Delayed Cell Death Research Articles

Electroporation Parameters for Human Cardiomyocyte Ablation In Vitro.

Cardiac ablation with irreversible electroporation (IRE) is quickly being established as a modality of choice for atrial fibrillation treatment. While it has not yet been optimised, IRE has the potential to significantly limit collateral damage and improve cell-specific targeting associated with other energy sources. However, more tissue and cell-specific evidence is required to demonstrate the selective threshold parameters for human cells. The aim here is to determine the optimal ablation threshold parameters related to lesion size for human cardiomyocytes in 2D culture. Conventional biphasic pulses of different field strengths and on-times were delivered in a monolayer culture system of human AC16 cardiomyocytes. The dynamics of cell death and lesion dimensions were examined at different time points. Human cardiomyocytes are susceptible to significant electroporation and cell death at a field strength of 750 V/cm or higher with 100 μs pulses. Increasing the IRE on-time from 3 ms to 60 ms reduces the effective field threshold to 250 V/cm. Using very short pulses of 2 μs and 5 μs also causes significant cell death, but only at fields higher than 1000 V/cm. A longer on-time results in more cell death and induced greater lesion area in 2D models. In addition, different forms of cell death are predicted based on the evolution of cell death over time. This study presents important findings on the ability of different IRE parameters to induce human cardiomyocyte cell death. Lesion size can be tuned by appropriate choice of IRE parameters and cardiomyocytes display an upregulation of delayed cell death 24 h after electroporation, which is an important consideration for clinical practice.

Effect of ferroptosis inhibitors oxindole-curcumin hybrid compound and N,N-dimethylaniline derivatives on rotenone-induced oxidative stress

Oxidative stress is common to multiple cell death pathways, including apoptosis. We recently identified several compounds that protect against ferroptosis, another cell death pathway associated with oxidative stress, suggesting potential efficacy against apoptosis. The present study assessed the protective efficacies of the ferroptosis inhibitors oxindole-curcumin hybrid compound GIF-2165X-G1, N,N-dimethylaniline derivatives GIF-2014 and GIF-2115, and ferrostatin-1 against rotenone-induced apoptosis. Treatment of mouse hippocampal HT22 cells with the mitochondrial transport chain inhibitor rotenone for 24 h reduced mitochondrial membrane potential, increased reactive oxygen species production, promoted nuclear fragmentation, and ultimately impaired cell viability, consistent with apoptosis. Ferroptosis inhibitor cotreatment did not prevent any of these rotenone-induced apoptotic processes but did suppress delayed cell death associated with loss of plasma membrane integrity. These results suggest that GIF-2165X-G1, GIF-2014, GIF-2115, and ferrostatin-1 are selective for ferroptosis and do not affect apoptosis. Thus, erastin-induced ferroptosis and rotenone-induced apoptosis are distinct cell death pathways despite the common involvement of mitochondrial oxidative stress. Further, the cytoprotective efficacies of chemical antioxidants may depend on the specific source of oxidative stress.

Experimental paradigms revisited: oxidative stress-induced tRNA fragmentation does not correlate with stress granule formation but is associated with delayed cell death.

tRNA fragmentation is an evolutionarily conserved molecular phenomenon. tRNA-derived small RNAs (tsRNAs) have been associated with many cellular processes, including improved survival during stress conditions. Here, we have revisited accepted experimental paradigms for modeling oxidative stress resulting in tRNA fragmentation. Various cell culture models were exposed to oxidative stressors followed by determining cell viability, the production of specific tsRNAs and stress granule formation. These experiments revealed that exposure to stress parameters commonly used to induce tRNA fragmentation negatively affected cell viability after stress removal. Quantification of specific tsRNA species in cells responding to experimental stress and in cells that were transfected with synthetic tsRNAs indicated that neither physiological nor non-physiological copy numbers of tsRNAs induced the formation of stress granules. Furthermore, the increased presence of tsRNA species in culture medium collected from stressed cells indicated that cells suffering from experimental stress exposure gave rise to stable extracellular tsRNAs. These findings suggest a need to modify current experimental stress paradigms in order to allow separating the function of tRNA fragmentation during the acute stress response from tRNA fragmentation as a consequence of ongoing cell death, which will have major implications for the current perception of the biological function of stress-induced tsRNAs.

