Propidium Iodide-positive Cells Research Articles

AT11-guided liposomes for oral cancer cells: From characterization towards in vitro evaluation

The increasing prevalence of oral cancers, particularly those attributed to human papillomavirus infection, along with the associated drawbacks of conventional therapies, has spurred research into more effective treatment modalities. Liposomes have emerged as a potential strategy to improve the delivery of anticancer molecules to the cells of interest. However, functionalizing these nanoparticles with targeting agents could significantly enhance their selectivity. A prospective approach involves the incorporation of aptamers, which facilitate drug accumulation within cancer cells. Among these, the AT11 aptamer has garnered attention due to its improved toxicity and high affinity to nucleolin, making it a promising targeting moiety. Therefore, we propose to use AT11 for liposomes’ functionalization, to enhance the selectivity of C8, a potential anticancer compound, specifically targeting oral cancer cells. Thus, we produced liposomes (empty or C8-associated) by ethanol injection method and, then, proceeded with their functionalization with AT11-TEG-Cholesteryl. The resulting liposomes were characterized by dynamic light scattering and hydrodynamic diameters of 130–136 nm were obtained. Additionally, the effect of the produced liposomes on the viability of squamous cell carcinoma of the tongue (UPCI-SCC-154) and nonmalignant (Het1A) cells was determined, by MTT assay. It was demonstrated that the viability of cells treated with empty liposomes was almost unaffected until the 53.6 μg/mL concentration. After treating the cells with C8-associated liposomes, both cell lines showed a dose-response effect. Moreover, the AT11-functionalization of the obtained C8-associated liposomes enhanced the selectivity of the liposomes towards oral cancer cell line, as observed by the MTT assay (43.2 % in UPCI-SCC-154 versus 79.4 % in Het1A) and confocal microscopy. Furthermore, the anticancer potential of AT11 C8-associated liposomes was evaluated in terms of proliferation (ki67 positive cells), cell death (propidium iodide internalization in non-permeabilized cells and caspase-3 activity), migration (cell scratch assay) and invasion. Overall, the produced liposomes decreased tongue cancer cell proliferation (53.6 % versus 99.4 %), migration (from 24.6 % to −3.8 %) and invasion (to 19 %) and induced cell death (11.6-fold increase in propidium iodide positive cells, and a 140 % of caspase-3 activity). These findings suggest that the AT11 C8-associated liposomes are promising drug carriers for oral cancer therapy.

Inhibitory action of methylene bisphosphonic acid on metabolic activity and viability of J774A.1 cells

Bisphosphonates (BPs) are primary agents in the current pharmacological arsenal against osteoclast-related bone loss due to osteoporosis, Paget’s disease and bone tumors. Due to the lack of complete understanding of the molecular mechanism of their action in bone tissue and the overlap of key properties between BPs of different generations, integral studies of BPs inhibitory and antiresorptive properties are relevant today. The present work was carried out to establish a comprehensive study of the inhibitory effects of methylene bisphosphonic acid (MBPA) on the mevalonate pathway, metabolic activity and cell death in vitro compared to zoledronic acid (Zol). Farnesyl pyrophosphate synthase activity of MBPA-treated J774A.1 cells was inhibited by 80%, compared with a 79% reduction in Zol-treated samples. The ability of MBPA to decrease the percentage of viable cells in culture is slightly lower compared with Zol. After 24 h of incubation with lowest concentration, the percentage of inhibition of metabolic activity was 10.6 and 25%, respectively. After 48 h, these values were 34.8 and 55.6%, respectively. The inhibitory effects of MBPA and Zol on the intensity of incorporation of radioactively labeled precursor [14C]-acetate to the cholesterol fraction were 76.2 and 59.1%, respectively. In the case of isoprenoid fraction, the inhibitory effects were 40.9% and 51.2%, respectively. MBPA and Zol differently induced apoptosis in the J774A.1 cells culture, increased count of apoptotic cells in 2.4 and 6.3 times, and also increased the number of propidium iodide-positive cells in 7.4 and 19 times, respectively. MBPA and Zol also increased the number of TUNEL-positive cells in macrophage culture in 2.6 and 5 times, respectively. Zoledronate significantly reduced carbonic anhydrase 2 and nuclear factor of activated T-cells 1 gene expression levels compared to the MBPA action. Thus, the use of MBPA in future research and therapy of both cancer and osteoporosis looks promising due to lower cytotoxicity, high efficiency of mevalonate pathway inhibition and the possibility of dosage variation. Keywords: cell death, cell viability, FPPS enzymatic activity, gene expression, J774A.1 macrophages, methylene bisphosphonic acid, radioisotopes., zoledronic acid

