Loss Of Plasma Membrane Integrity Research Articles

4-Quinolinylhydrazone analogues kill Leishmania (Leishmania) amazonensis by inducing apoptosis and mitochondria-dependent pathway cell death.

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 μM to promastigotes, and 14.31-61.98 μM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The invivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. Invivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.

Screening of amino acids as a safe energy source for isolated rat pancreatic acini.

Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acini. Adequate nutritional support is important for acute pancreatitis treatment. However, high concentrations of arginine and lysine may induce acute pancreatitis. The study aimed to identify the most suitable L-amino acids as safe energy sources for pancreatic acinar cells. Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1-20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell death was evaluated with fluorescent microscopy and DNA gel electrophoresis. Among the tested amino acids, glutamate, glutamine, alanine, lysine and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, while arginine, histidine and asparagine were not. Lysine, arginine and glutamine (20 mM) caused complete loss of plasma membrane integrity of acinar cells after 24 h of incubation. Glutamine also caused early (2-4 h) cell swelling and blebbing. Aspartate, asparagine and glutamate only moderately decreased the number of viable cells, while alanine and histidine were not toxic. DNA fragmentation assay and microscopic analysis of nuclei showed no evidence of apoptosis in cells treated with amino acids. Alanine and glutamate are safe and effective energy sources for mitochondria of pancreatic acinar cells.

Effect of levan polysaccharide on chronological aging in the yeast Saccharomyces cerevisiae

Levan is a fructose-based biopolymer with diverse applications in the medicinal, pharmaceutical, and food industries. However, despite its extensive biological and pharmacological actions, including antioxidant, anti-inflammatory, and antidiabetic properties, research on its anti-aging potential is limited. This study explored levan's impact on the chronological lifespan (CLS) of yeast Saccharomyces cerevisiae for the first time. The results show that levan treatment significantly extended the CLS of wild-type (WT) yeast by preventing the accumulation of oxidative stress markers (reactive oxygen species, malondialdehyde, and protein carbonyl content) and ameliorating apoptotic features such as reduced mitochondrial membrane potential, loss of plasma membrane integrity, and externalization of phosphatidylserine. By day 40 of the CLS, a significant increase in yeast viability of 6.8 % (p < 0.01), 11.9 % (p < 0.01), and 20.8 % (p < 0.01) was observed at 0.25, 0.5, and 1 mg/mL of levan concentrations, respectively, compared to control (0 %). This study's results indicate that levan treatment substantially modulates the expression of genes involved in the TORC1/Sch9 pathway. Moreover, levan treatment significantly extended the CLS of yeast antioxidant-deficient mutant sod2Δ and antiapoptotic gene-deficient mutant pep4Δ. Levan also extended the CLS of signaling pathway gene-deficient mutants such as pkh2Δ, rim15Δ, atg1, and ras2Δ, while not affecting the CLS of tor1Δ and sch9Δ.

Improved photocatalytic disinfection performance of graphitic carbon nitride through hybridization with humic acid /zinc peroxide: A synergistic generation of antimicrobial reactive oxygen species