CRISPR/Cas9-Based Deletion of SpvB Gene From Salmonella gallinarum Leads to Loss of Virulence in Chicken.

Salmonella Gallinarum causes fowl typhoid in poultry leading to a huge economic loss to the poultry industry. The large virulence plasmid of S. gallinarum has been associated with various systemic infections in poultry. A five-gene spanning region (spvRABCD) of 7.8 kb on the large plasmid mainly confers virulence to the bacteria. However, the exact role of these genes in virulence has not been elucidated yet. SpvB exhibits delayed cell death by preventing actin polymerization followed by apoptosis during intracellular infection. The specific role of SpvB in causing the disease is not known yet. In the current study, the SpvB gene was deleted through CRISPR/Cas9 method from a large virulent plasmid of locally isolated S. gallinarum strain (SG18). The homology-directed repair method was used for complete deletion of SpvB gene using the modified pCas9 plasmid. The SpvB-deleted S. gallinarum strain (ΔSpvB_SG18), when tested for its virulence in broiler chicken showed no diseases signs and mortality. In addition, the avirulent strain does not affect the bird’s weight and was rapidly cleared from the liver after infection. However, it cleared from the intestine only after 4–5 days, which suggests that the ΔSpvB_SG18 strain is unable to invade from the intestine to the liver. This is the first study to report a complete gene deletion from the S. gallinarum virulent plasmid and its effect. This method will be useful for the deletion of virulent genes from S. gallinarum, to study their role in pathogenesis, and to prepare an effective vaccine strain for controlling fowl typhoid in poultry.

Genetic and pharmacologic proteasome augmentation ameliorates Alzheimer's-like pathology in mouse and fly APP overexpression models.

The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer’s disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome’s increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.

Self-assembling Molecular Medicine for the Subacute Phase of Ischemic Stroke

Ischemic stroke leads to acute neuron death and forms an injured core, triggering delayed cell death at the penumbra. The impaired brain functions after ischemic stroke are hardly recovered because of the limited regenerative properties. However, recent rodent intervention studies manipulating the extracellular environments at the subacute phase shed new light on the regenerative potency of the injured brain. This review introduces the rational design of artificial extracellular matrix (ECM) mimics using supramolecular peptidic scaffolds, which self-assemble via non-covalent bonds and form hydrogels. The facile customizability of the peptide structures allows tuning the hydrogels' physical and biochemical properties, such as charge states, hydrophobicity, cell adhesiveness, stiffness, and stimuli responses. Supramolecular peptidic materials can create safer and more economical drugs than polymer materials and cell transplantation. We also discuss the importance of activating developmental programs for the recovery at the subacute phase of ischemic stroke. Self-assembling molecular medicine mimicking the ECMs and activating developmental programs may stand as a new drug modality of regenerative medicine in various tissues.

Involvement of phosphatidylserine receptors in the skeletal muscle regeneration: therapeutic implications.

