Search

Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health. [ABSTRACT FROM AUTHOR]

• Anthocyanins are major phytochemicals present in the aril extracts of Southern African P. granatum 'Wonderful' cultivar. • The methanolic extract induced the strongest cytotoxicity in breast cancer MCF-7 cell line. • By regulating the proteins involved in apoptotic pathway, the extract treatment of MCF-7 cells enhanced the cell death by inducing apoptosis. Due to its superior nutritional, health promoting and disease preventing qualities, the Punica granatum is referred to as a "Super Fruit" and has been traditionally used for its therapeutic properties including cancer prevention. Although this plant is grown globally, phytochemical profile and their biological properties varies based on the pedoclimatic variables, fruit's phenogenotype, stage of ripeness, pre- and post-harvest circumstances, and extractive solvent, even among the same cultivars grown under various conditions. Here, we investigated the phytochemical composition and anticancer potency of three different aril solvent extracts (methanol, chloroform, and ethyl acetate) of South African 'Wonderful' cultivar against human breast cancer MCF-7 cell line. The identification of major phytochemical constituents of three different extracts was performed by UHPLC-Q-TOF-MS2. Total phenolic and flavonoid content were determined using the Folin–Ciocalteau and aluminium chloride colorimetric methods, respectively. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and adenosine triphosphate (ATP) content assays. The apoptotic effects were determined by morphological analysis (phase contrast and LIVE DEAD assay) and FITC-Annexin/ Propidium Iodide flow cytometry assay, Cytochrome-c release, caspase activation assay and ratio of Bax/Bcl-2 expression. UHPLC-Q-TOF-MS2 analysis of extracts revealed the presence phenolic acids, flavonoids, anthocyanins, tannins, coumarins, gallotannins, lignan, and phthalide. The total phenolic and flavonoid content was determined to be highest for methanol extract (91.78±1.55 mg GAE/g DM and 43.03±2.63 mg QE/g DM respectively). This correlated with its highest cytotoxic activity against MCF-7 cells compared to other two solvent extracts with IC 50 concentration ∼289 and ∼229 μg/mL at 24 and 48 h respectively. After 48 h, the apoptotic cell population were significantly increased with 400 μg/mL methanol extract treatment as observed via morphological changes and FITC-Annexin/ Propidium Iodide assay. The methanol extract significantly induced apoptosis via the release of cytochrome-c, increase in ratio of Bax to Bcl-2 and caspase-8 and 9 activations. Based on the present findings, we conclude that South African P. granatum 'Wonderful' cultivar had the potential as cytotoxic and apoptosis inducing anticancer agent, which is worth investigating in detail. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is a highly aggressive and fatal primary brain tumor. The resistance of GBM to conventional treatments is attributed to factors such as the blood–brain barrier, tumor heterogeneity, and treatment-resistant stem cells. Current therapeutic efforts show limited survival benefits, emphasizing the urgent need for novel treatments. In this context, natural anti-cancer extracts and especially animal venoms have garnered attention for their potential therapeutic benefits. Bee venom in general and that of the Middle Eastern bee, Apis mellifera syriaca in particular, has been shown to have cytotoxic effects on various cancer cell types, but not glioblastoma. Therefore, this study aimed to explore the potential of A. mellifera syriaca venom as a selective anti-cancer agent for glioblastoma through in vitro and in vivo studies. Our results revealed a strong cytotoxic effect of A. mellifera syriaca venom on U87 glioblastoma cells, with an IC50 of 14.32 µg/mL using the MTT test and an IC50 of 7.49 µg/mL using the LDH test. Cells treated with the bee venom became permeable to propidium iodide without showing any signs of early apoptosis, suggesting compromised membrane integrity but not early apoptosis. In these cells, poly (ADP-ribose) polymerase (PARP) underwent proteolytic cleavage similar to that seen in necrosis. Subsequent in vivo investigations demonstrated a significant reduction in the number of U87 cells in mice following bee venom injection, accompanied by a significant increase in cells expressing caspase-3, suggesting the occurrence of cellular apoptosis. These findings highlight the potential of A. mellifera syriaca venom as a therapeutically useful tool in the search for new drug candidates against glioblastoma and give insights into the molecular mechanism through which the venom acts on cancer cells. [ABSTRACT FROM AUTHOR]

Infections caused by pathogenic Escherichia coli are a serious threat to human health, while conventional antibiotic susceptibility tests (AST) have a long turn-around time, and rapid antibiotic susceptibility methods are urgently needed to save lives in the clinic, reduce antibiotic misuse and prevent emergence of antibiotic-resistant bacteria. We optimized and validated the feasibility of a novel rapid AST based on SYBR Green I and Propidium Iodide (SGPI-AST) for E. coli drug susceptibility test. A total of 112 clinical isolates of E. coli were collected and four antibiotics (ceftriaxone, cefoxitin, imipenem, meropenem) were selected for testing. Bacterial survival rate of E. coli was remarkably linearly correlated with S value at different OD600 values. After optimizing the antibiotic concentrations, the sensitivity and specificity of SGPI-AST reached 100%/100%, 97.8%/100%, 100%/100% and 98.4%/99% for ceftriaxone, cefoxitin, imipenem and meropenem, respectively, and the corresponding concordances of the SGPI-AST with conventional AST were 1.000, 0.980, 1.000 and 0.979, respectively. The SGPI-AST can rapidly and accurately determine the susceptibility of E. coli clinical isolates to multiple antibiotics in 60 min, and has the potential to be applied to guide the precise selection of antibiotics for clinical management of infections caused by pathogenic E. coli. [ABSTRACT FROM AUTHOR]

The limited arsenal of antifungal drugs have prompted the search for novel molecules with biological activity. This study aimed to characterize the antifungal mechanism of action of Eugenia uniflora extract and its synergistic activity with commercially available antifungal drugs on the following Candida species: C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. dubliniensis. In silico analysis was performed to predict antifungal activity of the major compounds present in the extract. Minimal inhibitory concentrations (MICs) were determined in the presence of exogenous ergosterol and sorbitol. Yeast cells were grown in the presence of stressors. The loss of membrane integrity was assessed using propidium iodide staining (fluorescence emission). Synergism between the extract and antifungal compounds (in addition to time kill-curves) was determined. Molecular docking revealed possible interactions between myricitrin and acid gallic and enzymes involved in ergosterol and cell wall biosynthesis. Candida cells grown in the presence of the extract with addition of exogenous ergosterol and sorbitol showed 2 to 8-fold increased MICs. Strains treated with the extract revealed greater loss of membrane integrity when compared to their Fluconazole counterparts, but this effect was less pronounced than the membrane damage caused by Amphotericin B. The extract also made the strains more susceptible to Congo red and Calcofluor white. A synergistic action of the extract with Fluconazole and Micafungin was observed. The E. uniflora extract may be a viable option for the treatment of Candida infections. [ABSTRACT FROM AUTHOR]

