Membrane Cytochrome Research Articles

In vitro characterization of beta-cyclodextrin liposomal formulation of cisplatin nanomedicine

This study aims to develop a nanomedicine-based approach to protect healthy tissue during cancer treatment, improve patient compliance, reduce adverse drug reactions, and enhance the quality of life. By utilizing hydroxypropyl-β-cyclodextrin (BPCP) to increase drug hydrophilicity and liposomes for controlled release of cisplatin (CP), the hydroxypropyl-β-cyclodextrin-cisplatin liposomal formulation (BCDCPLP) demonstrated improved stability, targeted delivery, and reduced toxicity. The study confirmed that the above formulation had the highest cytotoxicity against HeLa cells, with significantly lower IC50 and IC90 values than cisplatin alone. This is attributed to improved drug stability and cellular uptake. Caspase-3 and cleaved PARP assays confirmed that BCDCPLP and CP-loaded liposomes induced substantial apoptosis, with BCDCPLP showing the highest increase in caspase-3 activity and PARP cleavage at 85 kDa. The ability of BCDCPLP to enhance apoptosis was further demonstrated through Western blot analysis, where the density of PARP cleavage bands was highest in BCDCPLP-treated cells, followed by CPLP and CP-treated cells. The study highlights that the cumulative release of PARP from CP-nanocarrier complexes leads to more efficient apoptosis than free CP. These findings align with the synergistic effects observed when combining CP with PARP inhibitors, which induce mitochondrial membrane permeabilization and cytochrome C release, activating the intrinsic apoptosis pathway. The potential of these nanomedicines, especially BCDCPLP, is in reducing the required dosage while maintaining therapeutic efficacy offers promising advances in cancer therapy.

Polymer nanodiscs support the functional extraction of an artificial transmembrane cytochrome

Polymer nanodiscs are an attractive alternative to surfactants for studying integral membrane proteins within their native lipid environment. Here, we investigate the use of such polymers to isolate a computationally-designed de novo membrane cytochrome named CytbX. We show that the block copolymers known as CyclAPols can efficiently extract CytbX directly from biomembranes and are compatible with the downstream purification and biophysical characterisation of this artificial protein. CyclAPol-solubilised CytbX is well-folded and highly robust with properties that are essentially identical to those observed for the same protein in a detergent micelle. However, electron transfer to CytbX from a diffusive flavoprotein is substantially faster in micelles than in the nanodisc system. Our results confirm that polymer nanodiscs will be a useful tool for the ongoing study and application of de novo membrane proteins.

Bifunctional sludge-derived redox carbon dots with photoelectron storage and delivery properties for ammonia production by photosensitized Shewanella oneidensis MR-1

Combining the light-harvesting capabilities of photosensitizers with microbial catalysis shows great potential in solar-driven biomanufacturing. However, little information is available about the effects of photosensitizers on the photoelectron transport during the dissimilatory nitrate reduction to ammonium (DNRA) process. Herein, redox carbon dots (CDs-500) were prepared from sludge via the pyrolysis-Fenton reaction and then used to construct a photosynthetic system with Shewanella oneidensis MR-1. The MR-1/CDs-500 photosynthetic system showed a 5.9-fold increase in ammonia production (4.9 mmol(NH3)·g−1(protein)·h−1) with a high selectivity of 94.0 %. The photoelectrons were found to be stored in CDs-500 and transferred into the cells. During the inward electron transport, the intracellular CDs-500 could be used as the direct photoelectron transfer stations between outer membrane cytochrome c and DNRA-related enzymes without the involvement of CymA and MtrA. This work provides a new method for converting waste into functional catalysts and increases solar-driven NH3 production to a greater extent.

ONC212, alone or in synergistic conjunction with Navitoclax (ABT-263), promotes cancer cell apoptosis via unconventional mitochondrial-independent caspase-3 activation

Mitochondria-targeting agents, known as mitocans, are emerging as potent cancer therapeutics due to pronounced metabolic and apoptotic adaptations in the mitochondria of cancer cells. ONC212, an imipridone-family compound initially identified as a ClpP agonist, is currently under investigation as a potential mitocan with demonstrated preclinical efficacy against multiple malignancies. Despite this efficacy, the molecular mechanism underlying the cell death induced by ONC212 remains unclear. This study systematically investigates the mitochondrial involvement and signaling cascades associated with ONC212-induced cell death, utilizing HeLa and A549 cancer cells. Treated cancer cells exhibited characteristic apoptotic features, such as annexin-V positivity and caspase-3 activation; however, these occurred independently of typical mitochondrial events like membrane potential loss (ΔΨm) and cytochrome c release, as well as caspase-8 activation associated with the extrinsic pathway. Additionally, ONC212 treatment increased the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, which impeded apoptosis, as the overexpression of Bcl-2-GFP and Bcl-xL-GFP significantly reduced ONC212-mediated cell death. Furthermore, combining a sub-lethal dose of the Bcl-2/Bcl-xL inhibitor Navitoclax with ONC212 markedly augmented caspase-3 activation and cell death, still without any notable ΔΨm loss or cytochrome c release. Moreover, inhibition of caspase-9 activity unexpectedly augmented, rather than attenuated, caspase-3 activation and the subsequent cell death. Collectively, our research identifies ONC212 as an atypical mitochondrial-independent, yet Bcl-2/Bcl-xL-inhibitable, caspase-3-mediated apoptotic cell death inducer, highlighting its potential for combination therapies in tumors with defective mitochondrial apoptotic signaling.

