Apoptosis-like Death Research Articles

Averrhoa carambola Leaf Extract Induces Apoptosis-Like Death with Increased ROS Generation in Leishmania donovani.

The parasitic disease leishmaniasis is responsible for high mortality and morbidity rates worldwide. The visceral form is the most severe form of leishmaniasis (or leishmaniosis), which is caused predominantly by Leishmania donovani. Currently, clinically recommended antileishmanial drugs are not convenient because of several medical complications and resistance issues. Phytocompounds are the best candidates in this regard. The present study aimed to evaluate the antileishmanial activity of Averrhoa carambola leaf extract. The antipromastigote activity and cytotoxicity were assessed using the MTT assay. Morphological distortions were determined using phase contrast microscopy and scanning electron microscopy (SEM). Reactive oxygen species (ROS) production, nonprotein thiol depletion and apoptotic death in promastigotes were determined via flow cytometry. UV-visible spectroscopy and energy dispersive X-ray (EDX) spectroscopy was performed for elemental analysis. Fourier-transform infrared spectroscopy (FTIR) and liquid chromatography‒mass spectrometry (LCMS) were used to characterize the phytocomponent(s) present in the extract. The chloroform extract of Averrhoa carambola leaf (ACCEX) (IC50 = 50.76 ± 1.7µg/mL) exhibited the highest activity, followed by the ethyl acetate, hexane, and methanol extracts. ACCEX has also exhibited lower toxicity towards host macrophages. ACCEX also induced morphological distortions in promastigotes, with significant generation of ROS and the concomitant apoptosis initiation followed by a decrease in the nonprotein thiol level. The major phytometabolites present in ACCEX were identified from the National Institute of Standards and Technology (NIST) database and from a literature review. This study suggested that Averrhoa carambola leaf extracts are rich in some classes of biologically active phytocompounds and exhibit good antileishmanial activity.

Novel Schiff's base-assisted synthesis of metal-ligand nanostructures for multi-functional applications: Detection of catecholamines/antibiotics, removal of tetracycline, and antifungal treatment against plant pathogens

The development of nanozymes (NZ) for the simultaneous detection of multiple target chemicals is gaining paramount attention in the field of food and health sciences, and waste management industries. Nanozymes (NZ) effectively compensate for the environmental vulnerability of natural enzymes. Considering the development gap of NZ with diverse applications, we synthesized versatile Schiff’s base ligands following a facile route and readily available starting reagents (glutaraldehyde, aminopyridines). DPDI, one of the synthesized ligands, readily reacted with transition metal ions (Cu+2, Ag+1, Zn+2 in specific) under ambient conditions, yielding the corresponding nanoparticles/MOF. The structures of ligands and their products were confirmed using various analytical techniques. The enzymatic efficacy of DPDI-Cu (km 0.25 mM=, Vmax = 10.75 µM/sec) surpassed Tremetese versicolor laccase efficacy (km 0. 5 mM=, Vmax = 2.15 µM/sec). Additionally, DPDI-Cu proved resilient to changing pH, temperature, ionic strength, organic solvent, and storage time compared to laccase and provided reusability. DPDI-Cu proved promising for colorimetric detection of dopamine, epinephrine, catechol, tetracycline, and quercetin. The mechanism of oxidative detection of TC was studied through LC/MS analysis. DPDI-Cu-bentonite composite efficiently adsorbed tetracycline with maximum Langmuir adsorption of 208 mg/g. Moreover, DPDI/Cu and DPDI-Ag nanoparticles possessed antifungal activity exhibiting a minimum inhibitory concentration of 400 µg/mL and 3.12 µg/mL against Aspergillus flavus. Florescent dye tracking and SEM/TEM analysis confirmed that DPDI-Ag caused disruption of the plasma membrane and triggered ROS generation and apoptosis-like death in fungal cells. The DPDI-Ag coating treatment of wheat seeds confirmed the non-phytotoxicity of Ag-NPs.