Purpose: Benzalkonium chloride (BAK) is a common preservative in ophthalmic formulations that causes cytotoxic damage to the corneal epithelial cells. This study aims to explore the role of mesenchymal stem cell (MSC)-derived conditioned medium in modulating the BAK-induced cytotoxic effects in cultured human corneal epithelial cells (HCECs) as a cell-free therapeutic agent. Methods: The in vitro cultured HCECs derived from a HCE cell line were treated with BAK (0.001% and 0.005%, diluted in DMEM/F12, v/v) for 15 min, washed with 1xPBS, and allowed to recover for 24 h in human bone marrow MSC-derived conditioned medium (MSC-CM: undiluted (100%) and diluted (50%, v/v)). On the other hand, HCECs were co-incubated with BAK (0.005%, v/v) and MSC-CM (100% and 50%, v/v) for 24 h. The HCEC-derived conditioned medium (HCE-CM) was used as an optimal control for MSC-CM, whereas HCECs cultured in DMEM/F12 were used as a control. The DMEM/F12 was used as the base medium for the culture of HCECs and preparation of HCE- and MSC-CM. The role of MSC-CM in modulating the metabolic activity, cell death, epithelial repair, and proliferation, in BAK-treated HCECs was evaluated using MTT assay, Propidium iodide staining, scratch assay, and Ki-67 staining, respectively. Results: Compared to the control, recovery of BAK-treated (0.001% and 0.005%, for 15 min) HCECs in MSC-CM showed significantly reduced cell death with enhanced metabolic activity, epithelial repair, and proliferation. However, in comparison with HCE-CM, the beneficial effects of MSC-CM were predominantly observed at lower BAK concentration (0.001%, for 15 min). Whereas the co-incubation of BAK (0.005%) and MSC-CM for a longer duration (24 h) was marginally beneficial. Conclusions: Our results suggest that the MSC-CM is effective in modulating the BAK-induced cell death, retardation of metabolic activity and proliferation in cultured HCECs, particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK. [ABSTRACT FROM AUTHOR]

Background: Ferroptosis is caused by iron‐dependent peroxidation of membrane phospholipids and chondrocyte ferroptosis contributes to osteoarthritis (OA) progression. Glutathione peroxidase 4 (GPX4) plays a master role in blocking ferroptosis. N6‐methyladenosine (m6A) is an epigenetic modification among mRNA post‐transcriptional modifications. This study investigated the effect of methyltransferase‐like 14 (METTL14), the key component of the m6A methyltransferase, on chondrocyte ferroptosis via m6A modification. Methods: An OA rat model was established through an intra‐articular injection of monosodium iodoacetate in the right knee. OA cartilages in rat models were used for gene expression analysis. Primary mouse chondrocytes or ADTC5 cells were stimulated with IL‐1β or erastin. The m6A RNA methylation quantification kit was used to measure m6A level. The effect of METTL14 and GPX4 on ECM degradation and ferroptosis was investigated through western blotting, fluorescence immunostaining, propidium iodide staining, and commercially available kits. The mechanism of METTL14 action was explored through MeRIP‐qPCR assays. Results: METTL14 and m6A expression was upregulated in osteoarthritic cartilages and IL‐1β‐induced chondrocytes. METTL14 depletion repressed the IL‐1β or erastin‐stimulated ECM degradation and ferroptosis in mouse chondrocytes. METTL14 inhibited GPX4 gene through m6A methylation modification. GPX4 knockdown reversed the si‐METTL14‐mediated protection in IL‐1β‐induced chondrocytes. Conclusion: METTL14 depletion inhibits ferroptosis and ECM degradation by suppressing GPX4 mRNA m6A modification in injured chondrocytes. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells can differentiate into specific cell lineages in the tissue repair process. Photobiomodulation with laser and LED is used to treat several comorbidities, can interfere in cell proliferation and viability, in addition to promoting responses related to the physical parameters adopted. Evaluate and compare the effects of laser and LED on mesenchymal cells, with different energy doses and different wavelengths, in addition to viability and wound closure. Mesenchymal stem cells derived from human adipocytes were irradiated with laser (energy of 0.5 J, 2 J and 4 J, wavelength of 660 nm and 830 nm), and LED (energy of 0.5 J, 2 J and 4 J, where lengths are 630 nm and 850 nm). The wound closure process was evaluated through monitoring the reduction of the lesion area in vitro. Viability was determined by analysis with Hoechst and Propidium Iodide markers, and quantification of viable and non-viable cells respectively Data distributions were analyzed using the Shapiro–Wilk test. Homogeneity was analyzed using Levene's test. The comparison between the parameters used was analyzed using the Two-way ANOVA test. The T test was applied to data relating to viability and lesion area. For LED photobiomodulation, only the 630 nm wavelength obtained a significant result in 24, 48 and 72 h (p = 0,027; p = 0,024; p = 0,009). The results related to the in vitro wound closure test indicate that both photobiomodulation with laser and LED demonstrated significant results considering the time it takes to approach the edges (p < 0.05). Considering the in vitro experimental conditions of the study, it is possible to conclude that the physical parameters of photobiomodulation, such as energy and wavelength, with laser or LED in mesenchymal stem cells, can play a potential role in cell viability and wound closure. [ABSTRACT FROM AUTHOR]

The availability of metamizole varies greatly around the world. There are countries such as the USA, UK, or Australia where the use of metamizole is completely forbidden, and there are also countries where this drug is available only on prescription (e.g., Greece, Italy, Spain, etc.) and those in which it is sold OTC—over the counter (e.g., most Asian and South American countries). Metamizole, as a drug with a strong analgesic effect, is used as an alternative to other non-steroidal anti-inflammatory drugs, alone or in combination with opioid drugs. Risedronate sodium is a third-generation bisphosphonate commonly used in orthopaedic and metabolic diseases of the musculoskeletal system, including hypercalcemia, postmenopausal osteoporosis, Paget's disease, etc. The aim of this study was to check whether there were any pharmacological interactions between metamizole and risedronate sodium in in vitro studies. Cell viability was assessed using the MTT method, the number of apoptotic cells was assessed using the labelling TUNEL method, and the cell cycle assessment was performed with a flow cytometer and propidium iodide. This was a pilot study, which is why only two cancer cell lines were tested: D-17 of canine osteosarcoma and U-2 OS of human osteosarcoma. Exposure of the canine osteosarcoma cell line to a combination of risedronate sodium (100 µg/mL) and metamizole (50, 5, and 0.5 µg/mL) resulted in the complete abolition of the cytoprotective activity of metamizole. In the human osteosarcoma cell line, the cytotoxic effect of risedronate sodium was entirely eliminated in the presence of 50 µg/mL of metamizole. The cytoprotective and anti-apoptotic effect of metamizole in combination with risedronate sodium in the tested human and canine osteosarcoma cell lines indicates an urgent need for further in vivo studies to confirm or disprove the potential dose-dependent undesirable effect of such a therapy. [ABSTRACT FROM AUTHOR]

Background: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. Methods: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. Results: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. Conclusion: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells. [ABSTRACT FROM AUTHOR]

Bacteriocins are membrane-acting peptides and generally kill closely related bacteria using pore formation. In this study, we have studied a bacteriocin, weissellicin LM85 from Weissella confusa LM85 to monitor its antimicrobial activity against Escherichia coli ATCC 25922. It was purified from cell-free supernatant of W. confusa LM85 with molecular weight ~ 6.5 kDa and showed minimum inhibitory concentration, 138.3 µg/mL and minimum bactericidal concentration, 553.3 µg/mL against E. coli ATCC 25922. The loss of cell-viability, tested by staining with propidium iodide, suggested bactericidal effect of weissellicin LM85. There was efflux of potassium (K+) ions, dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) in bacteriocin-treated cells. The target cells were found swollen and ruptured when visualized under electron microscope. It inhibited range of Gram-positive and Gram-negative bacteria such as Lactiplantibacillus plantarum NRRL B-4496, Lpb. plantarum LD4, Lactobacillus acidophilus NRRL B-4495, Enterococcus faecium NRRL B-2354, E. hirae LD3, E. faecalis ATCC 29212, Pediococcus pentosaceus LB44, Vibrio sp., Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311, Shigella flexneri, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. The above results indicate weissellicin LM85 is a membrane-acting peptide with broad host-range of antimicrobial activity and may be used as alternative to clinical antibiotics. [ABSTRACT FROM AUTHOR]