Photocatalytic disinfection of bacteria using photosensitive nanomaterials is an efficient method for bacteria inactivation and is considered a beneficial alternative to chemical disinfectants that produce harmful by-products. However, due to the limited application scope of photocatalysts, the antibacterial efficiency remains unsatisfactory. To enhance the photocatalytic efficiency of the non-metal photosensitizer, graphitic carbon nitride (g-C3N4) nanosheets, they were composited with a metal-based photosensitizer, zinc peroxide (ZnO2) nanoparticles, with the aim of humic acid (HA). Besides the self-generating ability of H2O2 by ZnO2 in an aqueous medium, its deposition over g-C3N4 nanosheets enhances visible light harvesting and increases both surface area and active sites, thereby enhancing photocatalytic efficiency. The resulting composite, g-C3N4/ZnO2, produced more H2O2, •OH, and O2•- radicals than either component when exposed to visible light. Moreover, the photocatalyst demonstrated enhanced photocatalytic disinfection efficacy against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) under dark conditions compared to pure g-C3N4 nanosheets. However, a notably greater antibacterial activity was observed when the nanocomposites were subjected to light irradiation. FESEM images and live-dead assays revealed the physical contact of the nanocomposite with the surface of bacterial cells and the loss of cell membrane integrity. Furthermore, it was observed that there was greater cellular uptake of nanocomposites into cells and the generation of reactive oxygen species (ROS) under light irradiation compared to similar conditions in the dark, leading to an increased photoinactivation rate of bacteria. The enhanced photocatalytic and antibacterial activities of g-C3N4/ZnO2 nanocomposites make them attractive for a range of applications, including use in nanomedicine and environmental surface disinfection.

NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis.

Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.

Verteporfin induces lipid peroxidation and ferroptosis in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) has extremely poor prognosis, with a 5-year survival rate of approximately 11 %. Yes-associated protein (YAP) is a major downstream effector of the Hippo-YAP pathway and plays a pivotal role in regulation of cell proliferation and organ regeneration and tumorigenesis. Activation of YAP signaling has been associated with PDAC progression and drug resistance. Verteporfin (VP) is a photosensitizer used for photodynamic therapy and previous work showed that it can function as a YAP inhibitor. The efficacy of VP on human cancer are being tested in several trials. In this study, we examined the effect of VP on reactive oxygen species (ROS) and lipid peroxidation in pancreatic cancer cells, by using fluorescent molecular probes and by measuring the levels of malondialdehyde, a metabolic byproduct and marker of lipid peroxidation. We found that VP causes rapid increase of both overall ROS and lipid peroxide levels, independent of light activation. These effects were not dependent on YAP, as knockdown of YAP did not cause ROS or lipid peroxidation or enhance VP-induced ROS production. Temoporfin, another photodynamic drug, did not show similar activities. In addition, VP treatment led to loss of cell membrane integrity and reduction of viability. Notably, the activity of VP to induce lipid peroxidation was neutralized by ferroptosis inhibitors ferrostatin-1 or liproxstatin-1. VP treatment also reduced the levels of glutathione peroxidase 4 (GPX4), an enzyme that protects against lipid peroxidation. These results indicate that VP can induce lipid peroxidation and ferroptosis in the absence of light activation. Our findings reveal a novel mechanism by which VP inhibits tumor growth and provide insights into development of new therapeutic strategies for the treatment of pancreatic cancer.

Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line.

Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Using Cell Motility and Particle Tracking to Deduce Mechanisms and Kinetics Underlying Photocatalytic Water Disinfection in Real Time

Photocatalysis is a promising water disinfection method that has the potential to complement traditional approaches to water remediation. Large-scale implementation of photocatalysis for water remediation requires a two-fold improvement to the current state-of-the-art: understanding of the mechanisms underlying photocatalyst-pathogen interaction and pathogen inactivation during photocatalysis, and a process for rapid catalyst discovery that provides for evaluating photocatalyst efficacies in a reliable manner. Toward this goal, we will discuss the use of novel tools developed in our laboratory to track the viability loss of bacterial cells inactivated by photocatalytic treatment in real time. Using optical microscopy and particle-tracking algorithms, we observed that, when exposed to titanium dioxide (TiO2) nanowire-assisted photocatalytic stressors, the change in the motility of Escherichia coli (E. coli) cells tracks viability loss precisely. Furthermore, using phase and fluorescence optical microscopy, real-time observations of the interactions between the cells and the photocatalyst nanowires were also performed. Our findings suggest that these interactions occur through collisions between the nanowires and bacteria. Based on these observations, we developed a phenomenological model explaining the pseudo-first-order kinetics of E. coli inactivation. Through the use of fluorescence-based microscopy, we observed that the motility loss (and hence viability loss) was due to the dissipation of the proton motive force that powers motility, caused by cell membrane integrity loss. Our experiments show a good match between the kinetics of motility loss and viability loss for various operating conditions, showing that the methods presented here are versatile in applicability. Overall, our results indicate that these in situ methods offer significant time-savings for characterizing viability loss of pathogens over the traditional ex-situ methods. As indicated above, these methods will also help accelerate the evaluation of novel antibacterial agents, including, but not limited to novel photocatalysts, against emerging treatment-resistant pathogens, for applications ranging from water disinfection to equipment decontamination in healthcare facilities.

