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Chemico-biological Interactions Nov 2022Nuclear receptor pregnane X receptor (PXR) can induce significant liver enlargement through hepatocyte hypertrophy and proliferation. A previous report showed that...
Nuclear receptor pregnane X receptor (PXR) can induce significant liver enlargement through hepatocyte hypertrophy and proliferation. A previous report showed that during the process of PXR-induced liver enlargement, hepatocyte hypertrophy occurs around the central vein (CV) area while hepatocyte proliferation occurs around the portal vein (PV) area. However, the features of this spatial change remain unclear. Therefore, this study aims to explore the features of the spatial changes in hepatocytes in PXR-induced liver enlargement. PXR-induced spatial changes in hepatocyte hypertrophy and proliferation were confirmed in C57BL/6 mice. The liver was perfused with digitonin to destroy the hepatocytes around the CV or PV areas, and then the regional expression of proteins related to hepatocyte hypertrophy and proliferation was further measured. The results showed that the expression of PXR downstream proteins, such as cytochrome P450 (CYP) 3A11, CYP2B10, P-glycoprotein (P-gp) and organ anion transporting polypeptide 4 (OATP4) was upregulated around the CV area, while the expression of proliferation-related proteins such as cyclin B1 (CCNB1), cyclin D1 (CCND1) and serine/threonine NIMA-related kinase 2 (NEK2) was upregulated around the PV area. At the same time, the expression of cyclin-dependent kinase inhibitors such as retinoblastoma-like protein 2 (RBL2), cyclin-dependent kinase inhibitor 1B (CDKN1B) and CDKN1A was downregulated around the PV area. This study demonstrated that the spatial change in PXR-induced hepatocyte hypertrophy and proliferation is associated with the regional expression of PXR downstream targets and proliferation-related proteins and the regional distribution of triglycerides (TGs). These findings provide new insight into the understanding of PXR-induced hepatomegaly.
Topics: ATP Binding Cassette Transporter, Subfamily B; Animals; Anions; Cell Proliferation; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Digitonin; Hepatocytes; Hepatomegaly; Hypertrophy; Liver; Mice; Mice, Inbred C57BL; NIMA-Related Kinases; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Retinoblastoma-Like Protein p130; Serine; Threonine; Triglycerides
PubMed: 36030841
DOI: 10.1016/j.cbi.2022.110133 -
Molecules (Basel, Switzerland) Jul 2022The rhizomes of are commonly consumed as food and also used as medicine. However, the metabolic profile of has not been fully revealed yet. Recently, we developed a...
The rhizomes of are commonly consumed as food and also used as medicine. However, the metabolic profile of has not been fully revealed yet. Recently, we developed a novel evergreen species of P. sibiricum. The objectives of this study were to compare the metabolic profiles of two types of , i.e., the newly developed evergreen type (Gtype) and a wide-type (Wtype), by using UHPLC-Q-Orbitrap-MS-based untargeted metabolomics approach. A total of 263 and 258 compounds in the positive and negative modes of the mass spectra were tentatively identified. Distinctively different metabolomic profiles of these two types of were also revealed by principal component analysis (PCA) and principal coordinates analysis (PCoA). Furthermore, by using partial least squares discriminant analysis (PLS-DA) modeling, it was found that, as compared with Wtype, Gtype samples had significantly higher content of oxyberberine, proliferin, alpinetin, and grandisin. On the other hand, 15 compounds, including herniarin, kaempferol 7-neohesperidoside, benzyl beta-primeveroside, vanillic acid, biochanin A, neoschaftoside, benzyl gentiobioside, cornuside, hydroxytyrosol-glucuronide, apigenin-pentosyl-glucoside, obacunone, 13-alpha-(21)-epoxyeurycomanone, vulgarin, digitonin, and 3-formylindole, were discovered to have higher abundance in Wtype samples. These distinguishing metabolites suggest the different beneficial health potentials and flavor attributes of the two types of rhizomes.
Topics: Chromatography, High Pressure Liquid; Mass Spectrometry; Metabolomics; Polygonatum; Rhizome
PubMed: 35897876
DOI: 10.3390/molecules27154685 -
Journal of Pharmaceutical and... Sep 2022Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets....
Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.
Topics: Animals; Detergents; Electrophoresis, Polyacrylamide Gel; Humans; Hydrophobic and Hydrophilic Interactions; Mammals; Membrane Proteins; Mice; Octoxynol
PubMed: 35839578
DOI: 10.1016/j.jpba.2022.114926 -
RSC Advances Jun 2022There is a great demand for the rapid and non-invasive atherosclerosis screening method. Cholesterol content in the epidermis of the skin is an early biomarker for...