Oxidative stress can damage neuronal cells, greatly contributing to neurodegenerative diseases (NDs). In this study, the protective activity of arzanol, a natural prenylated α-pyrone-phloroglucinol heterodimer, was evaluated against the H2O2-induced oxidative damage in trans-retinoic acid-differentiated (neuron-like) human SH-SY5Y cells, widely used as a neuronal cell model of neurological disorders. The pre-incubation (for 2 and 24 h) with arzanol (5, 10, and 25 μM) significantly preserved differentiated SH-SY5Y cells from cytotoxicity (MTT assay) and morphological changes induced by 0.25 and 0.5 mM H2O2. Arzanol reduced the generation of reactive oxygen species (ROS) induced by 2 h oxidation with H2O2 0.5 mM, established by 2′,7′-dichlorodihydrofluorescein diacetate assay. The 2 h incubation of differentiated SH-SY5Y cells with H2O2 determined a significant increase in the number of apoptotic cells versus control cells, evaluated by propidium iodide fluorescence assay (red fluorescence) and NucView® 488 assay (green fluorescence). Arzanol pre-treatment (2 h) exerted a noteworthy significant protective effect against apoptosis. In addition, arzanol was tested, for comparison, in undifferentiated SH-SY5Y cells for cytotoxicity and its ability to protect against H2O2-induced oxidative stress. Furthermore, the PubChem database and freely accessible web tools SwissADME and pkCSM-pharmacokinetics were used to assess the physicochemical and pharmacokinetic properties of arzanol. Our results qualify arzanol as an antioxidant agent with potential neuroprotective effects against neuronal oxidative stress implicated in NDs. [ABSTRACT FROM AUTHOR]

Aim: Fibroblasts are common cells in subcutaneous tissue and they have an important role on healing process. Although patients who are receiving first-generation low molecular weight heparins are more likely to experience skin responses, there is no study evaluating the effect of bemiparin sodium on subcutaneous fibroblasts following administration. In the present study, we aimed to evaluate the effects of bemiparin sodium on fibroblast cell viability in cell culture model. Material and Methods: The L929 cells were incubated for 12 hours in 96-well culture dishes. Fresh medium containing different concentrations of bemiparin was added in five different concentrations (Dilution I: 3,500 IU; Dilution II: 1,750 IU; Dilution III: 875 IU; Dilution IV: 437.5 IU; Dilution V: 218.75 IU). A control group was established with drug-free culture medium. Cell viability analysis was performed after 48 hours of incubation by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The change in cell morphology was examined at each dilution by direct and acridine orange/propidium iodide staining. Results: The highest cytotoxic effect was observed in dilution I compared to the control group (p<0.05). There is a clear deviation from the fibroblastic morphology of the cells in dilution I. The cells have a round morphology with degeneration and nuclear condensation. Conclusion: Although it seems that high doses of bemiparin may have a negative effect on fibroblast cells in subcutaneous tissue, it can be considered that the cytotoxic effects of high concentrations of the drug at the application site will not be clinically significant due to using a single dose per day. [ABSTRACT FROM AUTHOR]

Abstract Infections caused by pathogenic Escherichia coli are a serious threat to human health, while conventional antibiotic susceptibility tests (AST) have a long turn-around time, and rapid antibiotic susceptibility methods are urgently needed to save lives in the clinic, reduce antibiotic misuse and prevent emergence of antibiotic-resistant bacteria. We optimized and validated the feasibility of a novel rapid AST based on SYBR Green I and Propidium Iodide (SGPI-AST) for E. coli drug susceptibility test. A total of 112 clinical isolates of E. coli were collected and four antibiotics (ceftriaxone, cefoxitin, imipenem, meropenem) were selected for testing. Bacterial survival rate of E. coli was remarkably linearly correlated with S value at different OD600 values. After optimizing the antibiotic concentrations, the sensitivity and specificity of SGPI-AST reached 100%/100%, 97.8%/100%, 100%/100% and 98.4%/99% for ceftriaxone, cefoxitin, imipenem and meropenem, respectively, and the corresponding concordances of the SGPI-AST with conventional AST were 1.000, 0.980, 1.000 and 0.979, respectively. The SGPI-AST can rapidly and accurately determine the susceptibility of E. coli clinical isolates to multiple antibiotics in 60 min, and has the potential to be applied to guide the precise selection of antibiotics for clinical management of infections caused by pathogenic E. coli.

The famous "light-switch" ruthenium complex [Ru(bpy)2(dppz)](PF6)2 (1) has been long known for its DNA binding properties in vitro. However, the biological utility of this compound has been hampered by its poor cellular uptake in living cells. Here we report a bioimaging application of 1 as cell viability probe in both 2D cells monolayer and 3D multi-cellular tumor spheroids of various human cancer cell lines (U87, HepG2, A549). When compared to propidium iodide, a routinely used cell viability probe, 1 was found to enhance the staining of dead cells in particular in tumor spheroids. 1 has high photostability, longer Stokes shift, and displays lower cytotoxicity compared to propidium iodide, which is a known carcinogenic. Finally, 1 was also found to displace the classical DNA binding dye Hoechst in dead cells, which makes it a promising dye for time-dependent imaging of dead cells in cell cultures, including multi-cellular tumor spheroids. [ABSTRACT FROM AUTHOR]

Biocolourants could be a sustainable option for dyes that require fossil-based chemicals in their synthesis. We studied the in vitro toxicity of anthraquinone aglycone extract obtained from Cortinarius sanguineus fungus and compared it to the toxicity of its two main components, emodin and previously studied dermocybin. Cell viability, cytotoxicity, and oxidative stress responses in HepG2 liver and THP-1 immune cell lines were studied along with skin sensitisation. In addition, genotoxicity was studied with comet assay in HepG2 cells. Cellular viability was determined by MTT, propidium iodide, and lactate dehydrogenase assays, which showed that the highest doses of both the aglycone extract and emodin affected the viability. However, the effect did not occur in all of the used assays. Notably, after both exposures, a dose-dependent increase in oxidative stress factors was observed in both cell lines as measured by MitoSOX and dihydroethidium assays. C. sanguineus extract was not genotoxic in the comet assay. Importantly, both emodin and the extract activated the skin sensitisation pathway in the KeratinoSens assay, suggesting that they can induce allergy in humans. As emodin has shown cytotoxic and skin-sensitising effects, it is possible that the adverse effects caused by the extract are also mediated by it since it is the main component present in the fungus. [ABSTRACT FROM AUTHOR]

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced. [ABSTRACT FROM AUTHOR]

Context: Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims: This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods: Spermatozoa were exposed to either isotonic (300 mOsm/kg) or hypotonic (180 mOsm/kg) media for 5 and 20 min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+/PI−) or with different degrees of plasma membrane alteration (SYBR14+/PI+ and SYBR14−/PI+). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results: The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P < 0.01) higher in spermatozoa losing membrane integrity. In the second strategy, three out of five subpopulations (SP2, SP3 and SP4) showed some degree of alteration in their plasma membrane with significant (P < 0.01) differences in EV. In both cluster analyses, SP5 (intact-membrane spermatozoa) presented the lowest EV. Besides, SP3 and SP4 (Strategy 1) and SP5 (Strategy 2) were found to be significantly (P < 0.05) correlated with sperm functional competence. Conclusions: Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications: Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation. Evaluation of sperm quality in farm animals and humans is crucial to predict reproductive efficiency, yet conventional tests are not sufficient to predict fertility. Clustering sperm using individual data from flow cytometry analysis provides more information than typical dot plots, which could better predict sperm fertility and cryotolerance. Herein, combining cluster analysis with flow cytometry led to the identification of five sperm subpopulations with differences in cell volume and membrane integrity, whereas typical dot plots just allowed for the identification of three. Image by the authors. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