β-cyclocitral induced rapid cell death of Microcystis aeruginosa

β-cyclocitral (BCC) is an odorous compound that can be produced by bloom-forming cyanobacteria, for example, Microcystis aeruginosa. BCC has been proposed to explain the rapid decline of cyanobacterial blooms in natural water bodies due to its lytic effects on cyanobacteria cells. However, few insights have been gained regarding the mechanisms of its lethality on cyanobacteria. In this study, M. aeruginosa was exposed to 0–300 mg/L BCC, and the physiological responses were comprehensively studied at the cellular, molecular, and transcriptomic levels. The result indicated that the lethal effect was concentration-dependent; 100 mg/L BCC only caused recoverable stress, while 150–300 mg/L BCC caused rapid rupture of cyanobacterial cells. Scanning electron microscope images suggested two typical morphological changes exposed to above 150 mg/LBCC: wrinkled/shrank with limited holes on the surface at 150 and 200 mg/L BCC exposure; no apparent shrinkage at the surface but with cell perforation at 250 and 300 mg/L BCC exposure. BCC can rapidly inhibit the photosynthetic activity of M. aeruginosa cells (40%∼100% decreases for 100–300 mg/L BCC) and significantly down-regulate photosynthetic system Ⅰ-related genes. Also, chlorophyll a (by 30%∼90%) and ATP (by ∼80%) contents severely decreased, suggesting overwhelming pressure on the energy metabolism in cells. Glutathione levels increased significantly, and stress response-related genes were upregulated, indicating the perturbation of intracellular redox homeostasis. Two cell death pathways were proposed to explain the lethal effect: apoptosis-like death as revealed by the upregulation of SOS response genes when exposed to 200 mg/L BCC and mazEF-mediated death as revealed by the upregulation of mazEF system genes when exposed to 300 mg/L BCC. Results of the current work not only provide insights into the potential role of BCC in inducing programmed cell death during bloom demise but also indicate the potential of using BCC for harmful algal control.

Supernatant of platelet-Klebsiella pneumoniae coculture induces apoptosis-like death in Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae strains, especially carbapenem-resistant K. pneumoniae, have become a rapidly emerging crisis worldwide, greatly limiting current therapeutic options and posing new challenges to infection management. Therefore, it is imperative to develop novel and effective biological agents for the treatment of multidrug-resistant K. pneumoniae infections. Platelets play an important role in the development of inflammation and immune responses. The main component responsible for platelet antibacterial activity lies in the supernatant stimulated by gram-positive bacteria. However, little research has been conducted on the interaction of gram-negative bacteria with platelets. Therefore, we aimed to explore the bacteriostatic effect of the supernatant derived from platelet-K. pneumoniae coculture and the mechanism underlying this effect to further assess the potential of platelet-bacterial coculture supernatant. We conducted this study on the gram-negative bacteria K. pneumoniae and CRKP and detected turbidity changes in K. pneumoniae and CRKP cultures when grown with platelet-K. pneumoniae coculture supernatant added to the culture medium. We found that platelet-K. pneumoniae coculture supernatant significantly inhibited the growth of K. pneumoniae and CRKP in vitro. Furthermore, transfusion of platelet-K. pneumoniae coculture supernatant alleviated the symptoms of K. pneumoniae and CRKP infection in a murine model. Additionally, we observed apoptosis-like changes, such as phosphatidylserine exposure, chromosome condensation, DNA fragmentation, and overproduction of reactive oxygen species in K. pneumoniae following treatment with the supernatant. Our study demonstrates that the platelet-K. pneumoniae coculture supernatant can inhibit K. pneumoniae growth by inducing an apoptosis-like death, which is important for the antibacterial strategies development in the future.IMPORTANCEWith the widespread use of antibiotics, bacterial resistance is increasing, and a variety of multi-drug resistant Gram-negative bacteria have emerged, which brings great challenges to the treatment of infections caused by Gram-negative bacteria. Therefore, finding new strategies to inhibit Gram-negative bacteria and even multi-drug- resistant Gram-negative bacteria is crucial for treating infections caused by Gram-negative bacteria, improving the abuse of antibiotics, and maintaining the balance between bacteria and antibiotics. K. pneumoniae is a common clinical pathogen, and drug-resistant CRKP is increasingly difficult to cure, which brings great clinical challenges. In this study, we found that the platelet-K. pneumoniae coculture supernatant can inhibit K. pneumoniae growth by inducing an apoptosis-like death. This finding has inspired the development of future antimicrobial strategies, which are expected to improve the clinical treatment of Gram-negative bacteria and control the development of multidrug-resistant strains.