Rapamycin ameliorates doxorubicin-induced cardiomyopathy through suppression of RIPK1-independent necroptosis

Abstract Background Although several strategies to mitigate doxorubicin-induced cardiomyopathy (DIC), such as early induction of neurohormonal regulators, have been attempted, there is no currently available therapy focusing on molecular mechanisms of DIC. Necroptosis is a novel type of regulated necrosis and has been implicated in the mechanisms of various diseases including heart failure. Activation of MLKL by its phosphorylation, leading to loss of plasma membrane integrity, is a hallmark of necroptosis, and RIPK1 activation is an upstream event in a canonical necroptotic pathway, whereas RIPK1-independent MLKL activation has been reported. There are no available necroptosis inhibitors in the clinical setting, but our experimental studies have shown that mTOR inhibitors suppress necroptosis in cardiomyocytes. Purpose This study aimed to determine the involvement of necroptosis and the effect of rapamycin in DIC. Methods and results C57BL/6N mice received intraperitoneal injection of doxorubicin (10 mg/kg, three times a week) and its vehicle to induce DIC. Rapamycin and necrostatin-1, a RIPK1 inhibitor, were intraperitoneally administered at 0.25 mg/kg and 6 mg/kg one hour before doxorubicin administration, respectively. Eight days after the commencement of doxorubicin injection, cardiac MRI analyses showed that left ventricular ejection fraction (LVEF) was significantly lower in the doxorubicin-treated mice than in the vehicle-treated mice (45.5±5.1% vs. 59.6±2.2%, p&amp;lt;0.05), indicating the development of DIC. The treatment of rapamycin significantly improved LVEF in DIC mice (65.4±4.2%, P&amp;lt;0.05). Kaplan-Meier survival curve analyses during a follow-up period of 12 days showed that the survival rate was significantly higher in the rapamycin-treated DIC mice than in vehicle-treated DIC mice (40% vs. 0%, p&amp;lt;0.001 by the log-rank test), but it was comparable in the necrostatin-1-treated DIC mice and the vehicle-treated DIC mice (both 0%). Quantitative immunohistochemical analyses showed that the level of phospho-Ser345-MLKL, an activated form of MLKL, was significantly higher by 1.5-fold in the doxorubicin-treated mice than in the vehicle-treated mice, which was reversed by the treatment with rapamycin, suggesting that rapamycin improves DIC through suppression of necroptosis. In the second series of experiments, the effect of doxorubicin on necroptosis was analyzed. H9c2 cardiomyoblasts received doxorubicin (5 µmol/L) or its vehicle for 24 h, and cell death was determined by LDH release (as % of total). LDH release was higher in the doxorubicin-treated cells than in the vehicle (45.5±6.7% vs. 8.4±2.2%), and doxorubicin-induced increase in LDH release was significantly attenuated by siRNA-mediated knockdown of MLKL (36.5±3.9%) but not by pretreatment with necrostatin-1 (50 nmol/L, 49.9±3.2%). Conclusion RIPK1-independent necroptosis is involved in the pathogenesis of DIC, and rapamycin prevents the development of DIC through the reduction in MLKL activity.

ATP functions as a primary alarmin in allergen-induced type 2 immunity.

Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from damage-associated molecular patterns (DAMPs), which typically escape from cells after loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review, we highlight examples of alarmin release and discuss their roles in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification, where "primary" alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of "secondary" alarmins that include proinflammatory cytokines, such as IL-33, as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity, and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

In Situ Time‐Controllable Chemical Plasma Membrane Injury by Microfluidic Probe Reveals Self‐Repair Ability of Single Cells

AbstractThe loss of plasma membrane integrity poses a serious threat to cells, and the plasma membrane repair mechanism is vital to cell survival. To investigate the plasma membrane repair mechanism, a method for causing controllable plasma membrane injury on single cells and the visualization of the plasma membrane repair process are proposed. A microregion with harmful chemicals is created by open microfluidic probe, which is applied to target single cells for injury. The injury time is precisely controlled by simply moving the target cells in and out of the microregion. Wound‐repair process caused by injury is analyzed by fluorescence imaging in real time, revealing the repair kinetics at the single cell level. Using this method, the effect of external conditions (Ca2+, Mg2+, glucose) on plasma membrane repair in single cells is studied and the dynamics of intracellular species (Ca2+) is monitored. Meanwhile, cellular heterogeneity of plasma membrane repair is revealed. This method provides a precise way to induce chemical plasma membrane injury at the single‐cell level, which is a promising tool to study the mechanism of plasma membrane repair.

Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.

Analysis of the main bioactive compounds from Ocimum basilicum for their antimicrobial and antioxidant activity.

The interest in bioactives especially from botanicals to treat vancomycin-resistant enterococcal (VRE) infections is increased. Many species of Ocimum have a long history in folk medicinal and food industries. Nevertheless, their bioactive compounds remain unexplored. This study is aimed to assess the antimicrobial and antioxidant activity of basil leaf extract prepared using ethanol, methanol, and water. The ethanol and methanol extract have all the phytochemicals (alkaloids, flavonoids, phenolic compounds, tannins, saponins, quinones, carbohydrates, and proteins) except steroids and terpenoids. In addition to steroids and terpenoids, tannin was also absent in the aqueous extract. Total phenolic and flavonoid content was high in ethanol and followed by methanol and aqueous extract. Similarly, ethanol and methanol extract showed strong antimicrobial activity against VRE and MTCC strains at a concentration of 20mg/mL than aqueous extract. Among the 10 indicators, Staphylococcus aureus is highly susceptible to ethanol extract at a concentration of 8mg/mL and followed by other MTCC strains. Vancomycin-resistant enterococci pathogens were inhibited at the minimum inhibitory concentration of 14, 16, and 20mg/mL of ethanol, methanol, and aqueous extract. Further, on the basis of determining the absorbing material (nucleic acid and protein) at 260nm and scanning electron microscopic, it was confirmed that the loss of cell membrane integrity and cell membrane damage were the effective mechanisms of plant extract antimicrobial activity. All three solvents have shown remarkable antioxidant activity. Gas chromatography-mass spectrometry analysis of basil leaves ethanol extract identified 19 compounds with various therapeutic and food applications.

Real-Time Deduction of Mechanisms and Kinetics Underlying Photocatalytic Water Disinfection: Cell Motility and Particle Tracking.

The current methods used to study photocatalysis-assisted water disinfection at a laboratory scale may not lead to process scale-up for large-scale implementation. These methods do not capture the process complexity and address all the factors underlying disinfection kinetics, including the physical characteristics (e.g., shape and size) of the photocatalyst, the light intensity, the form of the catalyst (e.g., free-floating and immobilized), and the photocatalyst-microorganism interaction mode (e.g., collision mode and constant contact mode). This drawback can be overcome using in situ methods to track the interaction between the photocatalysts and the microorganisms (e.g., Escherichia coli) and thereby engineering the resulting disinfection kinetics. Contextually, this study employed microscopy and particle-tracking algorithms to quantify in situ cell motility of E. coli undergoing titanium dioxide (TiO2) nanowire-assisted photocatalysis, which was observed to correlate with cell viability closely. This experimentation also informed that the E. coli bacterium interacted with the photocatalysts through collisions (without sustained contact), which allowed for phenomenological modeling of the observed first-order kinetics of E. coli inactivation. Addition of fluorescent-tagging assays to microscopy revealed that cell membrane integrity loss is the primary mode of bacterial inactivation. This methodology is independent of the microorganism or the photocatalyst type and hence is expected to be beneficial for engineering disinfection kinetics.