Significant enhancement of anticancer effect of iridium (III) complexes encapsulated in liposomes

In this study, the ligand EIPP (5-ethoxy-2-(1H-imidazo[4,5-f] [1,10] phenanthrolin-2-yl)phenol) and [Ir(ppy)2(EIPP)](PF6)] (5a, ppy = 2-phenylpyridine) and [Ir(piq)2(EIPP)](PF6)] (5b, piq = 1-phenylisoquinoline) were synthesized and they were entrapped into liposomes to produce 5alipo and 5blipo. 5a and 5b were characterized via HRMS, NMR, UV–vis and IR. The cytotoxicity of 5a, 5b, 5alipo and 5blipo on cancer and non-cancer cells was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT assay demonstrated that 5a and 5b did not show any significant cellular activity but their liposome-encapsulated 5alipo and 5blipo had significant toxic effects. The mechanism of 5alipo, 5blipo-inducing apoptosis was explored by studying cellular uptake, mitochondrial localization, mitochondrial membrane potential, cytochrome C, glutathione (GSH), malondialdehyde (MDA) and protein immunoblotting. The results demonstrated that 5alipo and 5blipo caused a release of cytochrome C, downregulated the expression of Bcl-2, upregulated the expression of BAX, activated caspase 3, and downregulated PARP expression. It was shown that 5alipo and 5blipo could inhibit cancer cell proliferation in G2/M phase by regulating p53 and p21 proteins. Additionally, 5alipo and 5blipo induced autophagy through an adjustment from LC3-I to LC3-II and caused ferroptosis. The in vivo antitumor activity of 5alipo was examined in detail

PPARγ regulates osteoarthritis chondrocytes apoptosis through caspase-3 dependent mitochondrial pathway

Osteoarthritis (OA) is the most prevalent form of arthritis, characterized by a complex pathogenesis. One of the key factors contributing to its development is the apoptosis of chondrocytes triggered by oxidative stress. Involvement of peroxisome proliferator-activated receptor gamma (PPARγ) has been reported in the regulation of oxidative stress. However, there remains unclear mechanisms that through which PPARγ influences the pathogenesis of OA. The present study aims to delve into the role of PPARγ in chondrocytes apoptosis induced by oxidative stress in the context of OA. Primary human chondrocytes, both relatively normal and OA, were isolated and cultured for the following study. Various assessments were performed, including measurements of cell proliferation, viability and cytotoxicity. Additionally, we examined cell apoptosis, levels of reactive oxygen species (ROS), nitric oxide (NO), mitochondrial membrane potential (MMP) and cytochrome C release. We also evaluated the expression of related genes and proteins, such as collagen type II (Col2a1), aggrecan, inducible nitric oxide synthase (iNOS), caspase-9, caspase-3 and PPARγ. Compared with relatively normal cartilage, the expression of PPARγ in OA cartilage was down-regulated. The proliferation of OA chondrocytes decreased, accompanied by an increase in the apoptosis rate. Down-regulation of PPARγ expression in OA chondrocytes coincided with an up-regulation of iNOS expression, leading to increased secretion of NO, endogenous ROS production, and decrease of MMP levels. Furthermore, we observed the release of cytochrome C, elevated caspase-9 and caspase-3 activities, and reduction of the components of extracellular matrix (ECM) Col2a1 and aggrecan. Accordingly, utilization of GW1929 (PPARγ Agonists) or Z-DEVD-FMK (caspase-3 inhibitor) can protect chondrocytes from mitochondrial-related apoptosis and alleviate the progression of OA. During the progression of OA, excessive oxidative stress in chondrocytes leads to apoptosis and ECM degradation. Activation of PPARγ can postpone OA by down-regulating caspase-3-dependent mitochondrial apoptosis pathway.