Polymeric nanoparticles containing kojic acid induce structural alterations and apoptosis-like death in Leishmania (Leishmania) amazonensis.

Leishmaniasis encompasses a cluster of neglected tropical diseases triggered by kinetoplastid phatogens belonging to the genus Leishmania. Current therapeutic approaches are toxic, expensive, and require long-term treatment. Nanoparticles are emerging as a new alternative for the treatment of neglected tropical diseases. Silk Fibroin is a biocompatible and amphiphilic protein that can be used for formulating nanoemulsions, while kojic acid is a secondary metabolite with antileishmanial actions. Thus, this study evaluated the efficacy of a nanoemulsion, formulated with silk fibroin as the surfactant and containing kojic acid (NanoFKA), against promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. The NanoFKA had an average particle size of 176nm, Polydispersity Index (PDI) of 0.370, and a Zeta Potential of -32.3mV. It presented inhibitory concentration (IC50) values of >56μg/mL and >7μg/mL for the promastigote and amastigote forms, respectively. Ultrastructural analysis, cell cycle distribution and phosphatidylserine exposure showed that NanoFKA treatment induces apoptosis-like cell death and cell cycle arrest in L. (L.) amazonensis. In addition, NanoFKA exhibited no cytotoxicity against macrophages. Given these results, NanoFKA present leishmanicidal activity against L. (L.) amazonensis.

Activation and Denitrosylation of Procaspase-3 in KA-induced Excitotoxicity.

It has been reported that activation of glutamate kainate receptor subunit 2 (GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation, caspase-3 activation, subsequently results in delayed apoptosis-like neuronal death in hippocampus CA1 subfield after cerebral ischemia. Nitric oxide-mediated S-nitrosylation might inhibit the procaspase activation, whereas denitrosylation might contribute to cleavage and activation of procaspases. The study aimed to elucidate the molecular mechanisms underlying procaspase-3 denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat hippocampus. S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase (TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced apoptosis and cell death in hippocampal CA1 and CA3 subfields. KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR. Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process before becoming activated, providing valuable insights into the underlying mechanisms and possible treatment of excitotoxicity.

Beetroot (Beta vulgaris) Extract against Salmonella Typhimurium via Apoptosis-Like Death and Its Potential for Application in Cooked Pork.

Salmonella Typhimurium is a common foodborne pathogen in meat and meat products, causing significant harm and losses to producers and consumers. The aim of this study was to investigate the antibacterial activity and possible mechanisms of beetroot (Beta vulgaris) extract against S. Typhimurium, as well as the application potential in cooked pork. The results suggested beetroot extract could inhibit S. Typhimurium with a minimum inhibitory concentration (MIC) of 20 mg/mL. After treatment with beetroot extract (1 or 2 MIC), S. Typhimurium exhibited the characteristics of apoptotic-like death (ALD), such as membrane depolarization, phosphatidylserine (PS) externalization, caspase-like protein activation, and DNA fragmentation. Further research has shown that the ALD induced by beetroot extract in S. Typhimurium was caused by reactive oxygen species (ROS) consumption, which was different from most natural products. The treatment of cooked pork with beetroot extract could reduce the number of S. Typhimurium, lower pH, defer lipid oxidation, and improve the colour. These results indicate that beetroot extract can inhibit S. Typhimurium through the ALD mechanism and has potential as an antibacterial agent against S. Typhimurium in ready-to-eat meat products.