Imaging of cell death in malignancy: Targeting pathways or phenotypes?

Cell death is fundamental in health and disease and resisting cell death is a hallmark of cancer. Treatment of malignancy aims to cause cancer cell death, however current clinical imaging of treatment response does not specifically image cancer cell death but assesses this indirectly either by changes in tumor size (using x-ray computed tomography) or metabolic activity (using 2-[18F]fluoro-2-deoxy-glucose positron emission tomography). The ability to directly image tumor cell death soon after commencement of therapy would enable personalised response adapted approaches to cancer treatment that is presently not possible with current imaging, which is in many circumstances neither sufficiently accurate nor timely. Several cell death pathways have now been identified and characterised that present multiple potential targets for imaging cell death including externalisation of phosphatidylserine and phosphatidylethanolamine, caspase activation and La autoantigen redistribution. However, targeting one specific cell death pathway carries the risk of not detecting cell death by other pathways and it is now understood that cancer treatment induces cell death by different and sometimes multiple pathways. An alternative approach is targeting the cell death phenotype that is “agnostic” of the death pathway. Cell death phenotypes that have been targeted for cell death imaging include loss of plasma membrane integrity and dissipation of the mitochondrial membrane potential. Targeting the cell death phenotype may have the advantage of being a more sensitive and generalisable approach to cancer cell death imaging. This review describes and summarises the approaches and radiopharmaceuticals investigated for imaging cell death by targeting cell death pathways or cell death phenotype.

Silicon mediated redox homeostasis in the root-apex transition zone of rice plays a key role in aluminum tolerance

The supply of silicon (Si) mitigates the aluminum (Al) toxicity on plant root growth, while the underlying mechanism remains unknown. Transition zone (TZ) emerges as the Al-toxicity target of plant root apex. The objective of the study was to evaluate the effect of Si on redox homeostasis in root-apex TZ of rice seedlings under Al stress. Si alleviated Al toxicity as revealed by promotion of root elongation and less Al accumulation. In Si-deprived plants, treatment with Al altered the normal distribution of superoxide anion (O2·−) and hydrogen peroxide (H2O2) in root tip. Al induced a significant increase of reactive oxygen species (ROS) in root-apex TZ, resulting in the peroxidation of membrane lipid and loss of plasma membrane integrity in root-apex TZ. However, Si greatly increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and enzymes involved in ascorbate–glutathione (AsA–GSH) cycle in root-apex TZ under Al stress, and enhanced AsA and GSH contents, which reduced ROS and callose contents, thereby reducing malondialdehyde (MDA) content and Evans blue uptake. These results allow to precise the changes of ROS in root-apex TZ after exposure to Al, and the positive role of Si in maintaining redox balance in root-apex TZ.

P-224 Basonuclin 1 prevents intrauterine growth restriction via inhibiting necroptosis