Neuroprotective effects of polyacrylic acid (PAA) conjugated cerium oxide against hydrogen peroxide- and 6-OHDA-induced SH-SY5Y cell damage

Cerium oxide nanoparticles have been widely investigated against neurodegenerative diseases due to their antioxidant properties that aid in quenching reactive oxygen species. In this study, polyacrylic acid conjugated cerium oxide (PAA-CeO) nanoparticles were synthesized in a 50–60 nm size range with a zeta potential of − 35 mV. X-ray photoelectron spectroscopy analysis revealed a mixed valence state of Ce4+ and Ce3+. PAA-CeO nanoparticles were safe for undifferentiated (UN-) and retinoic acid-differentiated (RA-) human neuroblastoma SH-SY5Y cells and reduced the extent of cell damage evoked by hydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA). In the H2O2 model of cell damage PAA-CeO did not affect the caspase-3 activity (apoptosis marker) but attenuated the number of propidium iodide-positive cells (necrosis marker). In the 6-OHDA model, nanoparticles profoundly reduced necrotic changes and partially attenuated caspase-3 activity. However, we did not observe any impact of PAA-CeO on intracellular ROS formation induced by H2O2. Further, the flow cytometry analysis of fluorescein isothiocyanate-labeled PAA-CeO revealed a time- and concentration-dependent cellular uptake of nanoparticles. The results point to the neuroprotective potential of PAA-CeO nanoparticles against neuronal cell damage induced by H2O2 and 6-OHDA, which are in both models associated with the inhibition of necrotic processes and the model-dependent attenuation of activity of executor apoptotic protease, caspase-3 (6-OHDA model) but not with the direct inhibition of ROS (H2O2 model).

RIP3/MLKL regulates necroptosis via activating 4EBP1-eIF4E pathway.

Necroptosis is a cell death type mediated by receptor interacting protein 3 (RIP3)/mixed lineage kinase domain-like protein (MLKL). It has been reported that mammalian target of rapamycin plays a regulatory role in necroptosis. Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)-eukaryotic initiation factor 4E (eIF4E) pathway is a key down streamer of mammalian target of rapamycin. However, whether 4EBP1-eIF4E pathway is involved in necroptosis is still unknown. This study aims to investigate the changes of 4EBP1-eIF4E pathway in necroptosis. TNF-α/SM-164/Z-VAD-FMK (TSZ), a necroptosis inducer, was used to induce necroptosis in murine fibroblastoid cell line L929. Cell necrosis was observed under an optical microscope. Then, TSZ was added to L929 cells with RIP3 and MLKL gene knockout. Propidium iodide (PI) staining was used to observe cell necrosis. Real-time fluorescence quantitative PCR and Western blotting were used to determine the mRNA and protein expression of 4EBP1 and eIF4E, respectively. After treating L929 cells with TSZ, the number of necrotic cells was increased, the mRNA and protein expression levels of 4EBP1 were significantly downregulated, and the ratio of phosphorylated 4EBP1 (p-4EBP1) to 4EBP1 was increased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly upregulated, and the ratio of phosphorylated eIF4E (p-eIF4E) to eIF4E was increased (both P<0.01). After knocking out RIP3 and MLKL in L929 cells, PI positive necrotic cells were significantly reduced, the mRNA and protein expression levels of 4EBP1 were significantly upregulated, and the ratio of p-4EBP1 to 4EBP1 was decreased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly downregulated, and the ratio of p-eIF4E to eIF4E was decreased (both P<0.01). 4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis.