Honokiol induces apoptosis-like death in Cryptocaryon irritans Tomont

BackgroundCryptocaryon irritans, a common parasite in tropical and subtropical marine teleost fish, has caused serious harm to the marine aquaculture industry. Honokiol was proven to induce C. irritans tomont cytoplasm shrinkage and death in our previous study, but the mechanism by which it works remains unknown.MethodsIn this study, the changes of apoptotic morphology and apoptotic ratio were detected by microscopic observation and AnnexinV-FITC/PI staining. The effects of honokiol on intracellular calcium ([Ca2+]i) concentration, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), quantity of DNA fragmentations (QDF) and caspase activities were detected by Fluo-3 staining, JC-1 staining, DCFH-DA staining, Tunel method and caspase activity assay kit. The effects of honokiol on mRNA expression levels of 61 apoptosis-related genes in tomonts of C. irritans were detected by real-time PCR.ResultsThe results of the study on the effects of honokiol concentration on C. irritans tomont apoptosis-like death showed that the highest levels of prophase apoptosis-like death rate (PADR), [Ca2+]i concentration, ROS, the activities of caspase-3/9 and the lowest necrosis ratio (NER) were obtained at a concentration of 1 μg/ml, which was considered the most suitable for inducing C. irritans tomont apoptosis-like death. When C. irritans tomonts were treated with 1 μg/ml honokiol, the [Ca2+]i concentration began to increase significantly at 1 h. Following this, the ROS, QDF and activities of caspase-3/9 began to increase significantly, and the ΔΨm began to decrease significantly at 2 h; the highest PADR was obtained at 4 h. The mRNA expression of 14 genes was significantly upregulated during honokiol treatment. Of these genes, itpr2, capn1, mc, actg1, actb, parp2, traf2 and fos were enriched in the pathway related to apoptosis induced by endoplasmic reticulum (ER) stress.ConclusionsThis article shows that honokiol can induce C. irritans tomont apoptosis-like death. These results suggest that honokiol may disrupt [Ca2+]i homeostasis in ER and then induce C. irritans tomont apoptosis-like death by caspase cascade or mitochondrial pathway, which might represent a novel therapeutic intervention for C. irritans infection.Graphical abstract

Polyoxometalate K6[P2Mo18O62] Inactivates Escherichia coli O157:H7 by Inducing recA Expression and Apoptosis-like Bacterial Death.

Polyoxometalates have emerged as promising bactericidal agents. In the current study, the bactericidal activity of polyoxometalate K6[P2Mo18O62] against Escherichia coli (E. coli) O157:H7 and its possible underlying mechanisms were explored. The obtained results demonstrated that K6[P2Mo18O62] could effectively kill E. coli O157:H7 at millimolar levels. Moreover, K6[P2Mo18O62] treatment also induced significant increases in recA protein expression and further triggered characteristic apoptosis-like bacterial death events such as DNA fragmentation and phosphatidylserine exposure. In conclusion, polyoxometalate K6[P2Mo18O62] possesses a desirable antibacterial activity, and induction of bacterial apoptosis-like death might be involved in its underlying bactericidal mechanisms.

Beetroot (Beta vulgaris) extract exerts an antibacterial effect by inducing apoptosis-like death in Bacillus cereus

In this study, we assessed the phytochemicals of beetroot (Beta vulgaris) extract (BE), and examined the antibacterial activity and possible mechanism of BE against Bacillus cereus. The phytochemicals in BE were analyzed by UPLC-ESI-MS-based widely targeted metabolomics analysis, and identified a total of 745 phytochemicals. Nine polyphenols and 19 flavonoids with growth-inhibiting activity against B. cereus were identified. The minimum inhibitory concentration (MIC) of BE against B. cereus was 15 mg/mL. The intracellular adenosine 5′-triphosphate (ATP) levels were decreased and the B. cereus exhibited the features of apoptosis-like death (ALD) [cell membrane depolarization, caspase-like protein activation, phosphatidylserine (PS) translocation, and DNA fragmentation] by depleting reactive oxygen species (ROS) after BE treatment. These results suggest BE is rich in bioactive substances and induces ALD in B. cereus. Thus, the phytochemicals diversity makes BE as a source of bioactive ingredients, especially as a promising potential natural preservative to reduce B. cereus contamination.