The use of dead probiotics and their cellular metabolites seems to exhibit immunomodulatory and anti-inflammatory properties, providing protection against pathogens. These inanimate microorganisms, often referred to as tyndallized or heat-killed bacteria, are a new class of probiotics employed in clinical practice. Safety concerns regarding the extensive use of live microbial cells have increased interest in inactivated bacteria, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. Culture-dependent methods are not suitable for the quality assessment of these products, and alternative methods are needed for their quantification. To date, bacterial counting chambers and microscopy have been used for tyndallized bacteria enumeration, but no alternative validated methods are now available for commercial release. The aim of the present study is to design a new method for the qualitative and quantitative determination of tyndallized bacterial cells using flow cytometric technology. Using a live/dead viability assay based on two nucleic acid stains, thiazole orange (TO) and propidium iodide (PI), we optimized a workflow to evaluate bacterial viability beyond the reproduction capacity that provides information about the structural properties and metabolic activities of probiotics on FACSVerse without using beads as a reference. The data obtained in this study represent the first analytical application that works effectively both on viable and non-viable cells. The results provided consistent evidence, and different samples were analyzed using the same staining protocol and acquisition settings. No significant discrepancies were highlighted between the declared specification of commercial strain and the analytical data obtained. For the first time, flow cytometry was used for counting tyndallized bacterial cells as a quality control assessment in probiotic production. This aspect becomes important if applied to medical devices where we cannot boast metabolic but only mechanical activities. [ABSTRACT FROM AUTHOR]

Introduction: Natural bioactive substances have become increasingly noticeable for their capability to eliminate and counteract cancer throughout time. Curcumin, a bioactive compound derived from the rhizomes of turmeric, is well known for its therapeutic effect in inducing anti-inflammatory, anti-migration, and anti-proliferation activities. However, curcumin encounters several limitations that prevent it from reaching its maximum capabilities. One of the curcuminoid analogues, 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC), was synthesized by removing the unstable β-diketone moiety and changing into double bonds while retaining the hydroxyl group to improve the curcumin’s bioavailability. It is aims to investigate the cytotoxicity of BHMC especially on the proliferation and migration effects towards human liver cancer, HepG2 cells. Methods: MTT assay was performed to determine the cytotoxicity of BHMC and curcumin on HepG2 and Hs27 cells. Next, Hoechst 33342 and Propidium Iodide staining were executed to observe the morphological changes on HepG2 cells treated with BHMC and curcumin. Further analysis on the migration rate of HepG2 cells upon treatment with BHMC and curcumin was measured using scratch assay. Results: At lower concentration, BHMC demonstrated approximately 3-7 times higher toxicity effect towards HepG2 cells compared to curcumin. BHMC also specifically targets HepG2 cells with a selectivity index of up to 6 units which clearly demonstrate its cytotoxic selectivity towards Hs27 cells. Further examination reveals that BHMC induces cytotoxicity via late-stage apoptosis. BHMC also enhanced the inhibition of the migration effects by 4.2, 7.2, and 7.6% throughout incubation period compared to the untreated and curcumin. Conclusion: Despite the pronounced toxicity of BHMC on HepG2 cells, BHMC was demonstrated more selective cytotoxic on Hs27. [ABSTRACT FROM AUTHOR]

The plate count agar (PCA) method is a common technique used to count microorganisms in dairy products, but this method is time‐consuming and highly selective by the medium. This study adopted Lab158 and PF2 as the universal probes for hybridising lactic acid bacteria and yeasts, respectively, and investigated the effects of fixation time, hybridisation time, hybridisation temperature, probe concentration and propidium iodide (PI) concentration on hybridisation efficiency in the samples. The results showed that the highest hybridisation efficiency for lactic acid bacteria and yeast, along with the best double staining results, was observed under the following conditions: a fixation time of 80 min, a hybridisation time of 180 min, a hybridisation temperature of 48°C, a probe volume of 5 μL for lactic acid bacteria and 3 μL for yeast, and a PI volume of 5 μL for lactic acid bacteria and 3 μL for yeast. The fluorescence in situ hybridisation‐flow cytometry (FISH‐FCM) and PCA counting results showed a significant positive correlation (r > 0.9). The FISH‐FCM and PCA counting results showed a significant positive correlation (r > 0.9). The FISH‐FCM counting method can serve as a quick and accurate method for the counting of lactic acid bacteria and yeast in dairy products. [ABSTRACT FROM AUTHOR]

Cancer is a disease that encompasses multiple and different malignant conditions and is among the leading causes of death in the world. Therefore, the search for new pharmacotherapeutic options and potential candidates that can be used as treatments or adjuvants to control this disease is urgent. Natural products, especially those obtained from plants, have played an important role as a source of specialized metabolites with recognized pharmacological properties against cancer, therefore, they are an excellent alternative to be used. The objective of this research was to evaluate the action of the monoterpene isoespintanol (ISO) against the human tumor cell lines MDA-MB-231, A549, DU145, A2780, A2780-cis and the non-tumor line MRC-5. Experiments with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescence with propidium iodide (PI), 4′,6-diamidino-2-phenylindole dilactate (DAPI) and green plasma revealed the cytotoxicity of ISO against these cells; furthermore, morphological and chromogenic studies revealed the action of ISO on cell morphology and the inhibitory capacity on reproductive viability to form colonies in MDA-MB-231 cells. Likewise, 3D experiments validated the damage in these cells caused by this monoterpene. These results serve as a basis for progress in studies of the mechanisms of action of these compounds and the development of derivatives or synthetic analogues with a better antitumor profile. [ABSTRACT FROM AUTHOR]

The properties of six commonly used, commercially available, fluorescent dyes were compared in staining right-handed B-DNA and left-handed Z-DNA. All showed different degree of fluorescence turn-on in the presence of B-DNA, but very little in the presence of Z-DNA. The optimal range of dye-DNA ratios of DNA was determined. While these dyes do not provide a turn-on type probe for Z-DNA, staining between B- and Z-DNA using dyes such as SYBR Green I was shown to be useful in tracking the kinetics of conformational changes between these two forms of DNA. Finally, SYBR Green I showed unique circular dichroism patterns in 4 M NaCl that change in the presence of double stranded DNA, both in the visible and UV range. [ABSTRACT FROM AUTHOR]

Background: Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. Methods: The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA‐seq, CRISPR‐Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit‐8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V‐FITC staining and/or propidium iodide staining. Results: PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild‐type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild‐type nucleotide. Comparing the HCC cells with wild‐type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. Conclusions: PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. Key points: TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC.The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3.Combined inhibition of PLK1 and Smad3 showed a cooperative anti‐tumor effect in TERT mutant HCC cells. [ABSTRACT FROM AUTHOR]

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages. [ABSTRACT FROM AUTHOR]

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus. [ABSTRACT FROM AUTHOR]

Objective: PANoptosis, a new form of regulated cell death, concomitantly manifests hallmarks for pyroptosis, apoptosis, and necroptosis. It has been usually observed in macrophages, a class of widely distributed innate immune cells in various tissues, upon pathogenic infections. The second-generation curaxin, CBL0137, can trigger necroptosis and apoptosis in cancer-associated fibroblasts. This study aimed to explore whether CBL0137 induces PANoptosis in macrophages in vitro and in mouse tissues in vivo. Methods: Bone marrow-derived macrophages and J774A.1 cells were treated with CBL0137 or its combination with LPS for indicated time periods. Cell death was assayed by propidium iodide staining and immunoblotting. Immunofluorescence microscopy was used to detect cellular protein distribution. Mice were administered with CBL0137 plus LPS and their serum and tissues were collected for biochemical and histopathological analyses, respectively. Results: The results showed that CBL0137 alone or in combination with LPS induced time- and dose-dependent cell death in macrophages, which was inhibited by a combination of multiple forms of cell death inhibitors but not each alone. This cell death was independent of NLRP3 expression. CBL0137 or CBL0137 + LPS-induced cell death was characterized by simultaneously increased hallmarks for pyroptosis, apoptosis and necroptosis, indicating that this is PANoptosis. Induction of PANoptosis was associated with Z-DNA formation in the nucleus and likely assembly of PANoptosome. ZBP1 was critical in mediating CBL0137 + LPS-induced cell death likely by sensing Z-DNA. Moreover, intraperitoneal administration of CBL0137 plus LPS induced systemic inflammatory responses and caused multi-organ (including the liver, kidney and lung) injury in mice due to induction of PANoptosis in these organs. Conclusions: CBL0137 alone or plus inflammatory stimulation induces PANoptosis both in vitro and in vivo, which is associated with systemic inflammatory responses in mice. [ABSTRACT FROM AUTHOR]