Abstract 6283: Evaluating the potency of a first-in-class covalent antagonist of the H3K9me3 reader protein MPP8 in bladder cancer

Abstract Background: Bladder cancer (BC) is a common and deadly disease, and despite recent treatment advances, metastatic BC (mBC) remains incurable. Mutations in genes that encode epigenetic/chromatin modifier proteins are common in mBC, with &amp;gt;90% of tumors harboring at least one inactivating mutation. The epigenetic reader protein MPP8 recognizes the histone-3-lysine-9-trimethyl (H3K9me3) region of target gene promoters, and recruits transcription factors associated with cell proliferation and metastasis. UNC7713 was developed as a covalent antagonist to disrupt MPP8 binding to H3K9me3. Here, we explored whether UNC7713 potently inhibits cell proliferation, migration, and viability in preclinical models of BC. Methods: 5637 cells were treated with ascending concentrations of UNC7713 (0.1 nM-100 µM), negative control compound UNC7716, or 0.1% DMSO negative control. Cell viability was measured using CellTiter-Glo™ after 48 h and 96h incubations. IC50 values were calculated using a four-parameter non-linear regression model in GraphPad. Cells were stained with Annexin V and propidium iodide (PI) to evaluate apoptosis versus necrosis signaling by flow cytometry after 48 h incubation of UNC7713. Flow cytometry experiments were performed on a Thermo Attune NxT, and data were analyzed using FlowJo. 5637 cells were grown in a monolayer, treated with UNC7713 (25 nM-100 nM), and then subjected to a wound healing assay to evaluate cell migration after 48 h. To evaluate effects on cell proliferation, 5637 cells were treated with four doses of UNC7713 (25 nM-100 nM), and colony formation was evaluated after 10 days using crystal violet and analyzed by Fiji. For wound healing, images were captured by an Olympus IX83 inverted microscope and analyzed by Fiji. Results: UNC7713 achieved submicromolar potency for reduction of cell viability in 5637 cells and was nearly 200x more potent than UNC7716 at 48 h (IC50: 0.28 μM vs. 56.03 μM). Potency was also maintained at 96 h (0.18 µM). Concentrations of UNC7713 as low as 75 nM caused over 2.5x greater apoptotic signaling than 20 µM UNC7716 after 48 h (18.5% vs. 7.3% Annexin V and PI positive cells). Next, cells treated with UNC7713 did not migrate to initiate wound closure, but instead caused cell death, increasing the size of the initial wound. Cells treated with UNC7713 at concentrations as low as 50 nM caused dramatic wound expansion compared to 0.1% DMSO and 20 µM UNC7716 (70% wound expansion for UNC7713 vs. 100% wound closure for both controls). Last, after 10 days &amp;gt;90% fewer colonies were detected in cells treated with UNC7713 (25 nM-100 nM) when compared to 20 µM UNC7716 and 0.1% DMSO controls. Conclusions: These preliminary data support further inquiry into the role of MPP8 in BC. Future studies will focus on identifying molecular mechanisms that underlie UNC7713’s ability to inhibit cell proliferation, migration, and viability in preclinical models of BC. Citation Format: Stephany Gonzalez Tineo, Ryan M. Kemper, Surya K. Tripathi, Peter H. Buttery, William Y. Kim, Lindsey I. James, Daniel J. Crona. Evaluating the potency of a first-in-class covalent antagonist of the H3K9me3 reader protein MPP8 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6283.

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

The Anti-Tumor and Immunomodulatory Effects of PLGA-Based Docetaxel Nanoparticles in Lung Cancer: The Potential Involvement of Necroptotic Cell Death through Reactive Oxygen Species and Calcium Build-Up.

