Antimicrobial resistance is a global health issue, in which microorganisms develop resistance to antimicrobial drugs, making infections more difficult to treat. This threatens the effectiveness of standard medical treatments and necessitates the urgent development of new strategies to combat resistant microbes. Studies have increasingly explored natural sources of new antimicrobial agents that harness the rich diversity of compounds found in plant species. This pursuit holds promise for the discovery of novel treatments for combating antimicrobial resistance. In this context, the chemical composition, antibacterial, and antibiofilm activities of the essential oil from Croton urticifolius Lam. leaves (CuEO) were evaluated. CuEO was extracted via hydrodistillation, and its chemical constituents were identified via gas chromatography-mass spectrometry (GC/MS). The antibacterial activity of CuEO was evaluated in a 96-well plate via the microdilution method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined. The effect of CuEO on biofilm formation was assessed by quantifying the biomass using crystal violet staining and viable cell counting. In addition, alterations in the cellular morphology of biofilms treated with CuEO were examined using scanning electron microscopy (SEM) and laser confocal microscopy. GC/MS analysis identified 26 compounds, with elemicine (39.72%); eucalyptol (19.03%), E-caryophyllene (5.36%), and methyleugenol (4.12%) as the major compounds. In terms of antibacterial activity, CuEO showed bacteriostatic effects against Staphylococcus aureus ATCC 700698, S. aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, and Escherichia coli ATCC 11303, and bactericidal activity against S. aureus ATCC 700698. In addition, CuEO significantly inhibited bacterial biofilm formation. Microscopic analysis showed that CuEO damaged the bacterial membrane by leaching out the cytoplasmic content. Therefore, the results of this study show that the essential oil of C. urticifolius may be a promising natural alternative for preventing infections caused by bacterial biofilms. This study is the first to report the antibiofilm activity of C. urticifolius essential oil.

Developing T1-weighted magnetic resonance imaging (MRI) contrast agents with enhanced biocompatibility and targeting capabilities is crucial owing to concerns over current agents\' potential toxicity and suboptimal performance. Drawing inspiration from \"biomimetic camouflage,\" we isolated cell membranes (CMs) from human glioblastoma (T98G) cell lines via the extrusion method to facilitate homotypic glioma targeting. At an 8:1 mass ratio of ferric chloride hexahydrate to gallic acid (GA), the resulting iron (Fe)-GA nanoparticles (NPs) proved effective as a T1-weighted MRI contrast agent. T98G CM-coated Fe-GA NPs demonstrated improved homotypic glioma targeting, validated through Prussian blue staining and in vitro MRI. This biomimetic camouflage strategy holds promise for the development of targeted theranostic agents in a safe and effective manner.

Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.

Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.

The peroxisome is a versatile organelle that performs diverse metabolic functions. PEX3, a critical regulator of the peroxisome, participates in various biological processes associated with the peroxisome. Whether PEX3 is involved in peroxisome-related redox homeostasis and myocardial regenerative repair remains elusive. We investigate that cardiomyocyte-specific PEX3 knockout (Pex3-KO) results in an imbalance of redox homeostasis and disrupts the endogenous proliferation/development at different times and spatial locations. Using Pex3-KO mice and myocardium-targeted intervention approaches, the effects of PEX3 on myocardial regenerative repair during both physiological and pathological stages are explored. Mechanistically, lipid metabolomics reveals that PEX3 promotes myocardial regenerative repair by affecting plasmalogen metabolism. Further, we find that PEX3-regulated plasmalogen activates the AKT/GSK3β signaling pathway via the plasma membrane localization of ITGB3. Our study indicates that PEX3 may represent a novel therapeutic target for myocardial regenerative repair following injury.

According to single-molecule localisation microscopy almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell single molecule localisation microscopy using the membrane probe DiI alongside either the transferrin receptor or the GPI-anchored protein CD59, we found that pair correlation analysis reported both proteins and DiI as being clustered, as did its derivative pair correlation-photoactivation localisation microscopy and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations can make membrane molecules appear clustered and present a straightforward remedy suitable as the first step in the cluster analysis pipeline.