The fig‐milk dessert, a traditional and nutritionally rich treat infused with bioactive compounds, was subjected to a comprehensive analysis in this study. The novelty of this research lies in the investigation of the in vitro antioxidant, anticancer, and antimicrobial potential of the fig‐milk dessert. This was accomplished through the utilization of the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay, Annexin/propidium iodide staining, microtiter plate‐based assay and agar well diffusion, respectively, for the first time. Additionally, the study assessed the total phenols and flavonoid content of the extract using the Folin–Ciocalteu assay and the aluminum chloride method, respectively. The findings revealed that the cooking method exerted a significant influence on the bioactive properties and nutritional composition of the dessert. Among the samples analyzed, CM1, consisting of figs steamed for 2 min and milk heated to 70°C, exhibited remarkable characteristics. This sample demonstrated the highest peptide concentration (1290 mg/L), superior antioxidant and anticancer activities, and favorable sensory attributes. Specifically, CM1 induced apoptosis in 84% of AGS cells and inhibited 68% of free radicals in the DPPH assay. It is noteworthy that the fig‐milk dessert did not exhibit any antibacterial properties. These discerning results carry substantial implications for the development of functional dairy products endowed with both nutritional and potential therapeutic properties. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the in vitro antimicrobial and antibiofilm properties and the immune modulatory activity of cannabidiol (CBD) and cannabigerol (CBG) on oral bacteria and periodontal ligament fibroblasts (PLF). Methods: Cytotoxicity was assessed by propidium iodide flow cytometry on fibroblasts derived from the periodontal ligament. The minimum inhibitory concentration (MIC) of CBD and CBG for S. mutans and C. albicans and the metabolic activity of a subgingival 33-species biofilm under CBD and CBG treatments were determined. The Quantification of cytokines was performed using the LEGENDplex kit (BioLegend, Ref 740930, San Diego, CA, USA). Results: CBD-treated cell viability was greater than 95%, and for CBG, it was higher than 88%. MIC for S. mutans with CBD was 20 µM, and 10 µM for CBG. For C. albicans, no inhibitory effect was observed. Multispecies biofilm metabolic activity was reduced by 50.38% with CBD at 125 µg/mL (p = 0.03) and 39.9% with CBG at 62 µg/mL (p = 0.023). CBD exposure at 500 µg/mL reduced the metabolic activity of the formed biofilm by 15.41%, but CBG did not have an effect. CBG at 10 µM caused considerable production of anti-inflammatory mediators such as TGF-β and IL-4 at 12 h. CBD at 10 µM to 20 µM produced the highest amount of IFN-γ. Conclusion: Both CBG and CBD inhibit S. mutans; they also moderately lower the metabolic activity of multispecies biofilms that form; however, CBD had an effect on biofilms that had already developed. This, together with the production of anti-inflammatory mediators and the maintenance of the viability of mammalian cells from the oral cavity, make these substances promising for clinical use and should be taken into account for future studies. [ABSTRACT FROM AUTHOR]

Background and Aim: One of the most prevalent cancers worldwide is lung cancer with the second top fatality rate. The purpose of this work is to disclose the anti-proliferative ability of hydroalcoholic Ficus benghalensis bark extract against lung cancer cell line (A549). Materials and Methods: Antioxidant property of Ficus benghalensis bark extract was studied using a, a-diphenyl-ß-picrylhydrazyl and Hydrogen Peroxide assays. The investigation of anti-proliferative property of Ficus benghalensis hydroalcoholic bark extract was carried out through tetrazolium salt-based cytotoxicity assay, cell viability assessment using trypan blue dye and morphometric analysis. The cytotoxic potency was analysed by propidium iodide staining, oxidative stress markers determination and expression of apoptotic gene markers such as Bax, c-MYC and PARP. Results: The anti-oxidant assays revealed the great radical scavenging property of Ficus benghalensis bark extract. Ficus benghalensis hydroalcoholic bark extract showed 50.12% inhibition at 50 µg/mL which is also confirmed by viability assay. In morphometric analysis the distortion of cells were noted in treated groups. The nuclear staining and gene expression studies further confirms the anti-cancer activity of Ficus benghalensis hydroalcoholic bark extract. The estimation of release of nitric oxide and lipid peroxidation was high in treated groups which also supports the previous results. Conclusion: The study emphasize the anti proliferative and anti oxidant property of Ficus benghalensis hydroalcoholic bark extract against A549 cell line. [ABSTRACT FROM AUTHOR]

Plumbago zeylanica L., a wild shrub, is a vital natural source of plumbagin, a potent 1,4-naphthoquinone renowned for its anti-cancer properties, notably effective against breast, prostate, and ovarian cancers. Traditional plumbagin extraction, involving root uprooting and plant destruction, raises ecological concerns. The primary objective of this study is to enhance plumbagin production by incorporating the elicitation process into in vitro cultivation with regenerated plants that retain all of their intact organs. Seven different elicitors categorized into three distinct groups were employed to stimulate plumbagin content. Among the various elicitors used, this study marks the first application of biogenic silver nanoparticles (AgNPs) from Curcuma amada in stimulating plumbagin production in this plant. The maximum plumbagin content, recorded at 8.98 ± 0.24 mg/g dry weight basis, was found in the roots when elicited with AgNPs at a concentration of 15 mg/l. In addition to that, biotic elicitors (yeast extract, chitosan and casein hydrolysate) and heavy metals (lead, cobalt and nickel) also successfully elicit plumbagin in the root and aerial parts of the plants, quantified through High Performance Liquid Chromatography (HPLC). In our study, we found that certain elicitors induced root browning and tissue necrosis, as confirmed by propidium iodide (PI) staining. The most significant browning effects were observed with chitosan from biotic sources and lead from heavy metals, while no such effects were associated with AgNPs at any concentration. Utilizing intact, entire plants as the subjects for elicitation in our study is a valuable aspect. This approach closely replicates the natural process occurring in intact plants, enhancing the relevance of our findings to practical situations.Key message: Utilizing silver nanoparticles alongside diverse biotic and heavy metal elicitors applied to entire plants in liquid cultures facilitates the sustainable production of plumbagin from various plant organs. [ABSTRACT FROM AUTHOR]

Flow cytometry techniques utilizing dual staining with annexin V and propidium iodide (PI) provide a robust method for quantitatively analyzing apoptosis induction. Annexin V binds phosphatidylserine exposed on the outer leaflet of the plasma membrane during early apoptosis, while PI permeates late apoptotic/necrotic cells. Simultaneous staining allows differentiation of viable, early apoptotic, and late apoptotic/necrotic populations. This approach can be enhanced by using fluorochrome-conjugated antibodies to stain specific proteins, enabling the simultaneous tracking of protein expression changes in defined cell subpopulations during apoptosis. This multiparametric approach provides key insights into signaling regulation and the mechanisms underlying the apoptotic response to cytotoxic treatments. Here we present a protocol that combines annexin V-FITC/PI staining with APC-conjugated antibody labeling in MDA-MB-231 breast cancer cells treated with doxorubicin. This protocol enables both the quantitative assessment of apoptosis induction and the tracking of decreased CD44 expression from viable to apoptotic cells. This protocol also provides guidelines for appropriate filter selection, compensation controls, gating strategies, and troubleshooting. This robust protocol holds significant potential for elucidating signaling networks involved in apoptosis and therapeutic resistance across various cellular models.