Abstract Study question Is basonuclin 1 (BNC1) involved in intrauterine growth restriction (IUGR), and what’s the underlying mechanism? Summary answer BNC1 is significantly decreased in the placenta of IUGR patients. BNC1 deficiency engenders IUGR in Bnc1 truncation mutation (Bnc1tr/tr) mice model by inducing placental necroptosis. What is known already IUGR is among the leading causes of perinatal morbidity and mortality. IUGR occurs due to multiple factors, including genetic, placental, fetal, and maternal factors. Among which genetic factors remain largely unknown. Our previous exploration found that Bnc1 mutationmice exhibit low birth weight and survival rates, indicating BNC1 may play a potential role in fetal and placental development. Necroptosis is a recently recognized cell death pathway that may contribute to placental pathophysiology in IUGR patients. While the underlying mechanism remains largely unknown. Study design, size, duration A clinical retrospective cohort study of 40 IUGR patients (diagnosed by Hadlock ultrasound measurements) and 40 controls were included (2017–2022) for placenta collection. Bnc1 targeted mutation mouse was on a C57BL/6 J background. Male and female mice of the Bnc1+/tr genotype were mated to produce Bnc1+/+, Bnc1+/tr, and Bnc1tr/tr mice. Placenta and fetuses were obtained on the 15th and 18th days of pregnancy. Participants/materials, setting, methods The following analyses were performed: (i) Immunohistochemistry for detecting the location and expression of BNC1 in the placenta of humans and mice, (ii) placental and fetal weights for evaluation of IUGR development, (iii) morphometric evaluation of fetal utilizing Alcian blue and alizarin red staining, (iv) morphometric evaluation of placental compartments utilizing H&amp;E staining, TUNEL staining, and TEM analysis, (v) RNA sequencing (RNA-seq) of placental tissues. Main results and the role of chance BNC1 was specifically highly expressed in trophoblasts and endothelial cells in the early and mature placental development stages of humans and mice. The expression of BNC1 was significantly decreased in the placenta of IUGR patients compared to the control group (P &amp;lt; 0.0001). In the mice model, the number of Bnc1tr/tr fetuses was significantly decreased compared with wild-type Bnc1+/+ fetuses on the 18th day of pregnancy. While the number of Bnc1tr/tr fetuses and Bnc1+/+ fetuses was similar on the 15th day of pregnancy, indicating the loss of Bnc1tr/tr fetuses between the two stages. Besides, both the Bnc1tr/tr fetal and placental weights were significantly decreased on the 15th day of pregnancy compared with Bnc1+/+ fetuses and placenta (P &amp;lt; 0.01). The Bnc1 truncation mutation caused poor development of the placental labyrinthine layer with an increased number of TUNEL-positive foci. Transmission electron microscopy also revealed a disorganized labyrinthine layer with defects in cytoplasm translucence, loss of plasma membrane integrity, swollen mitochondria, and decondensed chromatin. Finally, RNA sequencing of placental tissues indicated BNC1 deficiency-induced IUGR may be modulated by necroptosis. In conclusion, we found a relationship between the disturbed BNC1 expression and the occurrence of IUGR through necroptosis. Limitations, reasons for caution This study used only Bnc1 truncation mutation mice model, the other animal model with placenta-conditional knockout mice was needed to consolidate the results. Besides, further mechanism exploration should be conducted in both human and mouse cell lines. Wider implications of the findings Our work suggests a new gene related to the development of IUGR, which provides early diagnosis and treatment for managing IUGR, thus improving outcomes for both fetal and gravida. Trial registration number not applicable

Calcium as a master regulator of ferroptosis and other types of regulated necrosis

Regulation of proliferation and cell death is fundamental for organismal development and for restoring tissue homeostasis after biological stress. During the last years, several forms of regulated cell death have been discovered that share the loss of plasma membrane integrity as a common hallmark and that are collectively known as regulated necrosis (RN) pathways. During RN, plasma membrane damage is sensed by the cell by increases in the levels of intracellular calcium. Interestingly, cytosolic calcium influx can either lead to cell death or survival, given the versatile role of this ion in regulating multiple signaling processes. Among them, membrane repair enables the cells to tolerate the injury and, even in some conditions, survive. Here, we review calcium signaling in the context of RN pathways, with a focus on ferroptosis, a type of RN in which plasma membrane damage is elicited by the accumulation of oxidized lipids. In contrast, other forms of RN such as necroptosis and pyroptosis require dedicated pore-forming proteins for plasma membrane damage and cell death. We first focus on the current knowledge regarding the contribution of calcium to ferroptosis, and then illustrate the similarities and differences in calcium signaling with necroptosis and pyroptosis. Calcium signaling emerges as a key event in the cellular responses to membrane damage and in the regulation of cell death.

Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis. Here we address whether ATR inhibition in combination with irradiation may increase the presentation of hallmark factors of immunogenic cell death (ICD), and to what extent caspase activation regulates this response. Human lung cancer and osteosarcoma cell lines (SW900, H1975, H460, U2OS) were treated with X-rays and ATR inhibitors (VE822; AZD6738) in the absence and presence of a pan-caspase inhibitor. The ICD hallmarks HMGB1 release, ATP secretion and calreticulin surface-presentation were assessed by immunoblotting of growth medium, the CellTiter-Glo assay and an optimized live-cell flow cytometry assay, respectively. To obtain accurate measurement of small differences in the calreticulin signal by flow cytometry, we included normalization to a barcoded control sample. Extracellular release of HMGB1 was increased in all the cell lines at 72 hours after the combined treatment with radiation and ATR inhibitors, relative to mock treatment or cells treated with radiation alone. The HMGB1 release correlated largely - but not strictly - with loss of plasma membrane integrity, and was suppressed by addition of the caspase inhibitor. However, one cell line showed HMGB1 release despite caspase inhibition, and in this cell line caspase inhibition induced pMLKL, a marker for necroptosis. ATP secretion occurred already at 48 hours after the co-treatment and did clearly not correlate with loss of plasma membrane integrity. Addition of pan-caspase inhibition further increased the ATP secretion. Surface-presentation of calreticulin was increased at 24-72 hours after irradiation, but not further increased by either ATR or caspase inhibition. These results show that ATR inhibition can increase the presentation of two out of three ICD hallmark factors from irradiated human cancer cells. Moreover, caspase activation distinctly affects each of the hallmark factors, and therefore likely plays a dual role in tumor immunogenicity by promoting both immunostimulatory and -suppressive effects.

Epicatechin gallate: A promising agent isolated from Sargentodoxa cuneata for the treatment of ichthyophthiriasis in goldfish (Carassius auratus)

Ichthyophthirius multifiliis is a pathogenic parasite of freshwater fish that threatens the sustainability of aquaculture industry globally. Our previous studies have identified Sargentodoxa cuneata ethanol extracts exhibited potent antiparasitic activity against I. multifiliis. However, the active ingredients in S. cuneata remain unknown. In this study, we isolated an active compound from S. cuneata by bioassay-guided fractionation and identified it as epicatechin gallate (ECG) by NMR, FT-IR and HR-MS spectroscopy. In vitro assays revealed that ECG was effective against all the free-living stages of I. multifiliis. ECG could cause 100% mortality of theronts at 0.3 mg/L within 30.6 ± 0.4 min, and terminate reproduction of protomonts and tomonts at concentrations of 2.0 and 10.0 mg/L, respectively. Morphologically, 2.0 mg/L ECG treatment for 2 h induced the loss of plasma membrane integrity and leakage of intracellular contents of the protomont. Moreover, an in vivo parasite‑goldfish infection model was applied to evaluate the efficacy of ECG on prevention and treatment of ichthyophthiriasis. The results showed that 2.0 mg/L ECG treatment for 12 days could cure goldfish ichthyophthiriasis and significantly increase the survival rate of the infected goldfish by 86.7%. Acute toxicity test revealed that ECG was relatively safe to goldfish, with a median lethal concentration (LC50) value of 63.7 mg/L, which was >30 times higher than effective concentration against I. multifiliis in vivo. Overall, our data indicated that ECG had the potential as a novel antiparasitic agent for the treatment of ichthyophthiriasis in aquaculture.