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Pathogens (Basel, Switzerland) Oct 2022In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen...
In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen peroxide (100 μM) showed two-fold and five-fold viability when compared to epimastigotes grown in LIT medium supplemented with two different glucose concentrations (20 mM and 1.5 mM), respectively. Similar results were obtained when exposing epimastigotes from all treatments to methylene blue 30 μM. Additionally, through differential centrifugation and the selective permeabilization of cellular membranes with digitonin, we found that phosphoglucomutase activity (a key enzyme in galactose metabolism) occurs predominantly within the cytosolic compartment. Furthermore, after partially permeabilizing epimastigotes with digitonin (0.025 mg × mg of protein), intact glycosomes treated with 20 mM galactose released a higher hexose phosphate concentration to the cytosol in the form of glucose-1-phosphate, when compared to intact glycosomes treated with 20 mM glucose, which predominantly released glucose-6-phosphate. These results shine a light on 's galactose metabolism and its interplay with mechanisms that enable resistance to oxidative stress.
PubMed: 36297231
DOI: 10.3390/pathogens11101174 -
Chemical Research in Toxicology Nov 2022Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the...
Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the medium () can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log -0.07-6.84), in the culture medium () and cells () after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished mass balance model that combined relevant equations from similar models published by others. The total for all chemicals except tamoxifen (TAM) were similar to the . This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) for the low/no protein binding chemicals were similar to the , whereas values of all moderately to highly protein-bound chemicals were less than 30% of the . Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the was the same as the . The for all other chemicals tended to increase over time and were all 2- to 274-fold higher than . Measurements of , using a digitonin method to release cytosol, compared well with (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total within 30% of the measured values for 11 chemicals. The free of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between and total and free and , which were also relatively well predicted by the mass balance model.
Topics: Mice; Animals; Acetaminophen; Hydrophobic and Hydrophilic Interactions; Protein Binding; Cell Culture Techniques
PubMed: 36264934
DOI: 10.1021/acs.chemrestox.2c00128 -
Computational and Structural... 2022Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and...
Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and -mutant high-throughput screening (HTS) data. We matched essential genes from text-mining and mutant genes from the COSMIC and CCLE HTS datasets to build a prediction model of SL gene pairs. CCLE gene expression data were used to enrich the essential-mutant SL gene pairs using Spearman's correlation coefficient and literature mining. In total, 223 essential trigger terms were extracted and ranked. The threshold of the essential gene score ( ) was set to 10. We identified 586 genes essential for the SL prediction model of colon cancer. Seven essential -mutant SL gene pairs were identified in our model, including -/----/ and - gene pairs. Using -mutant HTS data validation, we identified two potential SL gene pairs, including the (essential gene)- (mutant gene) pair and - pair in the DLD-1 colon cancer cell line (Spearman's correlation values = 0.004786 and 0.00249, respectively). Based on further annotations by PubChem, we observed that digitonin targeted the complex comprising , especially in -mutated HCT116 cancer cells. Moreover, we experimentally demonstrated that exhibited selective vulnerability in -mutant colorectal cancer. We used literature mining and HTS data to identify candidates for SL targets for mutant colon cancer.
PubMed: 36212540
DOI: 10.1016/j.csbj.2022.09.025 -
International Journal of Pharmaceutics Nov 2022Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the...
Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the quality of life. As self-medication practice, oral administration of triptans is the most common option, despite its relatively slow therapeutic onset and low drug bioavailability. To overcome these issues, here we present, to the best of our knowledge, the first study on the possibility of oral transmucosal delivery of one of the safest triptans, namely eletriptan hydrobromide (EB). Based on a comprehensive set of in vitro and ex vivo experiments, we highlight the conditions required for oral transmucosal delivery, potentially giving rise to similar, or even higher, drug plasma concentrations expected from conventional oral administration. With histology and tissue integrity studies, we conclude that EB neither induces morphological changes nor impairs the integrity of the mucosal barrier following 4 h of exposure. On a cellular level, EB is internalized in human oral keratinocytes within the first 5 min without inducing toxicity at the relevant concentrations for transmucosal delivery. Considering that the pK of EB falls within the physiologically range, we systematically investigated the effect of pH on both solubility and transmucosal permeation. When the pH is increased from 6.8 to 10.4, the drug solubility decreases drastically from 14.7 to 0.07 mg/mL. At pH 6.8, EB gave rise to the highest drug flux and total permeated amount across mucosa, while at pH 10.4 EB shows greater permeability coefficient and thus higher ratio of permeated drug versus applied drug. Permeation experiments with model membranes confirmed the pH dependent permeation profile of EB. The distribution of EB in different cellular compartments of keratinocytes is pH dependent. In brief, high drug ionization leads to higher association with the cell membrane, suggesting ionic interactions between EB and the phospholipid head groups. Moreover, we show that the chemical permeation enhancer DMSO can be used to enhance the drug permeation significantly (i.e., 12 to 36-fold increase). Taken together, this study presents important findings on transmucosal delivery of eletriptan via the oral cavity and paves the way for clinical investigations for a fast and safe migraine treatment.
Topics: Humans; Quality of Life; Dimethyl Sulfoxide; Tryptamines; Administration, Oral; Pharmaceutical Preparations; Migraine Disorders; Phospholipids
PubMed: 36155795
DOI: 10.1016/j.ijpharm.2022.122222 -
Bioluminescent test systems based on firefly luciferase for studying stress effects on living cells.Biophysical Reviews Aug 2022The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly... (Review)
Review
The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly luciferase. The possibilities of using this reaction to study the influence of external effectors of a physical and chemical nature (temperature exposure, additions of drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes) are considered. Examples of the use of test systems based on living cells producing thermostable firefly luciferase for monitoring cellular homeostasis are given. The study of the kinetics of changes in the concentration of ATP and luciferase inside and outside cells made it possible to determine in dynamics the metabolic activity, cytotoxicity, and survival of cells under conditions of cellular stress, to study the processes of ATP synthesis/hydrolysis, and to evaluate the effectiveness of lytic agents in changing the permeability of the cell membrane.
PubMed: 36124280
DOI: 10.1007/s12551-022-00978-y -
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 -
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