Taxanes, microtubule stabilizing agents, are extensively used in the treatment of non-small cell lung cancer (NSCLC). However, their clinical effectiveness remains restricted owing to significant adverse effects and drug resistance. Nanotechnology may guide chemotherapeutic drugs directly and selectively to malignant cells, improving their therapeutic efficiency. In the present study, we synthesized polylactic-co-glycolic acid (PLGA) based nanoparticles encapsulating docetaxel and evaluated their efficacy in non-small cell lung carcinoma (A549) cells and primary immune cells derived from humans. Docetaxel-PLGA nanoparticles (PLGA-Dtx) were synthesized and characterized using distinct methods. Moreover, the cytotoxicity of free docetaxel (Dtx) and Dtx-conjugated nanoparticles (PLGA-Dtx) was studied in A549 cells and peripheral blood mononuclear cells derived from humans. Furthermore, annexin V-FITC/PI staining was used to assess the mode of cell death. Additionally, human peripheral blood mononuclear cells (PBMCs) were used for assessing the associated immune response and cytokine profile following PLGA-Dtx treatment. Spherical PLGA-Dtx nanoparticles with a 150 ± 10 nm diameter and 70% encapsulation efficiency (EE) were synthesized. The MTT assay showed that the IC50 of PLGA-Dtx nanoparticles was significantly lower than free docetaxel in A549 cells. Cytotoxicity data also revealed the selective nature of PLGA-Dtx with no significant effects in normal human bronchial epithelial cells (BEAS-2B) and PBMCs derived from healthy donors. Interestingly, PLGA-Dtx exerted an improved effect and tempted both apoptosis and necroptosis, as evidenced by annexin V and propidium iodide-positive cells. Further, PLGA-Dtx-exposed A549 cells showed increased Cas-3, Cas-9, RIP-1, and RIP-3, indicating apoptosis and necroptosis. An increased pro-inflammatory response manifested from the enhancement of IFN-γ and TNF-α in PLGA-Dtx-exposed PBMCs, posed by the occurrence of necroptosis and the immune stimulatory effect of PLGA-Dtx. In conclusion, PLGA-Dtx has a selective anticancer potential and better immunostimulatory potential. Therefore, PLGA-Dtx may be useful for the treatment of non-small cell lung carcinoma.

Trimethylamine oxide induces pyroptosis of vascular endothelial cells through ALDH2/ROS/NLRP3/GSDMD pathway.

Trimethylamine oxide (TMAO) is a metabolite of intestinal flora and is known to promote the progression of atherosclerotic plaques. However, how TMAO works, including its effect on vascular endothelial cells, is not fully understood. This study aims to explore the biological role of TMAO in human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Cell pyroptosis and the loss of plasma membrane integrity were induced under TMAO stimulation in HUVECs. The plasma membrane integrity of the cells was measured by Hoechst 33342/propidium iodide (PI) staining and lactate dehydrogenase leakage assay, and the changes in cell morphology were observed by atomic force microscope. The expression of proteins related to pyroptosis was determined by Western blotting or immunofluorescence. Mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) activity in HUVECs was measured by the ALDH2 activity assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. TMAO induced pyroptotic cell death, manifesting by the presence of propidium iodide-positive cells, the leakage of lactate dehydrogenase, the production of N-terminal gasdermin D (GSDMD-N), and the formation of plasma membrane pores. Moreover, TMAO induced elevated expression of inflammasome components, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 in cells. TMAO significantly inhibited ALDH2 activity and increased intracellular ROS production. However, the activation of ALDH2 by pharmacological manipulation attenuated TMAO-induced inflammasome activation and GSDMD-N production. TMAO induces pyroptosis of vascular endothelial cells through the ALDH2/ROS/NLRP3/GSDMD signaling pathway, which may be a potential therapeutic target for improving the treatment of atherosclerosis.

Mitotane Targets Lipid Droplets to Induce Lipolysis in Adrenocortical Carcinoma.

IntroductionAdrenocortical carcinoma (ACC) is a rare aggressive cancer with low overall survival. Adjuvant mitotane improves survival but is limited by poor response rates and resistance. Mitotane’s efficacy is attributed to the accumulation of toxic free cholesterol, predominantly through cholesterol storage inhibition. However, targeting this pathway has proven unsuccessful. We hypothesize that mitotane-induced free-cholesterol accumulation is also mediated through enhanced breakdown of lipid droplets.MethodologyATCC-H295R (mitotane-sensitive) and MUC-1 (mitotane-resistant) ACC cells were evaluated for lipid content using specific BODIPY dyes. Protein expression was evaluated by immunoblotting and flow cytometry. Cell viability was measured by quantifying propidium iodide-positive cells following mitotane treatment and pharmacological inhibitors of lipolysis.ResultsH295R and MUC-1 cells demonstrated similar neutral lipid droplet numbers at baseline. However, evaluation of lipid machinery demonstrated distinct profiles in each model. Analysis of intracellular lipid droplet content showed H295R cells preferentially store cholesteryl esters, whereas MUC-1 cells store triacylglycerol. Decreased lipid droplets were associated with increased lipolysis in H295R and in MUC-1 at toxic mitotane concentrations. Pharmacological inhibition of lipolysis attenuated mitotane-induced toxicity in both models.ConclusionWe highlight that lipid droplet breakdown and activation of lipolysis represent a putative additional mechanism for mitotane-induced cytotoxicity in ACC. Further understanding of cholesterol and lipids in ACC offers potential novel therapeutic exploitation, especially in mitotane-resistant disease.

