -
ACS Biomaterials Science & Engineering Jun 2024Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip...
Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip platforms. Barrier resistance is typically measured by trans-endothelial electrical resistance (TEER), but TEER is invasive and cannot accurately measure isolated monolayer resistance in coculture or most organ-on-chip devices. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here, we advanced the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation with TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 μm within the same insert. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest being the most sensitive ( < 0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation ( < 0.001). Barrier disruption with both EGTA and thrombin was detectable by all electrode sizes. Resistances measured by PM-ECIS vs TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes ( > 0.9; < 0.0001), but only with 750 μm electrodes for endothelial monolayers ( = 0.71; = 0.058). These data inform the design and selection of PM-ECIS electrodes for specific applications and support PM-ECIS as a promising alternative to conventional TEER for direct, noninvasive, real-time assessment of cells cultured on porous membranes in conventional and organ-on-chip barrier models.
PubMed: 38943620
DOI: 10.1021/acsbiomaterials.3c01898 -
Journal of Leukocyte Biology Jun 2024Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen...
Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen stimulation triggers mast cell granule exocytosis, releasing granule contents in a process known as degranulation. We have shown that Rho GTPase signaling is an essential component of granule exocytosis, however the proteins that regulate Rho GTPases during this process are not well-defined. Here we examined the role of Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) in regulating Rho GTPase signaling using RBL-2H3 cells as a mast cell model. We found that RBL-2H3 cells express two RhoGDI isoforms which are primarily localized to the cytosol. Knockdown of RhoGDI1 and RhoGDI2 greatly reduced the levels of all Rho GTPases tested: RhoA, RhoG, Rac1, Rac2 and Cdc42. The reduction in Rho GTPase levels was accompanied by an increase in their membrane-localized fraction and an elevation in the levels of active Rho GTPases. All RhoGDI knockdown strains had altered resting cell morphology, although each strain was activation competent when stimulated. Live cell imaging revealed that the RhoGDI1/2 double knockdown strain maintained its activated state for prolonged periods of time compared to the other strains. Only the RhoGDI1/2 double knockdown strain showed a significant increase in granule exocytosis. Conversely, RhoGDI overexpression in RBL-2H3 cells did not noticeably affect Rho GTPases or degranulation. Based on these results, RhoGDIs act as negative regulators of Rho GTPases during mast cell degranulation, and inhibit exocytosis by sequestering Rho GTPases in the cytosol.
PubMed: 38943612
DOI: 10.1093/jleuko/qiae150 -
Molekuliarnaia Biologiia 2024Many viruses, including SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, enter host cells through a process of cell-viral membrane fusion that is...
Many viruses, including SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, enter host cells through a process of cell-viral membrane fusion that is activated by proteolytic enzymes. Typically, these enzymes are host cell proteases. Identifying the proteases that activate the virus is not a simple task but is important for the development of new antiviral drugs. In this study, we developed a bioinformatics method for identifying proteases that can cleave viral envelope glycoproteins. The proposed approach involves the use of predictive models for the substrate specificity of human proteases and the application of a structural analysis method for predicting the vulnerability of protein regions to proteolysis based on their 3D structures. Specificity models were constructed for 169 human proteases using information on their known substrates. A previously developed method for structural analysis of potential proteolysis sites was applied in parallel with specificity models. Validation of the proposed approach was performed on the SARS-CoV-2 spike protein, whose proteolysis sites have been well studied.
Topics: Spike Glycoprotein, Coronavirus; Humans; SARS-CoV-2; Computational Biology; Proteolysis; Substrate Specificity; Peptide Hydrolases; COVID-19; Pandemics; Models, Molecular; Betacoronavirus
PubMed: 38943589
DOI: No ID Found