Sarcopenia is a progressive loss of muscle mass and strength with a risk of adverse outcomes such as disability, poor quality of life, and death. Increasing evidence indicates that diminished ability of the muscle to activate satellite cell‐dependent regeneration is one of the factors that might contribute to its development. Skeletal muscle regeneration following myogenic cell death results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibres. Satellite cell differentiation is not a satellite cell‐autonomous process but depends on signals provided by the surrounding cells. Infiltrating macrophages play a key role in the process partly by clearing the necrotic cell debris, partly by producing cytokines and growth factors that guide myogenesis. At the beginning of the muscle regeneration process, macrophages are pro‐inflammatory, and the cytokines produced by them trigger the proliferation and differentiation of satellite cells. Following the uptake of dead cells, however, a transcriptionally regulated phenotypic change (macrophage polarization) is induced in them resulting in their transformation into healing macrophages that guide resolution of inflammation, completion of myoblast differentiation, myoblast fusion and growth, and return to homeostasis. Impaired efferocytosis results in delayed cell death clearance, delayed macrophage polarization, prolonged inflammation, and impaired muscle regeneration. Thus, proper efferocytosis by macrophages is a determining factor during muscle repair. Here we review that both efferocytosis and myogenesis are dependent on the cell surface phosphatidylserine (PS), and surprisingly, these two processes share a number of common PS receptors and signalling pathways. Based on these findings, we propose that stimulating the function of PS receptors for facilitating muscle repair following injury could be a successful approach, as it would enhance efferocytosis and myogenesis simultaneously. Because increasing evidence indicates a pathophysiological role of impaired efferocytosis in the development of chronic inflammatory conditions, as well as in impaired muscle regeneration both contributing to the development of sarcopenia, improving efferocytosis should be considered also in its management. Again applying or combining those treatments that target PS receptors would be expected to be the most effective, because they would also promote myogenesis. A potential PS receptor‐triggering candidate molecule is milk fat globule‐EGF‐factor 8 (MFG‐E8), which not only stimulates PS‐dependent efferocytosis and myoblast fusion but also promotes extracellular signal‐regulated kinase (ERK) and Akt activation‐mediated cell proliferation and cell cycle progression in myoblasts.

A Unique Cooperativity between Optineurin and CCL5 Triggers Early Cell Death during Herpes Simplex Virus-1 Infection of the Cornea

Abstract Induction of cell death is considered an effective strategy to control viral infections. Rapid killing of infected cells prevents propagation of viruses and thereby, effectively reduces the infection. Herpes Simplex Virus-1 (HSV-1) is known to cause ocular and nervous system infections. While studying an autophagy receptor protein, Optineurin (OPTN), we found evidence of a unique collaboration between OPTN and CCL5 to generate signal for early cell death in HSV-1 infected cells in the cornea. OPTN knockout mice tend to die due to rapid spread of HSV-1 infection to the brain causing encephalitis. Interestingly, the OPTN knockout animals also exhibit a marked reduction in CCL5 induction. Human transcriptomic profile dataset shows a strong positive correlation between CCL5 upregulation and OPTN expression during HSV infection. Our data shows that a well-coordinated activity of both, OPTN and CCL5, is needed to trigger an early cell death signal that mimics pyroptosis to prevent viral spread. Downregulation of CCL5 and/or OPTN causes interference with this signal and a corresponding delay in cell death. Our results provide new information on an anti-HSV1 chemokine signaling pathway that participates in innate immune control of HSV-1 spread and viral transmission.

Diverse injury responses of human oligodendrocyte to mediators implicated in multiple sclerosis.

Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche.

Legionella pneumophila, the causative agent of Legionnaires' disease, a severe pneumonia, injects via a type 4 secretion system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here. we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization, and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

Establishing Irreversible Electroporation Electric Field Potential Threshold in A Suspension In Vitro Model for Cardiac and Neuronal Cells.

Aims: Irreversible electroporation is an ablation technique being adapted for the treatment of atrial fibrillation. Currently, there are many differences reported in the in vitro and pre-clinical literature for the effective voltage threshold for ablation. The aim of this study is a direct comparison of different cell types within the cardiovascular system and identification of optimal voltage thresholds for selective cell ablation. Methods: Monophasic voltage pulses were delivered in a cuvette suspension model. Cell viability and live–dead measurements of three different neuronal lines, cardiomyocytes, and cardiac fibroblasts were assessed under different voltage conditions. The immediate effects of voltage and the evolution of cell death was measured at three different time points post ablation. Results: All neuronal and atrial cardiomyocyte lines showed cell viability of less than 20% at an electric field of 1000 V/cm when at least 30 pulses were applied with no significant difference amongst them. In contrast, cardiac fibroblasts showed an optimal threshold at 1250 V/cm with a minimum of 50 pulses. Cell death overtime showed an immediate or delayed cell death with a proportion of cell membranes re-sealing after three hours but no significant difference was observed between treatments after 24 h. Conclusions: The present data suggest that understanding the optimal threshold of irreversible electroporation is vital for achieving a safe ablation modality without any side-effect in nearby cells. Moreover, the evolution of cell death post electroporation is key to obtaining a full understanding of the effects of IRE and selection of an optimal ablation threshold.