Isocitrate dehydrogenase 1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis.

The present study intends to clarify the hypothesis that isocitrate dehydrogenase 1 (IDH1) mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis through the in vitro and in vivo experiments. Cholangiocarcinoma RBE cell line was transfected with IDH1 R132C mutation plasmids and treated with erastin to induce ferroptosis, which were then microscopically photographed. Cell viability rate was calculated by trypan blue staining. The lipid ROS level was determined by using flow cytometer. The BALB/c nude mice were injected subcutaneously with IDH1 knockout (KO), WT, or R132C mutation cell line, followed by injecting erastin intraperitoneally. The tumor tissue was surgically separated for the measurement of tumor volume and weight. The results showed that IDH1 mutant RBE cell line are sensitive to erastin-induced ferroptosis, evidenced by the increased number of propidium iodide-positive cells, the decreased cell viability, and increased lipid ROS level. However, current targeted inhibitors of IDH1 mutation (AG120 and IDH305) reversed these effects caused by IDH1 mutation. The in vivo experiment showed that IDH1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to erastin-induced ferroptosis. This study indicated that IDH1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to erastin-induced ferroptosis.

Reduction of Hexavalent Chromium [Cr(VI)] by Heavy Metal Tolerant Bacterium Alkalihalobacillus clausii CRA1 and Its Toxicity Assessment Through Flow Cytometry.

A chromate-resistant bacterial strain was isolated from tannery effluent; based on morphological, biochemical, and 16S rRNA gene sequencing, it was identified as Alkalihalobacillus clausii and designated A. clausii CRA1. It was found to be halophilic, alkaliphilic, and resistant to multiple heavy metals like Cr(VI), Cd(II), As(II), Pb(II), Ni(II), Hg(II), Cu(II), Zn(II), and Fe(II). The strain was found to reduce 72% of chromate in 6days in Cr(VI) spiked Luria Bertani medium with unaffected bacterial growth at an initial C(VI) concentration of 50mg L-1. Chromate reductase activity of culture supernatant (cultivated in LB broth) and cell lysate of the bacterium was found to be 23 and 43U, where 1U is µmol of Cr(VI) reduced/min/mg protein. Flow cytometry studies revealed that no significant effect of Cr(VI) on cell viability was observed till 12h of exposure at 100, 200, 400mg L-1 concentrations, indicated by non-significant cell death (propidium iodide positive cells). However, at 800 and 1000mg L-1 Cr(VI) concentration, toxicity (cell death) was observed after 12h of exposure. FACs studies also indicated that exposure to Cr(VI) increases cell size and cell granularity, which was also confirmed in SEM and TEM images of Cr(VI) treated cells. The presence of Cr(III) species in EDX spectra of Cr(VI) treated cells confirms that reduction of Cr(VI) to Cr(III) is the primary mechanism of Cr(VI) removal by the bacterium. Therefore, the bacterium A. clausii has potential for application in chromate removal from industrial waste effluents.

Extracellular Concentration of L-Cystine Determines the Sensitivity to System xc - Inhibitors.

Targeting the cystine/glutamate exchange transporter, system xc-, is a promising anticancer strategy that induces ferroptosis, which is a distinct form of cell death mediated by iron-dependent lipid peroxidation. The concentration of L-cystine in culture medium is higher than the physiological level. This study was aimed to evaluate the effects of L-cystine concentration on the efficacy of ferroptosis inducers in hepatocellular carcinoma cells. This study showed that treatment with sulfasalazine or erastin, a system xc- inhibitor, decreased the viability of Huh6 and Huh7 cells in a dose-dependent manner, and the degree of growth inhibition was greater in medium containing a physiological L-cystine concentration of 83 µM than in commercial medium with a concentration of 200 µM L-cystine. However, RSL3, a glutathione peroxidase 4 inhibitor, decreased cell viability to a similar extent in media containing both L-cystine concentrations. Sulfasalazine and erastin significantly increased the percentages of propidium iodide-positive cells in media with 83 µM L-cystine, but not in media with 200 µM L-cystine. Sulfasalazine- or erastin-induced accumulation of lipid peroxidation as monitored by C11-BODIPY probe was higher in media with 83 µM L-cystine than in media with 200 µM L-cystine. In contrast, the changes in the percentages of propidium iodide-positive cells and lipid peroxidation by RSL3 were similar in both media. These results showed that sulfasalazine and erastin, but not RSL3, were efficacious under conditions of physiological L-cystine concentration, suggesting that medium conditions would be crucial for the design of a bioassay for system xc- inhibitors.