There is a great demand for the rapid and non-invasive atherosclerosis screening method. Cholesterol content in the epidermis of the skin is an early biomarker for atherosclerosis. Risk assessment of atherosclerosis can be achieved by measuring cholesterol in the epidermis. Here, we synthesised a new fluorescent digitonin derivative (FDD) for the non-invasive detection of skin cholesterol. The results of fluorescence spectroscopy studies indicated that the probe exhibited desirable selectivity for cholesterol. The proof-of-concept preclinical study confirmed that FDD can detect different concentrations of skin cholesterol; patients diagnosed with atherosclerotic cardiovascular disease and the at-risk atherosclerosis group exhibited higher skin cholesterol content than the normal group. The area under the ROC curve for distinguishing the normal/disease group was 0.9228 (95% confidence interval, 0.8938 to 0.9518), and the area under the ROC curve for distinguishing the normal/risk group was 0.9422 (95% confidence interval, 0.9178 to 0.9665). We anticipate that this non-invasive skin cholesterol test may be used as a risk assessment tool for atherosclerosis screening in a large population for further examination and intervention in high-risk populations.
PubMed: 35799936
DOI: 10.1039/d2ra01982e -
Bio-protocol Jun 2022Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane...
Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane repair system to survive. However, the different cellular and molecular mechanisms behind plasma membrane repair have not been fully elucidated. Here, we present three complementary methods for plasma membrane wounding, and measurement of membrane repair and integrity. The first protocol is based on real time imaging of cell membrane repair kinetics in response to laser-induced injury. The second and third protocols are end point assays that provide a population-based measure of membrane integrity, after either mechanical injury by vortex mixing with glass beads, or by detergent-induced injury by digitonin in sublytic concentrations. The protocols can be applied to most adherent eukaryotic cells in culture, as well as cells in suspension.
PubMed: 35799909
DOI: 10.21769/BioProtoc.4437 -
Drug Delivery and Translational Research Sep 2022State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial...
State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.
Topics: Digitonin; Holography; Humans; In Vitro Techniques; Microscopy; Reproducibility of Results
PubMed: 35799027
DOI: 10.1007/s13346-022-01207-5 -
Synthetic Biology (Oxford, England) 2022Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species....
Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of . Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.
PubMed: 35774105
DOI: 10.1093/synbio/ysac008 -
Genome Biology Jun 2022The recently developed method TEA-seq and similar DOGMA-seq single cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but...
The recently developed method TEA-seq and similar DOGMA-seq single cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but independent evaluation is lacking. We explore the utility of DOGMA-seq compared to the bimodal CITE-seq assay in activated and stimulated human peripheral blood T cells. We find that single cell trimodal omics measurements after digitonin (DIG) permeabilization were generally better than after an alternative "low-loss lysis" (LLL) permeabilization condition. Next, we find that DOGMA-seq with optimized DIG permeabilization and its ATAC library provides more information, although its mRNA and cell surface protein libraries have slightly inferior quality, compared to CITE-seq.
Topics: Benchmarking; Gene Library; High-Throughput Nucleotide Sequencing; Humans; RNA, Messenger; Sequence Analysis, DNA; Single-Cell Analysis
PubMed: 35739535
DOI: 10.1186/s13059-022-02698-8 -
Biochimica Et Biophysica Acta.... Oct 2022Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene...
Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.
Topics: Cytostatic Agents; Ergosterol; Ginsenosides; Saccharomyces cerevisiae; Sterols; Sugars; Triterpenes
PubMed: 35724740
DOI: 10.1016/j.bbamem.2022.183993 -
Spectrochimica Acta. Part A, Molecular... Oct 2022The concentration of potassium ion is an important indicator for human health, and its abnormality is often accompanied by various diseases. However, most tools...
The concentration of potassium ion is an important indicator for human health, and its abnormality is often accompanied by various diseases. However, most tools currently used to study potassium ion transport are low throughput. Herein, we reported a new K fluorescent nanoprobe CP1-KS with high selectivity and sensitivity to K (fluorescence enhanced factor was up to 9.91 at 20 mM K). The polymeric fluorescent probe CP1-KS was composed of the small-molecular K indicator KS and amphiphilic copolymer CP1. This sensor can be easily and uniformly dispersed in cell culture medium and is suitable for high throughput analysis. To assess the utility of the probe CP1-KS in biological field, this probe was employed as an extracellular fluorescent probe to monitor the efflux of K from cells (E coli, B. Subtilis 168, Hela and MCF-7 cells) under various stimulation including lysozyme, nigericin, digitonin, and ATP. Results demonstrated that CP1-KS is an effective analysis tool for extracellular K concentration. We believe that the nanoprobe has great potential in antibacterial drug screening, K ionophore function, K channel activity, cell membrane permeability analysis or other K related field in the future.
Topics: Biological Assay; Escherichia coli; Fluorescent Dyes; Humans; Ionophores; Ions; Potassium
PubMed: 35653810
DOI: 10.1016/j.saa.2022.121435