Pharmacological Reduction of Mitochondrial Iron in AML Triggers a BAX/BAK Dependent Non-Canonical Cell Death Synergistic with Venetoclax

Although the BCL2 inhibitor venetoclax have been transformative in the management of AML, therapeutic resistance and relapse are frequently observed. In light of the urgent need to uncover novel therapeutic options in AML, we sought to study the potential role of ironomycin (AM5), a recently described small molecule that induces cell death through the sequestration of lysosomal iron.To evaluate the effects of ironomycin in AML, we chose a diverse panel of AML cell lines. These data showed a potent and dose-dependent effect, on proliferation, cell cycle progression and survival at a nanomolar range. In contrast to venetoclax, the cell death induced by ironomycin did not result in potent caspase activation or PARP1 cleavage. Neither the caspase inhibitor Z-VAD-fmk nor the necroptosis inhibitor necrostatin-1 did prevent cell death. Consistent with previous observations, we found that ironomycin accumulates in the lysosomes of AML cells leading to a sequestration of iron in this organelle but inhibitors of canonical ferroptosis, including ferrostatin-1 and liproxstatin-1 failed to prevent the activity of ironomycin.To gain greater insight into the molecular mechanism of ironomycin in AML cells, we performed a genome-wide positive-selection resistance screen under ironomycin selection pressure and collected several samples for sequencing. We found nine genes whose knock out conferred resistance to the drug. Interestingly, these data implicated key components of mitochondrial metabolic pathways, including phosphoglycolate phosphatase (PGP), a central phosphatase involved in glycolysis and pentose phosphate pathway (PPP) regulation and Hexokinase 2 (HK2), the first enzyme of glycolysis.Mass-spectrometry metabolomics analyses highlighted that ironomycin treatment significantly reduced key components of the TCA cycle and consequently the reducing agent nicotinamide adenine dinucleotide (NADH) and increased the intracellular concentration of amino acids. These data were corroborated with RNAseq showing a mitochondrial stress response mediated through the Activating Transcription Factor 4 (ATF4) and its paralog Activating Transcription Factor 5 (ATF5). As mitochondria are major hubs of iron utilization for oxidative respiration, we used Mass-spectrometry to measure mitochondrial iron load. We observed a rapid and dose-dependent decrease in mitochondrial iron after treatment mirroring the iron sequestration into the lysosomes and inducing the mitochondrial dysfunction.We next examined the ultrastructural appearance of mitochondria after ironomycin using transmission electron microscopy and observed a dramatic alteration of the structural integrity of mitochondria resulting in abnormal cristae, matrix density changes and mitochondrial membrane blebbing. In cells lacking BAX and BAK, the two main effectors of mitochondrial membrane permeabilization, structural changes and cell death were almost completely rescued but cell proliferation was still markedly affected, consistent with a BAX/BAK dependent cell death following mitochondrial iron deprivation. In vivo imaging confirmed that BAX activation occurred after 30 hours of treatment and preceded cell death, but we observed some major differences with canonical apoptosis induced by venetoclax. First, the structural alterations were clearly distinct. Next, delay between MOMP and cell death was significantly longer and caspase inhibitors weakly delayed cell death. Finally, BCL2 overexpression and P53 deletion did not rescue ironomycin cell death.These non-canonical features prompted us to assess the efficacy of the combination between ironomycin and venetoclax. In vitro experiment on AML cell lines found a high synergy between the two drugs. In vivo experiments on xenotransplanted mice confirmed the efficacy of the combination, which was associated with a significant increase in mice survival in comparison with the controls (Figure). Finally, primary AML samples from patients clinically resistant or refractory to venetoclax were sensitive to ironomycin in monotherapy and even more in combination with venetoclax.These results demonstrate that the novel mechanism of ironomycin action can be leveraged to resensitize AML cells to venetoclax and substitute for cytotoxic drugs as a more effective therapeutic combination in the salvage setting. [Display omitted] DisclosuresHuang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Wei: Novartis, Celgene, AbbVie, Servier, AstraZeneca, and Amgen: Research Funding; Novartis, Janssen, Amgen, Roche, Pfizer, Abbvie, Servier, BMS, Macrogenics, Agios, Gilead: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria.