Antimycotic Effects of 11 Essential Oil Components and Their Combinations on 13 Food Spoilage Yeasts and Molds.

Food safety is important to reduce food spoilage microorganisms and foodborne pathogens. However, food safety is challenging, as customers’ demand for natural preservatives is increasing. Essential oils (EOs) and their components (EOCs) are alternative antibacterial and antimycotic food additives. In this study, the minimal inhibitory concentrations (MIC) of 11 different EOCs against 13 food spoilage molds and yeasts were investigated via the microdilution method. Cinnamaldehyde (CA) revealed the lowest MIC for all tested strains and all EOCs (32.81–328.1 µg ml−1). However, CA is organoleptic and was therefore combined with other EOCs via the checkerboard method. Overall, 27 out of 91 combinations showed a synergistic effect, and both respective EOC concentrations could be reduced by maintaining MIC. Thereby, the combination with citral or citronellal showed promising results. The concentration-dependent effect of CA was studied in further detail on Saccharomyces cerevisiae, with CA causing delayed growth-kinetics and reduced total cell numbers. In addition, flow cytometric measurements combined with live–dead staining indicate the fungicidal effect of CA, due to decreasing total cell numbers and increasing relative amount of propidium iodide-positive cells. In this study, we demonstrated that CA is a potent candidate for the use as a natural preservative against food-relevant mold and yeasts showing fungistatic and fungicidal effects. Therefore, CA and EOC combinations with respective lower EOC concentrations reduce organoleptic reservations, which ease their application in the food industry.

Poloxamer 188 Attenuates Ischemia-Reperfusion-Induced Lung Injury by Maintaining Cell Membrane Integrity and Inhibiting Multiple Signaling Pathways

Background: Poloxamer 188 (P188) possesses anti-inflammatory properties and can help to maintain plasma membrane function. P188 has been reported to exert beneficial effects in the treatment of various disorders. However, the effects of P188 in ischemia/reperfusion (IR)-induced acute lung injury have not been examined. Methods: We investigated the ability of P188 to attenuate IR-induced acute lung injury in rats and hypoxia/reoxygenation (HR) injury in murine epithelial cells. Isolated perfused rat lungs were exposed to 40 min ischemia followed by 60 min reperfusion to induce IR injury. Results: IR led to lung edema, increased pulmonary arterial pressure, promoted lung tissue inflammation and oxidative stress, and upregulated the levels of TNF-α, IL-6 and CINC-1, and increased Lactic dehydrogenase (LDH) activity in bronchoalveolar lavage fluid. IR also downregulated the levels of inhibitor of κB (IκB-α), upregulated nuclear factor (NF)-κB (NF-κB), and promoted apoptosis in lung tissues. P188 significantly suppressed all these effects. In vitro, P188 also exerted a similar effect in murine lung epithelial cells exposed to HR. Furthermore, P188 reduced the number of propidium iodide-positive cells, maintained cell membrane integrity, and enhanced cell membrane repair following HR. Conclusion: We conclude that P188 protects against lung IR injury by suppressing multiple signaling pathways and maintaining cell membrane integrity.

Knockdowns of AMPK α Subunit Isoforms Result in Differential H9c2 Cell Responses to Doxorubicin Treatment