Depletion of reactive oxygen species induced by beetroot (Beta vulgaris) extract leads to apoptosis-like death in Cronobacter sakazakii

This research aimed to disclose the antibacterial activity of beetroot extract (Beta vulgaris) against Cronobacter sakazakii and its possible mechanisms. We evaluated its antibacterial activity by measuring the minimum inhibitory concentration (MIC) and time-kill kinetics. We also evaluated the intracellular ATP levels, bacterial apoptosis-like death (ALD), and reactive oxygen species (ROS) levels to reveal the possible antibacterial mechanisms. Our results showed that the MIC of beetroot extract against C. sakazakii was 25 mg/mL and C. sakazakii (approximately 8 log cfu/mL) was completely inhibited after treatment with 2 MIC of beetroot extract for 3 h. Beetroot extract reduced intracellular ATP levels and facilitated characteristics of ALD in C. sakazakii, such as membrane depolarization, increased intracellular Ca2+ levels, phosphatidylserine externalization, caspase-like protein activation, and DNA fragmentation. Additionally, and different from most bacterial ALD caused by the accumulation of ROS, beetroot extract reduced the intracellular ROS levels in C. sakazakii. Our experimental data provide a rationale for further research of bacterial ALD and demonstrate that beetroot extract can inhibit C. sakazakii in food processing environments.

An anti-leishmanial compound 4',7-dihydroxyflavone elicits ROS-mediated apoptosis-like death in Leishmania parasite.

The treatment for leishmaniasis is currently plagued by side effects such as toxicity, and the emergence of drug resistance to the available repertoire of drugs, as well as the expense of these drugs. Considering the rising concerns, we report here , the anti-leishmanial activity and mechanism of a flavone compound 4',7-dihydroxyflavone (TI 4). Four flavanoids were initially screened for anti-leishmanial activity and cytotoxicity. The results showed that the compound TI 4 exhibited higher activity and selectivity index while maintaining low cytotoxicity. Preliminary microscopic studies and FACS analysis reported that the parasite underwent apoptosis on TI 4 treatment. Further in-depth studies revealed high ROS production and thiol levels in the parasites suggesting ROS mediated apoptosis in the parasites upon TI 4 treatment. Other apoptotic indicators like intracellular Ca2+ and mitochondrial membrane potential also indicated the onset of apoptosis in the treated parasites. The mRNA expression levels signified that the redox metabolism genes were upregulated by 2-fold along with the apoptotic genes. In summary, the use of TI 4 on Leishmania parasites induces ROS-mediated apoptosis, therefore the compound has immense potential to be an anti-leishmanial drug. However, in vivo studies would be required to ascertain its safety and efficacy before we can exploit the compound against the growing leishmaniasis crisis.

Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses

The zebrafish (Danio rerio) represents an increasingly important model organism in virology. We evaluated its utility in the study of economically important viruses from the genus Cyprinivirus (anguillid herpesvirus 1, cyprinid herpesvirus 2 and cyprinid herpesvirus 3 (CyHV-3)). This revealed that zebrafish larvae were not susceptible to these viruses after immersion in contaminated water, but that infections could be established using artificial infection models in vitro (zebrafish cell lines) and in vivo (microinjection of larvae). However, infections were transient, with rapid viral clearance associated with apoptosis-like death of infected cells. Transcriptomic analysis of CyHV-3-infected larvae revealed upregulation of interferon-stimulated genes, in particular those encoding nucleic acid sensors, mediators of programmed cell death and related genes. It was notable that uncharacterized non-coding RNA genes and retrotransposons were also among those most upregulated. CRISPR/Cas9 knockout of the zebrafish gene encoding protein kinase R (PKR) and a related gene encoding a protein kinase containing Z-DNA binding domains (PKZ) had no impact on CyHV-3 clearance in larvae. Our study strongly supports the importance of innate immunity-virus interactions in the adaptation of cypriniviruses to their natural hosts. It also highlights the potential of the CyHV-3-zebrafish model, versus the CyHV-3-carp model, for study of these interactions.