Loss of pro-apoptotic Bax and Bak increases resistance to dihydroartemisinin-mediated cytotoxicity in normoxia but not in hypoxia in HCT116 colorectal cancer cells

Tumor hypoxia is a major biological factor that drives resistance to chemotherapy and radiotherapy. We previously demonstrated that the pro-oxidative drug dihydroartemisinin (DHA) efficiently targeted normoxic and hypoxic cancer cells. Although well studied in normoxia, the mechanism behind DHA-mediated cytotoxicity in hypoxia is insufficiently explored. Here, we analyzed the effect of DHA in HCT116 wild type (wt) cells and in HCT116 Bax−/−Baksh cells with a defective intrinsic apoptosis pathway.Normoxic HCT116 wt cells underwent apoptosis shortly after treatment with DHA. Autophagy-associated cell death contributes to short-term cytotoxicity of DHA in normoxia. These cells switched to an apoptosis- and autophagy-independent cell death after treatment with DHA in hypoxia and displayed similar long-term survival in response to DHA in normoxia and hypoxia. In HCT116 Bax−/−Baksh cells, DHA induced cell cycle arrest shortly after treatment irrespective of oxygen levels. Later, HCT116 Bax−/−Baksh cells induced a delayed cell death after treatment with DHA in hypoxia followed by return to normoxia, while treatment with DHA in normoxia was hardly toxic. We identified lower glutathione levels in hypoxic HCT116 cells which correlated with higher lipid peroxidation after treatment with DHA. Moreover, insufficient expression of Bax/Bak counteracted hypoxia-mediated downregulation of mitochondrial function, thereby adding to DHA-induced ROS production and lipid peroxidation in hypoxia.In summary, DHA-mediated cytotoxicity in normoxia depended on Bax/Bak expression, while cytotoxicity after treatment with DHA in hypoxia was regulated independently of Bax/Bak in HCT116 colorectal cancer cells.

Jasmonates modulate sphingolipid metabolism and accelerate cell death in the ceramide kinase mutant acd5.

Sphingolipids are structural components of the lipid bilayer that acts as signaling molecules in many cellular processes, including cell death. Ceramides, key intermediates in sphingolipid metabolism, are phosphorylated by the ceramide kinase ACCELERATED CELL DEATH5 (ACD5). The loss of ACD5 function leads to ceramide accumulation and spontaneous cell death. Here, we report that the jasmonate (JA) pathway is activated in the Arabidopsis (Arabidopsis thaliana) acd5 mutant and that methyl JA treatment accelerates ceramide accumulation and cell death in acd5. Moreover, the double mutants of acd5 with jasmonate resistant1-1 and coronatine insensitive1-2 exhibited delayed cell death, suggesting that the JA pathway is involved in acd5-mediated cell death. Quantitative sphingolipid profiling of plants treated with methyl JA indicated that JAs influence sphingolipid metabolism by increasing the levels of ceramides and hydroxyceramides, but this pathway is dramatically attenuated by mutations affecting JA pathway proteins. Furthermore, we showed that JAs regulate the expression of genes encoding enzymes in ceramide metabolism. Together, our findings show that JAs accelerate cell death in acd5 mutants, possibly by modulating sphingolipid metabolism and increasing ceramide levels.