Doxorubicin (DOX) is an anthracycline anticancer drug which unfortunately causes severe dose-dependent dilated cardiomyopathy in some patients through poorly defined mechanisms. AMP-activated protein kinase (AMPK) is an important cellular energy regulator which plays a protective role in conditions such as ischemic heart disease and diabetic cardiomyopathy when activated. AMPK activation is believed to be similarly protective against DOX-induced cardiomyopathy. However, there has been little to no investigation into the role of the different subunits of AMPK or their individual isoforms in the cellular response to DOX. Previous studies have demonstrated subunit- and isoform-specific differences in effect with other drugs that act on or via AMPK, suggesting the possibility that certain AMPK isoforms may either mediate or attenuate the cardiotoxic effects of DOX. In the present study, we investigated the effects of knocking down isoforms of the catalytic α subunit of AMPK on DOX-induced cytotoxicity. H9c2 cardioblast cells were treated with siRNA to knock down the expression of α1, α2, or both and then exposed to DOX. Cell death was determined by propidium iodide (PI) staining that indicates necrosis and Western blot analysis of the expression levels of cleaved caspase 3 that signals apoptosis. Concurrent knockdown of both α1 and α2 isoforms dramatically reduced DOX-induced H9c2 cell death as shown by the decreased number of PI positive cells and the amount of cleaved caspase 3. These data indicate that the AMPK signaling pathway may have contributed to DOX cardiotoxicity, in stark contrast to the predominant belief that AMPK is cardioprotective. More interestingly, whereas α1 knockdown decreased DOX-induced cell death to the same degree as knocking down both α1 and α2 isoforms, α2 knockdown alone had minimal effect on DOX cytotoxicity. These results clearly demonstrate the isoform-dependent differential effects of AMPK α on H9c2 cell response to DOX treatment. Further studies are warranted to elucidate the relative importance of AMPK α, β, and γ subunits and their isoforms in either mediating or attenuating DOX cardiotoxicity.

Anti-listerial activity of microalgal fatty acid methyl esters and their possible applications as chicken marinade

Fatty acid methyl esters (FAMEs) from marine microalgae have been reported to possess antimicrobial activities against several Gram positive and Gram negative bacteria, but a majority of them needs to be explored. The objective of this study was to investigate the antibacterial activity, mechanism of FAMEs from selected marine microalgae against Listeria monocytogenes, and to elucidate its efficacy in food model. The minimum inhibitory concentration of FAMEs was calculated to be 155 μg/mL for Chromulina sp. and 162 μg/mL for Nannochloropsis sp. against L. monocytogenes. Time-killing kinetics showed that FAMEs efficiently inhibited the growth of L. monocytogenes in a time and concentration dependent manner. The mechanism of action of FAMEs was studied by analysing its effects at a MIC on the cellular metabolism, membrane permeability, and membrane integrity of L. monocytogenes. Transmission Electron Microscopy (TEM) results showed that cells exposed to FAMEs showed damaged cell membrane structure with leakage of the internal contents in the cells of L. monocytogenes. Fluorescence microscopy images showed that L. monocytogenes cells treated with FAMEs showed high dead cell population corresponding with propidium iodide positive cells. Furthermore, FAMEs significantly down regulated quorum sensing and biofilm related genes (DegU, FlaE, and FlaD). In vivo therapeutic potential of FAMEs revealed improved Caenorhabditis elegans survival and reduced intestinal colonization during L. monocytogenes infection. Growth of listeria was abolished in chicken meat during the cold storage of 9 days when the samples were pre-treated with FAMEs. These results suggest anti-L. monocytogenes activity of FAMEs and elucidated its use in food control of chicken meat at refrigerated conditions.

RTN1-C mediates cerebral ischemia/reperfusion injury via modulating autophagy.

It has been widely accepted that autophagic cell death exacerbates the progression of cerebral ischemia/reperfusion (I/R). Our previous study revealed that overexpression of reticulon protein 1-C (RTN1-C) is involved in cerebral I/R injury. However, the underlying mechanisms have not been studied intensively. This study was designed to evaluate the effect of RTN1-C on autophagy under cerebral I/R. Using an in vitro oxygen-glucose deprivation followed by reoxygenation and a transient middle cerebral artery occlusion model in rats, we found that the expression of RTN1-C protein was significantly upregulated. We also revealed that RTN1-C knockdown suppressed overactivated autophagy both in vivo and in vitro, as indicated by decreased expressions of autophagic proteins. The number of Beclin-1/propidium iodide-positive cells was significantly less in the LV-shRTN1-C group than in the LV-shNC group. In addition, rapamycin, an activator of autophagy, aggravated cerebral I/R injury. RTN1-C knockdown reduced brain infarct volume, improved neurological deficits, and attenuated cell vulnerability to cerebral I/R injury after rapamycin treatment. Taken together, our findings demonstrated that the modulation of autophagy from RTN1-C may play vital roles in cerebral I/R injury, providing a potential therapeutic treatment for ischemic brain injury.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2',7'-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Integrins α4β1 and αVβ3 are Reduced in Endothelial Progenitor Cells from Diabetic Dyslipidemic Mice and May Represent New Targets for Therapy in Aortic Valve Disease