Effect of liquiritigenin on chloroquine accumulation in digestive vacuole leading to apoptosis-like death of chloroquine-resistant P. falciparum

BackgroundMalaria remains one of the major health concerns, especially in tropical countries. Although drugs such as artemisinin-based combinations are efficient for treating Plasmodium falciparum, the growing threat from multi-drug resistance has become a major challenge. Thus, there is a constant need to identify and validate new combinations to sustain current disease control strategies to overcome the challenge of drug resistance in the malaria parasites. To meet this demand, liquiritigenin (LTG) has been found to positively interact in combination with the existing clinically used drug chloroquine (CQ), which has become unfunctional due to acquired drug resistance. PurposeTo evaluate the best interaction between LTG and CQ against CQ- resistant strain of P. falciparum. Furthermore, the in vivo antimalarial efficacy and possible mechanism of action of the best combination was also assessed. MethodsThe in vitro anti-plasmodial potential of LTG against CQ- resistant strain K1 of P. falciparum was tested using Giemsa staining method. The behaviour of the combinations was evaluated using the fix ratio method and evaluated the interaction of LTG and CQ by calculating the fractional inhibitory concentration index (FICI). Oral toxicity study was carried out in a mice model. In vivo antimalarial efficacy of LTG alone and in combination with CQ was evaluated using a four-day suppression test in a mouse model. The effect of LTG on CQ accumulation was measured using HPLC and the rate of alkalinization of the digestive vacuole. Cytosolic Ca2+ level, mitochondrial membrane potential, caspase-like activity, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and Annexin V Apoptosis assay to assess anti-plasmodial potential. Proteomics analysis was evaluated by LC-MS/MS analysis. ResultsLTG possesses anti-plasmodial activity on its own and it showed to be an adjuvant of CQ. In in vitro studies, LTG showed synergy with CQ only in the ratio (CQ: LTG-1:4) against CQ-resistant strain (K1) of P. falciparum. Interestingly, in vivo studies, LTG in combination with CQ showed higher chemo-suppression and enhanced mean survival time at much lower concentrations compared to individual doses of LTG and CQ against CQ- resistant strain (N67) of Plasmodium yoelli nigeriensis. LTG was found to increase the CQ accumulation into digestive vacuole, reducing the rate of alkalinization, in turn increasing cytosolic Ca2+ level, loss of mitochondrial potential, caspase-3 activity, DNA damage and externalization of phosphatidylserine of the membrane (in vitro). These observations indicate the involvement of apoptosis-like death of P. falciparum that might be due to the accumulation of CQ. ConclusionLTG showed synergy with CQ in the ratio LTG: CQ, 4:1) in vitro and was able to curtail the IC50 of CQ and LTG. Interestingly, in vivo in combination with CQ, LTG showed higher chemo-suppression as well as enhanced mean survival time at a much lower concentrations of both the partners as compared to an individual dose of CQ and LTG. Thus, synergistic drug combination offers the possibility to enhance CQ efficacy in chemotherapy.

The Anti-Leishmania amazonensis and Anti-Leishmania chagasi Action of Copper(II) and Silver(I) 1,10-Phenanthroline-5,6-dione Coordination Compounds.

Leishmaniasis is a neglected disease caused by protozoa belonging to the Leishmania genus. Notably, the search for new, promising and potent anti-Leishmania compounds remains a major goal due to the inefficacy of the available drugs used nowadays. In the present work, we evaluated the effects of 1,10-phenanthroline-5,6-dione (phendione) coordinated to silver(I), [Ag(phendione)2]ClO4 (Ag-phendione), and copper(II), [Cu(phendione)3](ClO4)2·4H2O (Cu-phendione), as potential drugs to be used in the chemotherapy against Leishmania amazonensis and Leishmania chagasi. The results showed that promastigotes treated with Ag-phendione and Cu-phendione presented a significant reduction in the proliferation rate. The IC50 values calculated to Ag-phendione and Cu-phendione, respectively, were 7.8 nM and 7.5 nM for L. amazonensis and 24.5 nM and 20.0 nM for L. chagasi. Microscopical analyses revealed several relevant morphological changes in promastigotes, such as a rounding of the cell body and a shortening/loss of the single flagellum. Moreover, the treatment promoted alterations in the unique mitochondrion of these parasites, inducing significant reductions on both metabolic activity and membrane potential parameters. All these cellular perturbations induced the triggering of apoptosis-like death in these parasites, as judged by the (i) increased percentage of annexin-positive/propidium iodide negative cells, (ii) augmentation in the proportion of parasites in the sub-G0/G1 phase and (iii) DNA fragmentation. Finally, the test compounds showed potent effects against intracellular amastigotes; contrarily, these molecules were well tolerated by THP-1 macrophages, which resulted in excellent selective index values. Overall, the results highlight new selective and effective drugs against Leishmania species, which are important etiological agents of both cutaneous (L. amazonensis) and visceral (L. chagasi) leishmaniasis in a global perspective.