Methane-producing Archaea can be found in a variety of habitats, including the gastrointestinal tract, where they are linked to various diseases. The majority of current monitoring methods can be slow and laborious. To facilitate gut methanogenic Archaea detection, we investigated flow cytometry for rapid quantification based on the autofluorescent F420 cofactor, an essential coenzyme in methanogenesis. The methanogenic population was distinguishable from the SYBR green (SG) and SYBR green/propidium iodide (SGPI) stained background microbiome based on elevated 452 nm emission in Methanobrevibacter smithii spiked controls. As a proof-of-concept, elevated F420-autofluorescence was used to detect and quantify methanogens in 10 faecal samples and 241 in vitro incubated faecal samples. The methanogenic population in faeces, determined through Archaea-specific 16S rRNA gene amplicon sequencing, consisted of Methanobrevibacter and Methanomassiliicoccus. F420-based methanogen quantification in SG and SGPI-stained faecal samples showed an accuracy of 90 and 100% against Archaea proportions determined with universal primers. When compared to methane and Archaea presence, methanogen categorisation in in vitro incubated faeces exhibited an accuracy of 71 and 75%, with a precision of 42 and 70%, respectively. To conclude, flow cytometry is a reproducible and fast method for the detection and quantification of gut methanogenic Archaea.

Stomatal movement, initiated by specialized epidermal cells known as guard cells (GCs), plays a pivotal role in plant gas exchange and water use efficiency. Despite protocols existing for isolating GCs through proplasting for carrying out biochemical, physiological, and molecular studies, protocals for isolating GCs with their cell walls still intact have been lacking in the literature. In this paper, we introduce a method for the isolation of complete GCs from Vicia faba and show their membrane to remain impermeable through propidium iodide staining. This methodology enables further in-depth analyses into the cell wall composition of GCs, facilitating our understanding of structure-function relationship governing reversible actuation within cells. [ABSTRACT FROM AUTHOR]

Viability and vitality assays play a crucial role in assessing the effectiveness of novel therapeutic approaches, with stain-based methods providing speed and objectivity. However, their application in yeast research lacks consensus. This study aimed to assess the performance of four common dyes on C. parapsilosis planktonic cells as well as sessile cells that form well-structured biofilms (treated and not treated with amphotericin B). Viability assessment employed Syto-9 (S9), thiazole orange (TO), and propidium iodide (PI). Metabolic activity was determined using fluorescein diacetate (FDA) and FUN-1. Calcofluor white (CW) served as the cell visualization control. Viability/vitality percentage of treated samples were calculated for each dye from confocal images and compared to crystal violet and PrestoBlue results. Heterogeneity in fluorescence intensity and permeability issues were observed with S9, TO, and FDA in planktonic cells and biofilms. This variability, influenced by cell morphology, resulted in dye-dependent viability/vitality percentages. Notably, PI and FUN-1 exhibited robust C. parapsilosis staining, with FUN-1 vitality results comparable to PrestoBlue. Our finding emphasizes the importance of evaluating dye permeability in yeast species beforehand, incorporating cell visualization controls. An improper dye selection may lead to misinterpreting treatment efficacy. [ABSTRACT FROM AUTHOR]

Methane-producing Archaea can be found in a variety of habitats, including the gastrointestinal tract, where they are linked to various diseases. The majority of current monitoring methods can be slow and laborious. To facilitate gut methanogenic Archaea detection, we investigated flow cytometry for rapid quantification based on the autofluorescent F420 cofactor, an essential coenzyme in methanogenesis. The methanogenic population was distinguishable from the SYBR green (SG) and SYBR green/propidium iodide (SGPI) stained background microbiome based on elevated 452 nm emission in Methanobrevibacter smithii spiked controls. As a proof-of-concept, elevated F420-autofluorescence was used to detect and quantify methanogens in 10 faecal samples and 241 in vitro incubated faecal samples. The methanogenic population in faeces, determined through Archaea-specific 16S rRNA gene amplicon sequencing, consisted of Methanobrevibacter and Methanomassiliicoccus. F420-based methanogen quantification in SG and SGPI-stained faecal samples showed an accuracy of 90 and 100% against Archaea proportions determined with universal primers. When compared to methane and Archaea presence, methanogen categorisation in in vitro incubated faeces exhibited an accuracy of 71 and 75%, with a precision of 42 and 70%, respectively. To conclude, flow cytometry is a reproducible and fast method for the detection and quantification of gut methanogenic Archaea. [ABSTRACT FROM AUTHOR]

Shigellosis is a pathological condition that affects the digestive system and possibly causes diarrhoea. Shigella species, which are responsible for this disease, are highly contagious and spread through contaminated food and water. The increasing development of resistance by Shigella species necessitates the urgent need to search for new therapies against diarrhoea-causing shigellosis. The scientific validation of medicinal plants, such as Diospyros gilletii, which is used for the traditional treatment of diarrhoeal conditions is worthwhile. The present study aims to investigate the antibacterial activity of extracts from D. gilletii against selected Shigella species. Extracts from D. gilletii stem bark were prepared by maceration using various solvents. The antibacterial activity of D. gilletii extracts was evaluated in Shigella dysenteriae, S. flexneri, S. boydii, and S. sonnei using a microdilution method, whereas a cytotoxicity test was performed on Vero and Raw cells using resazurin-based colorimetric assays. Bacterial membrane-permeability studies were evaluated using propidium iodide (PI)- and 1-N-phenyl-naphthylamine (NPN)-uptake assays, whereas inhibition and eradication tests on bacterial biofilms were carried out by spectrophotometry. As a result, methanol, ethanol and hydroethanol (water: ethanol; 30:70, v/v) extracts of D. gilletii inhibited the growth of S. boydii, S. flexneri and S. sonnei, with minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg/mL, without toxicity to Vero and Raw cells. Time-kill kinetics revealed bactericidal orientation at 2 MIC and 4 MIC and a bacteriostatic outcome at 1/2 MIC. The mechanistic basis of antibacterial action revealed that D. gilletii extracts inhibited and eradicated Shigella biofilms and promoted the accumulation of NPN and PI within the inner and outer membranes of bacteria to increase membrane permeability, thereby causing membrane damage. This novel contribution toward the antibacterial mechanisms of action of D. gilletii extracts against Shigella species substantiates the use of this plant in the traditional treatment of infectious diarrhoea. [ABSTRACT FROM AUTHOR]