Maneb adducts human peroxiredoxin 3 through thiol interactions

The dithiocarbamate fungicide maneb (MB) has garnered increasing concern due to its association with Parkinson's disease (PD) and the overall negative human health effects of pesticides in agriculture. As one of several environmental toxicant models of PD, MB has been shown to induce global dehydrogenase inhibition, mitochondrial dysfunction, proteostasis disruption, and delayed cell death in neuronal in vitro models. Recently, our group has reported distinct alterations in bioenergetics and energy metabolism after an acute exposure of MB to neuroblastoma cells. Additionally, this acute MB exposure in neuroblastoma also caused a disruption in peroxiredoxin 3 (Prdx3) oxidation, uncovering significant protein thiol interactions. In the current study, human recombinant Prdx3 was evaluated to evaluate direct thiol-thiol interactions between MB and Prdx3 protein. We report a direct, concentration dependent modification of Prdx3 after MB exposure in our system. Further investigation revealed that MB directly adducts Prdx3 protein through disulfide chemistry, similar to the hyperoxidation or glutathionylation of critical cysteine residues. Computational simulations confirm the disulfide bonding on both reduced monomers and oxidized dimers of Prdx3 protein by a possible MB polymer. The data presented strengthens the argument that MB can preferentially target thiol regulatory switches present in many proteins, giving further credence to the MB model of PD in which many disease-associated proteins contain these functional groups, e.g. ubiquinol-cytochrome c reductase, aconitase, and aldehyde dehydrogenase (ALDH2). Furthermore, MB's ability to mimic hyperoxidation of Prdx3 protein introduces a novel dynamic of toxicity within the toxicant model that accounts for the oxidative distress that cannot be reversed by therapeutic anti-oxidants in PD.

Neurotoxicity of zearalenone’s metabolites and beauvericin mycotoxins via apoptosis and cell cycle disruption

Cell cycle progression and programmed cell death are imposed by pathological stimuli of extrinsic or intrinsic including the exposure to neurotoxins, oxidative stress and DNA damage. All can cause abrupt or delayed cell death, inactivate normal cell survival or cell death networks. Nevertheless, the mechanisms of the neuronal cell death are unresolved. One of the cell deaths triggers which have been wildly studied, correspond to mycotoxins produced by Fusarium species, which have been demonstrated cytotoxicity and neurotoxicity through impairing cell proliferation, gene expression and induction of oxidative stress. The aim of present study was to analyze the cell cycle progression and cell death pathway by flow cytometry in undifferentiated SH-SY5Y neuronal cells exposed to α-zearalenol (α-ZEL), β-zearalenol (β-ZEL) and beauvericin (BEA) over 24 h and 48 h individually and combined at the following concentration ranges: from 1.56 to 12.5 μM for α-ZEL and β-ZEL, from 0.39 to 2.5 μM for BEA, from 1.87 to 25 μM for binary combinations and from 3.43 to 27.5 μM for tertiary combination. Alterations in cell cycle were observed remarkably for β-ZEL at the highest concentration in all treatments where engaged (β-ZEL, β-ZEL + BEA and β-ZEL + α-ZEL), for both 24 h and 48 h. by activating the cell proliferation in G0/G1 phase (up to 43.6 %) and causing delays or arrests in S and G2/M phases (up to 19.6 %). Tertiary mixtures revealed increases of cell proliferation in subG0 phase by 4-folds versus control. Similarly, for cell death among individual treatments β-ZEL showed a significant growth in early apoptotic cells population at the highest concentration assayed as well as for all combination treatments where β-ZEL was involved, in both early apoptotic and apoptotic/necrotic cell death pathways.

Neonatal Mesenchymal Stem Cell Treatment Improves Myelination Impaired by Global Perinatal Asphyxia in Rats.