Antibacterial Mode of Action of β-Amyrin Promotes Apoptosis-Like Death in Escherichia coli by Producing Reactive Oxygen Species.

β-Amyrin is a pentacyclic triterpene widely distributed in leaves and stems worldwide. The ability of β-amyrin to induce the production of reactive oxygen species (ROS) in microorganisms suggests its potential as an antimicrobial agent. Thus, this study aimed to elucidate the antibacterial mode of action of β-amyrin. We treated Escherichia coli cells with β-amyrin and found that it triggered ROS accumulation. Excessive stress caused by ROS, particularly hydroxyl radicals, induces glutathione (GSH) dysfunction. GSH protects cells from oxidative and osmotic stresses; thus, its dysfunction leads to membrane depolarization. The resultant change in membrane potential leads to the release of apoptotic proteins, such as caspases. The activated caspases-like protein promotes the cleavage of DNA into single strands, which is a hallmark of apoptosis-like death in bacteria. Apoptotic cells usually undergo events such as DNA fragmentation and phosphatidylserine exposure, differentiating them from necrotic cells, and the cells treated with β-amyrin in this study were positive for annexin V and negative for propidium iodide, indicating apoptosis-like death. In conclusion, our findings suggest that the antibacterial mode of action of β-amyrin involves the induction of ROS, which resulted in apoptosis-like death in E. coli.

Platelets Inhibit Methicillin-Resistant Staphylococcus aureus by Inducing Hydroxyl Radical-Mediated Apoptosis-Like Cell Death.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria and poses a significant threat to human health. Due to the emergence of multidrug resistance, limited drugs are available for the treatment of MRSA infections. In recent years, platelets have been reported to play important roles in inflammation and immune responses, in addition to their functions in blood hemostasis and clotting. We and other researchers have previously reported that platelets can inhibit Staphylococcus aureus growth. However, it remained unclear whether platelets have the same antibacterial effect on drug-resistant strains. In this study, we hypothesized that platelets may also inhibit the growth of MRSA; the results confirmed that platelets significantly inhibited the growth of MRSA in vitro. In a murine model of MRSA infection, we found that a platelet transfusion alleviated the symptoms of MRSA infection; in contrast, depletion of platelets aggravated infective symptoms. Moreover, we observed an overproduction of hydroxyl radicals in MRSA following platelet treatment, which induced apoptosis-like death of MRSA. Our findings demonstrate that platelets can inhibit MRSA growth by promoting the overproduction of hydroxyl radicals and inducing apoptosis-like death.IMPORTANCE The widespread use of antibiotics has led to the emergence of drug-resistant bacteria, particularly multidrug-resistant bacteria. MRSA is the most common drug-resistant bacterium that causes suppurative infections in humans. As only a limited number of drugs are available to treat the infections caused by drug-resistant pathogens, it is imperative to develop novel and effective biological agents for treating MRSA infections. This is the first study to show that platelets can inhibit MRSA growth in vitro and in vivo. Our results revealed that platelets enhanced the production of hydroxyl radicals in MRSA, which induced a series of apoptosis hallmarks in MRSA, including DNA fragmentation, chromosome condensation, phosphatidylserine exposure, membrane potential depolarization, and increased intracellular caspase activity. These findings may further our understanding of platelet function.