Background: The development of radioresistance seriously hinders the efficacy of radiotherapy in lung cancer. However, the underlying mechanisms by which radioresistance occurs are still incompletely understood. The N6-Methyladenosine (m6A) modification of RNA is involved in cancer progression, but its role in lung cancer radioresistance remains elusive. This study aimed to identify m6A regulators involved in lung cancer radiosensitivity and further explore the underlying mechanisms to identify therapeutic targets to overcome lung cancer radioresistance. Methods: Bioinformatic mining was used to identify the m6A regulator IGF2BP2 involved in lung cancer radiosensitivity. Transcriptome sequencing was used to explore the downstream factors. Clonogenic survival assays, neutral comet assays, Rad51 foci formation assays, and Annexin V/propidium iodide assays were used to determine the significance of FBW7/IGF2BP2/SLC7A5 axis in lung cancer radioresistance. Chromatin immunoprecipitation (ChIP)-qPCR analyses, RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)-qPCR analyses, RNA pull-down analyses, co-immunoprecipitation analyses, and ubiquitination assays were used to determine the feedback loop between IGF2BP2 and SLC7A5 and the regulatory effect of FBW7/GSK3β on IGF2BP2. Mice models and tissue microarrays were used to verify the effects in vivo. Results: We identified IGF2BP2, an m6A "reader", that is overexpressed in lung cancer and facilitates radioresistance. We showed that inhibition of IGF2BP2 impairs radioresistance in lung cancer both in vitro and in vivo. Furthermore, we found that IGF2BP2 enhances the stability and translation of SLC7A5 mRNA through m6A modification, resulting in enhanced SLC7A5-mediated transport of methionine to produce S-adenosylmethionine. This feeds back upon the IGF2BP2 promoter region by further increasing the trimethyl modification at lysine 4 of histone H3 (H3K4me3) level to upregulate IGF2BP2 expression. We demonstrated that this positive feedback loop between IGF2BP2 and SLC7A5 promotes lung cancer radioresistance through the AKT/mTOR pathway. Moreover, we found that the ubiquitin ligase FBW7 functions with GSK3β kinase to recognize and degrade IGF2BP2. Conclusions: Collectively, our study revealed that the m6A "reader" IGF2BP2 promotes lung cancer radioresistance by forming a positive feedback loop with SLC7A5, suggesting that IGF2BP2 may be a potential therapeutic target to control radioresistance in lung cancer. [ABSTRACT FROM AUTHOR]

Gerontology research on anti-aging interventions with drugs could be an answer to age-related diseases, aiming at closing the gap between lifespan and healthspan. Here, we present two methods for assaying chronological lifespan in human cells: (1) a version of the classical outgrowth assay with quantitative assessment of surviving cells and (2) a version of the PICLS method (propidium iodide fluorescent-based measurement of cell death). Both methods are fast, simple to conduct, cost-effective, produce quantitative data for further analysis and can be used with diverse human cell lines. Whereas the first method is ideal for validation and testing the post-intervention reproductive potential of surviving cells, the second method has true high-throughput screening potential. The new technologies were validated with known anti-aging compounds (2,5-anhydro-d-mannitol and rapamycin). Using the high-throughput screening method, we screened a library of 162 chemical entities and identified three compounds that extend the longevity of human cells. [ABSTRACT FROM AUTHOR]

Furanocoumarins are naturally occurring compounds in the plant world, characterized by low molecular weight, simple chemical structure, and high solubility in most organic solvents. Additionally, they have a broad spectrum of activity, and their properties depend on the location and type of attached substituents. Therefore, the aim of our study was to investigate the anticancer activity of furanocoumarins (imperatorin, isoimperatorin, bergapten, and xanthotoxin) in relation to human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cell lines. The tested compounds were used for the first time in combination with LY294002 (PI3K inhibitor) and sorafenib (Raf inhibitor). Apoptosis, autophagy, and necrosis were identified microscopically after straining with Hoechst 33342, acridine orange, and propidium iodide, respectively. The levels of caspase 3 and Beclin 1 were estimated by immunoblotting and for the blocking of Raf and PI3K kinases, the transfection with specific siRNA was used. The scratch test was used to assess the migration potential of glioma cells. Our studies showed that the anticancer activity of furanocoumarins strictly depended on the presence, type, and location of substituents. The obtained results suggest that achieving higher pro-apoptotic activity is determined by the presence of an isoprenyl moiety at the C8 position of the coumarin skeleton. In both anaplastic astrocytoma and glioblastoma, imperatorin was the most effective in induction apoptosis. Furthermore, the usage of imperatorin, alone and in combination with sorafenib or LY294002, decreased the migratory potential of MOGGCCM and T98G cells. [ABSTRACT FROM AUTHOR]

[Display omitted] Hypothesis : Hydrophobic ion pairs (HIPs) between two fluorescent components and incorporation into nanoemulsions (NE) allows tracking in cellular uptake studies. Experiments : HIPs were formed between propidium iodide and 1,2-dioleoyl- sn - glycero -3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-PE), azure A chloride and NBD-PE or coumarin 343 and 4-(4-dihexadecylaminostyryl)- N -methylpyridinium iodide) (DiA). Fluorescence spectra of the resulting complexes were recorded. HIPs were loaded into zwitterionic NE and their size, stability in different media, haemolytic properties and cytotoxicity were evaluated. Furthermore, cellular uptake at 37 °C and 4 °C was investigated via flow cytometry and confocal microscopy. Findings: HIP-formation increased lipophilicity of the hydrophilic model drugs. NE exhibited a size between 80 and 150 nm and were not toxic in concentrations up to 0.1 % but showed high haemolytic properties. Cellular uptake of propidium, azure A and coumarin 343 were 8-fold, 115-fold and 1.3-fold improved by the formation of HIPs and up to 59-fold, 120-fold and 50-fold by incorporating these HIPs in NE, respectively. Lower uptake was observed at 4 °C. In case of propidium/ NBD-PE and azure A/ NBD-PE HIPs, propidium and azure A were delivered into the cytosol, whereas NBD-PE was unable to enter cells. In case of coumarin 343/ DiA HIPs, both components accumulated in the cell membrane. Therefore, HIPs between two fluorescent compounds are a powerful tool to investigate cellular uptake of hydrophobic complexes via nanocarriers by visualization of their cellular distribution. [ABSTRACT FROM AUTHOR]

In an ongoing project on the Iranian Helichrysum Mill. species (Asteraceae), the phytochemical profiles of the extracts obtained from the flower and leaves of Helichrysum oligocephalum were investigated utilizing ultra‐performance liquid chromatography–mass spectrometry (MS) coupled with quadrupole time of flight. In addition, the cytotoxic activity of the extracts was evaluated against different cancerous cell lines by AlamarBlue assay. The apoptotic effects of the fractions were measured by propidium iodide staining and flow cytometry. HPLC‐based activity profiling was used to track the cytotoxic constituents. The MS and MSn data were analyzed by MZmine3. Clustering and visualization of the MS data were established by an online platform FreeClust. Concentration‐dependent cytotoxicity was observed for dichloromethane fractions. The active constituents were annotated as pyrone and phloroglucinol derivatives by comparing their MS and MSn profiles to an in‐house library, available databases, Dictionary of Natural Products, and literature. Additionally, plausible MS fragmentation pathways were suggested for the compounds; this can help in the dereplication process of similar compounds from the genus. In conclusion, this plant is rich in valuable phloroglucinol and pyrone compounds and can be used as a source of active phytochemicals. However, further phytochemical and pharmacological studies are necessary. [ABSTRACT FROM AUTHOR]

Irinotecan (IRI), an anticancer drug to treat colon cancer patients, causes cytotoxic effects on normal cells. Phenethyl isothiocyanate (PEITC), rich in common cruciferous plants, has anticancer activities (induction of cell apoptosis) in many human cancer cells, including colon cancer cells. However, the anticancer effects of IRI combined with PEITC on human colon cancer cells in vitro were unavailable. Herein, the aim of this study is to focus on the apoptotic effects of the combination of IRI and PEITC on human colon cancer HCT 116 cells in vitro. Propidium iodide (PI) exclusion and Annexin V/PI staining assays showed that IRI combined with PEITC decreased viable cell number and induced higher cell apoptosis than that of IRI or PEITC only in HCT 116 cells. Moreover, combined treatment induced higher levels of reactive oxygen species (ROS) and Ca2+ than that of IRI or PEITC only. Cells pre‐treated with N‐acetyl‐l‐cysteine (scavenger of ROS) and then treated with IRI, PEITC, or IRI combined with PEITC showed increased viable cell numbers than that of IRI or PEITC only. IRI combined with PEITC increased higher caspase‐3, ‐8, and ‐9 activities than that of IRI or PEITC only by flow cytometer assay. IRI combined with PEITC induced higher levels of ER stress‐, mitochondria‐, and caspase‐associated proteins than that of IRI or PEITC treatment only in HCT 116 cells. Based on these observations, PEITC potentiates IRI anticancer activity by promoting cell apoptosis in the human colon HCT 116 cells. Thus, PEITC may be a potential enhancer for IRI in humans as an anticolon cancer drug in the future. [ABSTRACT FROM AUTHOR]

Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 μm. It features an array of micro-holes ranging from 40 μm to 80 μm, spaced apart by 50 μm to 150 μm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 μm outer diameter, 3 μm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and β-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: This research evaluated the anti-Candida albicans effect of Mexican propolis from Chihuahua. Chemical composition of the ethanolic extract of propolis was determined by GC-MS, HPLC-DAD, and HPLC-MS. The presence of anthraquinone, aromatic acid, fatty acids, flavonoids, and carbohydrates was revealed. Results: The anti-Candida activity of propolis was determined. The inhibitions halos were between 10.0 to 11.8 mm; 25% minimum inhibitory concentration (0.5 mg/ml) was fungistatic, and 50% minimum inhibitory concentration (1.0 mg/ml) was fungicidal. The effect of propolis on the capability of C. albicans to change its morphology was evaluated. 25% minimum inhibitory concentration inhibited to 50% of germ tube formation. Staining with calcofluor-white and propidium iodide was performed, showing that the propolis affected the integrity of the cell membrane. INT1 gene expression was evaluated by qRT-PCR. Propolis significantly inhibited the expression of the INT1 gene encodes an adhesin (Int1p). Chihuahua propolis extract inhibited the proliferation of Candida albicans, the development of the germ tube, and the synthesis of adhesin INT1. Conclusions: Given the properties demonstrated for Chihuahua propolis, we propose that it is a candidate to be considered as an ideal antifungal agent to help treat this infection since it would not have the toxic effects of conventional antifungals. [ABSTRACT FROM AUTHOR]

This study was performed to explore the antifungal and antibiofilm effects of polyphyllin I (PPI) on Candida albicans. Microdilution assay was performed to determine the minimal inhibitory concentrations (MIC) of PPI against Candida species. Adhesion assay, hyphal growth assay, biofilm formation, and development were used to test the impacts of PPI on C. albicans virulence factors. Propidium iodide staining was performed to test whether the permeability of cell membrane was influenced by PPI. PPI showed significant antifungal activities against several Candida species, with MIC below or equal to 6.25 μM. PPI also inhibited the adhesion to polystyrene surfaces, hyphal growth, and biofilm formation. PPI significantly increased the permeability of C. albicans cell membrane. In sum, PPI can suppress the planktonic growth and biofilm of C. albicans and its mechanism involves the increased permeability of cell membrane. [ABSTRACT FROM AUTHOR]

Featured Application: The presented analysis provides a concise depiction of the fluid bed drying process of probiotics. It also introduces a new method of evaluation for probiotics, based on the actual number of biologically useful cells. This factor is used to evaluate and economically justify the introduction of technological procedures, such as sublethal stresses and coating. Probiotic bacteria confer a range of health benefits and are a focus of a growing number of studies. One of the main issues is their stability during drying and storage, which is why techniques, such as fluid bed drying and coating or treatment with stress factors during culturing, are utilized. The methods of the evaluation of probiotic viability and quality are, however, lacking and we need a way of distinguishing between different subpopulations of probiotic bacteria. To address this issue, imaging flow cytometry (IFC) has been utilized to assess cells after simulated in vitro digestion of dried and coated preparations treated with pH stress and heat shock. Samples were analyzed fresh and after 12 months of storage using RedoxSensor green and propidium iodide dyes to assess metabolic activity and cell membrane integrity of the cells. The results were then used to design a drying process on an industrial scale and evaluate the economic factors in the SuperPro Designer v13 software. Based on the number of biologically active and beneficial cells obtained utilizing tested methods, the coating process and treatment with heat shock and pH stress have been the most effective and up to 10 times cheaper to produce than only by drying. Additionally, samples after 12 months of storage have shown an increase in the proportion of cells with intermediate metabolic activity and small amounts of cell membrane damage, which are still viable in probiotic products. This subpopulation of bacteria can still be considered live in probiotic products but is not necessarily effectively detected by pour plate counts. [ABSTRACT FROM AUTHOR]

Background: Myocardial infarction is one of the leading causes of mortality worldwide; hence, there is an urgent need to discover novel cardioprotective strategies. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway, has been previously reported to have cardioprotective effects. However, the mechanisms by which KYNA may be protective are still unclear. The current study addressed this issue by investigating KYNA's cardioprotective effect in the context of myocardial ischemia/reperfusion. Methods: H9C2 cells and rats were exposed to hypoxia/reoxygenation or myocardial infarction, respectively, in the presence or absence of KYNA. In vitro, cell death was quantified using flow cytometry analysis of propidium iodide staining. In vivo, TTC-Evans Blue staining was performed to evaluate infarct size. Mitochondrial respiratory chain complex activities were measured using spectrophotometry. Protein expression was evaluated by Western blot, and mRNA levels by RT-qPCR. Results: KYNA treatment significantly reduced H9C2-relative cell death as well as infarct size. KYNA did not exhibit any effect on the mitochondrial respiratory chain complex activity. SOD2 mRNA levels were increased by KYNA. A decrease in p62 protein levels together with a trend of increase in PARK2 may mark a stimulation of mitophagy. Additionally, ERK1/2, Akt, and FOXO3α phosphorylation levels were significantly reduced after the KYNA treatment. Altogether, KYNA significantly reduced myocardial ischemia/reperfusion injuries in both in vitro and in vivo models. Conclusion: Here we show that KYNA-mediated cardioprotection was associated with enhanced mitophagy and antioxidant defense. A deeper understanding of KYNA's cardioprotective mechanisms is necessary to identify promising novel therapeutic targets and their translation into the clinical arena. [ABSTRACT FROM AUTHOR]

BACKGROUND: Phytophthora infestans causes late blight, threatening potato production. The tropane alkaloid scopolamine from some industrial plants (Datura, Atropa, etc.) has a broad‐spectrum bacteriostatic effect, but its effect on P. infestans is unknown. RESULTS: In the present study, scopolamine inhibited the mycelial growth of phytopathogenic oomycete P. infestans, and the half‐maximal inhibitory concentration (IC50) was 4.25 g L−1. The sporangia germination rates were 61.43%, 16.16%, and 3.99% at concentrations of zero (control), 0.5 IC50, and IC50, respectively. The sporangia viability of P. infestans was significantly reduced after scopolamine treatment through propidium iodide and fluorescein diacetate staining, speculating that scopolamine destroyed cell membrane integrity. The detached potato tuber experiment demonstrated that scopolamine lessened the pathogenicity of P. infestans in potato tubers. Under stress conditions, scopolamine showed good inhibition of P. infestans, indicating that scopolamine could be used in multiple adverse conditions. The combination effect of scopolamine and the chemical pesticide Infinito on P. infestans was more effective than the use of scopolamine or Infinito alone. Moreover, transcriptome analysis suggested that scopolamine leaded to a downregulation of most P. infestans genes, functioning in cell growth, cell metabolism, and pathogenicity. CONCLUSION: To our knowledge, this is the first study to detect scopolamine inhibitory activity against P. infestans. Also, our findings highlight the potential of scopolamine as an eco‐friendly option for controlling late blight in the future. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.