Ionic liquids (ILs) are interesting chemical compounds that have a wide range of industrial and scientific applications. They have extraordinary properties, such as the tunability of many of their physical properties and, accordingly, their activities; and the ease of synthesis methods. Hence, they became important building blocks in catalysis, extraction, electrochemistry, analytics, biotechnology, etc. This study determined antifungal activities of various imidazolium-based ionic liquids against yeast Saccharomyces cerevisiae via minimum inhibitory concentration (MIC) estimation method. Increasing the length of the alkyl group attached to the imidazolium cation, enhanced the antifungal activity of the ILs, as well as their ability of the disruption of the cell membrane integrity. FTIR studies performed on the S. cerevisiae cells treated with the ILs revealed alterations in the biochemical composition of these cells. Interestingly, the alterations in fatty acid content occurred in parallel with the increase in the activity of the molecules upon the increase in the length of the attached alkyl group. This trend was confirmed by statistical analysis and machine learning methodology. The classification of antifungal activities based on FTIR spectra of S. cerevisiae cells yielded a prediction accuracy of 83%, indicating the pharmacy and medicine industries could benefit from machine learning methodology. Furthermore, synthesized ionic compounds exhibit significant potential for pharmaceutical and medical applications.

PRL1 and PRL3, members of the protein tyrosine phosphatase family, have been associated with cancer metastasis and poor prognosis. Despite extensive research on their protein phosphatase activity, their potential role as lipid phosphatases remains elusive. Methods: We conducted comprehensive investigations to elucidate the lipid phosphatase activity of PRL1 and PRL3 using a combination of cellular assays, biochemical analyses, and protein interactome profiling. Functional studies were performed to delineate the impact of PRL1/3 on macropinocytosis and its implications in cancer biology. Results: Our study has identified PRL1 and PRL3 as lipid phosphatases that interact with phosphoinositide (PIP) lipids, converting PI(3,4)P2 and PI(3,5)P2 into PI(3)P on the cellular membranes. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis, facilitating nutrient extraction, cell migration, and invasion, thereby contributing to tumor development. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis. Additionally, we found a correlation between PRL1/3 expression and glioma development, suggesting their involvement in glioma progression. Conclusions: Combining with the knowledge that PRLs have been identified to be involved in mTOR, EGFR and autophagy, here we concluded the physiological role of PRL1/3 in orchestrating the nutrient sensing, absorbing and recycling via regulating macropinocytosis through its lipid phosphatase activity. This mechanism could be exploited by tumor cells facing a nutrient-depleted microenvironment, highlighting the potential therapeutic significance of targeting PRL1/3-mediated macropinocytosis in cancer treatment.

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.1.

Currently, specific cancer-responsive fluorogenic probes with activatable imaging and therapeutic functionalities are in great demand in the accurate diagnostics and efficient therapy of malignancies. Herein, an all-in-one strategy is presented to realize fluorescence (FL) imaging-guided and synergetic chemodynamic-photodynamic cancer therapy by using a multifunctional alkaline phosphatase (ALP)-response aggregation-induced emission (AIE) probe, TPE-APP. By responding to the abnormal expression levels of an ALP biomarker in cancer cells, the phosphate groups on the AIE probe are selectively hydrolyzed, accompanied by in situ formation of strong emissive AIE aggregates for discriminative cancer cell imaging over normal cells and highly active quinone methide species with robust chemodynamic-photodynamic activities. Consequently, the activated AIE probes can efficiently destroy cancer cell membranes and lead to the death of cancer cells within 30 min. A superior efficacy in cancer cell ablation is demonstrated in vitro and in vivo. The cancer-associated biomarker response-derived discriminative FL imaging and synergistic chemodynamic-photodynamic therapy are expected to provide a promising avenue for precise image-guided cancer therapy.