Antibacterial mechanism of beetroot (Beta vulgaris) extract against Listeria monocytogenes through apoptosis-like death and its application in cooked pork

Due to riching in polyphenolic compounds, betalains, and ascorbic acid, beetroot (Beta vulgaris) extract exhibits an antibacterial activity. However, its effect on Listeria monocytogenes is unclear. In this study, the antibacterial activity of beetroot extract against L. monocytogenes was assessed by measuring its minimum inhibitory concentration (MIC) and the time-kill curve. Results revealed that the MIC of beetroot extract against L. monocytogenes was 20 mg/mL and bacterial survival decreased as beetroot extract concentration increased. The antibacterial mechanism of beetroot extract involved the reduction of intracellular 5ʹ-adenosine triphosphate levels of L. monocytogenes after treatment. L. monocytogenes exhibiting membrane depolarization, caspase-like protein activation, phosphatidylserine externalization, and DNA fragmentation, demonstrated that L. monocytogenes underwent apoptosis-like death (ALD) following beetroot extract treatment. Furthermore, the reason of ALD in L. monocytogenes is reactive oxygen species depletion. Beetroot extract decreased the number of L. monocytogenes cells, pH values, and lipid oxidation in cooked pork. Meanwhile, cooked pork become darker and redder than the control samples. It suggested that beetroot extract could be used as a natural preservative in cooked meat production to inhibit the growth of L. monocytogenes.

The Natural Alkaloid Tryptanthrin Induces Apoptosis-like Death in Leishmania spp.

Leishmaniasis is a vector-borne disease against which there are no approved vaccines, and the treatment is based on highly toxic drugs. The alkaloids consist of a chemical class of natural nitrogen-containing substances with a long history of antileishmanial activity. The present study aimed at determining the antileishmanial activity and in silico pharmacokinetic and toxicological potentials of tryptanthrin alkaloid. The anti-Leishmania amazonensis and anti-L. infantum assays were performed against both promastigotes and intracellular amastigotes. Cellular viability was determined by parasites’ ability to grow (promastigotes) or differentiate (amastigotes) after incubation with tryptanthrin. The mechanisms of action were explored by mitochondrion dysfunction and apoptosis-like death evaluation. For the computational pharmacokinetics and toxicological analysis (ADMET), tryptanthrin was submitted to the PreADMET webserver. The alkaloid displayed anti-promastigote activity against L. amazonensis and L. infantum (IC50 = 11 and 8.0 μM, respectively). Tryptanthrin was active against intracellular amastigotes with IC50 values of 75 and 115 μM, respectively. Mitochondrial membrane depolarization was observed in tryptanthrin-treated promastigotes. In addition, parasites undergoing apoptosis-like death were detected after 18 h of exposure. In silico ADMET predictions revealed that tryptanthrin has pharmacokinetic and toxicological properties similar to miltefosine. The results presented herein demonstrate that tryptanthrin is an interesting drug candidate against leishmaniasis.

Naringin generates three types of reactive oxygen species contributing differently to apoptosis-like death in Escherichia coli

AimsNaringin is a flavonoid with a polyphenolic structure which induces formation of reactive oxygen species (ROS). Although the antibacterial effect of naringin has been demonstrated, the mechanism underlying this effect has not yet been elucidated. We focused on investigating the antibacterial mode of action of naringin in Escherichia coli following ROS generation. The contributions of ROS, hydroxy radicals (OH−), super oxide (O2−), and hydrogen peroxide (H2O2) were investigated. Main methodsROS accumulation was detected using fluorescence dyes, and all experiments were conducted using the scavenger including tiron, sodium pyruvate, and thiourea to assess the contribution of each ROS. Western blotting assays were used to observe the activation of the SOS response for DNA repair. DNA fragmentation, membrane depolarization, and phosphatidylserine exposure were estimated using TUNEL, DiBAC4(3), and Annexin V/PI. Key findingsAccumulation of ROS was observed in Escherichia coli after treatment with naringin. Oxidative stress induced cellular dysfunction including DNA damage, which results in SOS response activation. Eventually, apoptosis-like death occurred in cells treated with naringin. The cells had different contributions of each ROS and accompanying apoptotic factors. The ROS most destructive to E. coli was OH−, followed by H2O2 and O2−. SignificanceDue to its efficacy, naringin is a useful antimicrobial agent. An initial investigation into the antibacterial mode of action of naringin is presented in this paper. The contribution of each ROS to apoptosis-like cell death (ALD) was investigated, and the results enhanced our understanding of the correlation between the SOS response and oxidative stress in bacteria.