Diabetes reduces the number and induces dysfunction in circulating endothelial progenitor cells (EPCs) by mechanisms that are still uncovered. This study aims to evaluate the number, viability, phenotype, and function of EPCs in dyslipidemic mice with early diabetes mellitus and EPC infiltration in the aortic valve in order to identify possible therapeutic targets in diabetes-associated cardiovascular disease. A streptozotocin-induced diabetic apolipoprotein E knock-out (ApoE−/−) mouse model was used to identify the early and progressive changes, at 4 or 7 days on atherogenic diet after the last streptozotocin or citrate buffer injection. Blood and aortic valves from diabetic or nondiabetic ApoE−/− animals were collected. EPCs were identified as CD34 and vascular endothelial growth factor receptor 2 positive monocytes, and the expression levels of α4β1, αVβ3, αVβ5, β1, αLβ2, α5 integrins, and C-X-C chemokine receptor type 4 chemokine receptor on EPC surface were assessed by flow cytometry. The number of CD34 positive cells in the aortic valve, previously found to be recruited progenitor cells, was measured by fluorescence microscopy. Our results show that aortic valves from mice fed 7 days with atherogenic diet presented a significantly higher number of CD34 positive cells compared with mice fed only 4 days with the same diet, and diabetes reversed this finding. We also show a reduction of circulatory EPC numbers in diabetic mice caused by cell senescence and lower mobilization. Dyslipidemia induced EPC death through apoptosis regardless of the presence of diabetes, as shown by the higher percent of propidium iodide positive cells and higher cleaved caspase-3 levels. EPCs from diabetic mice expressed α4β1 and αVβ3 integrins at a lower level, while the rest of the integrins tested were unaffected by diabetes or diet. In conclusion, reduced EPC number and expression of α4β1 and αVβ3 integrins on EPCs at 4 and 7 days after diabetes induction in atherosclerosis-prone mice have resulted in lower recruitment of EPCs in the aortic valve.

Kanglexin, a novel anthraquinone compound, protects against myocardial ischemic injury in mice by suppressing NLRP3 and pyroptosis.

Pyroptosis is a form of inflammatory cell death that could be driven by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation following myocardial infarction (MI). Emerging evidence suggests the therapeutic potential for ameliorating MI-induced myocardial damages by targeting NLRP3 and pyroptosis. In this study, we investigated the myocardial protection effect of a novel anthraquinone compound (4,5-dihydroxy-7-methyl-9,10-anthraquinone-2-ethyl succinate) named Kanglexin (KLX) in vivo and in vitro. Male C57BL/6 mice were pre-treated either with KLX (20, 40 mg· kg−1per day, intragastric gavage) or vehicle for 7 consecutive days prior to ligation of coronary artery to induce permanent MI. KLX administration dose-dependently reduced myocardial infarct size and lactate dehydrogenase release and improved cardiac function as compared to vehicle-treated mice 24 h after MI. We found that MI triggered NLRP3 inflammasome activation leading to conversion of interleukin-1β (IL-1β) and IL-18 into their active mature forms in the heart, which could expand the infarct size and drive cardiac dysfunction. We also showed that MI induced pyroptosis, as evidenced by increased DNA fragmentation, mitochondrial swelling, and cell membrane rupture, as well as increased levels of pyroptosis-related proteins, including gasdermin D, N-terminal GSDMD, and cleaved caspase-1. All these detrimental alterations were prevented by KLX. In hypoxia- or lipopolysaccharide (LPS)-treated neonatal mouse ventricular cardiomyocytes, we showed that KLX (10 μM) decreased the elevated levels of terminal deoxynucleotidyl transferase dUTP nick end labeling- and propidium iodide-positive cells, and pyroptosis-related proteins. We conclude that KLX prevents MI-induced cardiac damages and cardiac dysfunction at least partly through attenuating NLRP3 and subsequent cardiomyocyte pyroptosis, and it is worthy of more rigorous investigations for its potential for alleviating ischemic heart disease.