The effect of perinatal asphyxia (PA) on oligodendrocyte (OL), neuroinflammation, and cell viability was evaluated in telencephalon of rats at postnatal day (P)1, 7, and 14, a period characterized by a spur of neuronal networking, evaluating the effect of mesenchymal stem cell (MSCs)-treatment. The issue was investigated with a rat model of global PA, mimicking a clinical risk occurring under labor. PA was induced by immersing fetus-containing uterine horns into a water bath for 21 min (AS), using sibling-caesarean-delivered fetuses (CS) as controls. Two hours after delivery, AS and CS neonates were injected with either 5 μL of vehicle (10% plasma) or 5 × 104 MSCs into the lateral ventricle. Samples were assayed for myelin-basic protein (MBP) levels; Olig-1/Olig-2 transcriptional factors; Gglial phenotype; neuroinflammation, and delayed cell death. The main effects were observed at P7, including: (i) A decrease of MBP-immunoreactivity in external capsule, corpus callosum, cingulum, but not in fimbriae of hippocampus; (ii) an increase of Olig-1-mRNA levels; (iii) an increase of IL-6-mRNA, but not in protein levels; (iv) an increase in cell death, including OLs; and (v) MSCs treatment prevented the effect of PA on myelination, OLs number, and cell death. The present findings show that PA induces regional- and developmental-dependent changes on myelination and OLs maturation. Neonatal MSCs treatment improves survival of mature OLs and myelination in telencephalic white matter.

UV-induced DNA damage disrupts the coordination between replication initiation, elongation and completion.

Replication initiation, elongation and completion are tightly coordinated to ensure that all sequences replicate precisely once each generation. UV-induced DNA damage disrupts replication and delays elongation, which may compromise this coordination leading to genome instability and cell death. Here, we profiled the Escherichia coli genome as it recovers from UV irradiation to determine how these replicational processes respond. We show that oriC initiations continue to occur, leading to copy number enrichments in this region. At late times, the combination of new oriC initiations and delayed elongating forks converging in the terminus appear to stress or impair the completion reaction, leading to a transient over-replication in this region of the chromosome. In mutants impaired for restoring elongation, including recA, recF and uvrA, the genome degrades or remains static, suggesting that cell death occurs early after replication is disrupted, leaving partially duplicated genomes. In mutants impaired for completing replication, including recBC, sbcCD xonA and recG, the recovery of elongation and initiation leads to a bottleneck, where the nonterminus region of the genome is amplified and accumulates, indicating that a delayed cell death occurs in these mutants, likely resulting from mis-segregation of unbalanced or unresolved chromosomes when cells divide.

A resin cyanoacrylate nanoparticle as an acute cell death inducer to broad spectrum of microalgae

We examined the potency of resin nanoparticles (NPs) to induce cell mortality in diverse non-green algal species (e.g., species in SAR and Hacrobia clades) and the difference in sensitivity to NPs among the species. In total, 18 non-green algal species were co-incubated with laboratory-prepared NPs (mean diameter, 180 nm) composed of poly (isobutylcyanoacrylate) resin NPs (iBCA-NPs).Exposure to 100 mg L−1 iBCA-NPs for 24 h induced cell death in two of three Bacillariophyceae species, all three Cryptophyceae species, four of six Dinophyta species, three of four Haptophyta strains, and all three Raphidophyceae species. However, exposure at an increased concentration of 1 g L−1 induced cell death in all the examined species. Observation of NP trails showed that they occasionally collided with the cells and bounced off but did not adhere to the cell surface. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine substantially delayed cell death induction. This finding strongly suggests that ROS generation is a direct cause of induced cell death.The cells of Raphidophyceae species that lack covering structures and a Haptophyta strain bearing no coccoliths were more sensitive than species bearing covering structures. Moreover, iBCA-NPs pre-coated with bovine serum albumin or skim milk had severely reduced cell death-inducing potency. We propose a hypothetical mechanism of cell death induction by iBCA-NPs in a wide range of algal species, focusing on cell covering structures and ROS generation.