Engineering hybrid conductive electrochemically active biofilms enable efficient interfacial electron transfer and syntrophic carbon metabolism

Supply of carbon-based nanomaterials (e.g., carbon nanotubes, CNTs) to develop highly conductive electrochemically active biofilms (EABs) is a potential strategy for facilitating extracellular electron transfer (EET) in bioelectrochemical systems (BESs). Understanding of the underlying CNTs-mediated EET behaviors is helpful to further advance the practical application of BESs. Here, the cognitive influence of CNTs on bioelectrocatalytic activity and electron transfer efficiency of EABs were elucidated. CNTs can be embedded into EABs to form hybrid conductive biofilms (CNTs/EABs), achieving a high current density (7.4 ± 1.40 A m−2) and excellent coulombic recovery (46.0 ± 2.70 %) over 100 days of steady operation. The supply of CNTs can mitigate the dependence of exoelectrogens (such as Geobacter) on outer membrane cytochromes (OMCs) and conductive pili due to their down-regulated genes expression in CNTs/EABs, but it can significantly improve microbial carbon metabolism because physically high-conductive CNTs can establish rapid EET pathways, which may reduce the necessity for cells to invest metabolic energy in producing conductive pili and cytochromes that are required in the absence of CNTs. Such enhancement in electron transfer rate may be caused by the interfacial interaction between OMCs and CNTs, resulting in an order of magnitude higher than in the control (5.5 ± 1.60 s−1vs. 0.28 ± 0.04 s−1) and without compromising of mass diffusion. This study provides comprehensive insight into the role of carbon-based nanomaterials in provoking interfacial electron transfer and renewable energy recovery.

Electromagnetic Field Drives the Bioelectrocatalysis of γ-Fe2O3-Coated Shewanella putrefaciens CN32 to Boost Extracellular Electron Transfer.

The microbial hybrid system modified by magnetic nanomaterials can enhance the interfacial electron transfer and energy conversion under the stimulation of a magnetic field. However, the bioelectrocatalytic performance of a hybrid system still needs to be improved, and the mechanism of magnetic field-induced bioelectrocatalytic enhancements is still unclear. In this work, γ-Fe2O3 magnetic nanoparticles were coated on a Shewanella putrefaciens CN32 cell surface and followed by placing in an electromagnetic field. The results showed that the electromagnetic field can greatly boost the extracellular electron transfer, and the oxidation peak current of CN32@γ-Fe2O3 increased to 2.24 times under an electromagnetic field. The enhancement mechanism is mainly due to the fact that the surface modified microorganism provides an elevated contact area for the high microbial catalytic activity of the outer cell membrane's cytochrome, while the magnetic nanoparticles provide a networked interface between the cytoplasm and the outer membrane for boosting the fast multidimensional electron transport path in the magnetic field. This work sheds fresh scientific light on the rational design of magnetic-field-coupled electroactive microorganisms and the fundamentals of an optimal interfacial structure for a fast electron transfer process toward an efficient bioenergy conversion.

Vibrio natriegens as a superior host for the production of c-type cytochromes and difficult-to-express redox proteins

C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens Vmax X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in Vmax X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, Vmax X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins.

Chrysophanol induces apoptosis and ferroptosis of gastric cancer cells by targeted regulation of mTOR.

Programmed cell death (PCD) induction is a promising strategy for killing gastric cancer cells. In this study, we investigated the effects of chrysophanol on apoptosis and ferroptosis in gastric cancer cells. Chrysophanol in concentrations ranging from 0 to 100 μM were used to treat GES-1, HGC-27 and AGS cells. Cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine staining, flow cytometry, JC-1 probe insertion, dihydroethidium staining and western blotting were performed. The effects of chrysophanol on gastric cancer cells were evaluated invivo using a xenograft mouse model. Chrysophanol had no cytotoxic effects on GES-1 cells. Chrysophanol with concentrations higher than 25 μM inhibited gastric cancer cell colony formation and proliferation. Chrysophanol induces gastric cancer cell apoptosis in a dose-dependent manner, accompanied by mitochondrial membrane potential dysfunction and cytochrome c release. Additionally, chrysophanol increased the levels of reactive oxygen species, total iron, and Fe2+ in HGC-27 and AGS cells, in a dose-dependent manner. Treatment of cells with the ferroptosis inhibitor ferrostatin-1 attenuated the effects of chrysophanol on cell survival and the expression of ferroptosis markers SLC7A11 and GPX4. Screening by GEO software indicated that the mTOR signalling pathway is possibly regulated by chrysophanol. Furthermore, mTOR overexpression significantly reversed the inhibitory effects of chrysophanol on gastric cancer cells. In gastric cancer xenograft mouse models, chrysophanol treatment inhibited tumour growth and downregulated SLC7A11 and GPX4. Chrysophanol induces apoptosis and ferroptosis, making it a potential candidate for killing gastric cancer cells. The beneficial effects of chrysophanol may be attribute to the targeted regulation of mTOR.

Disrupting Development: Unraveling the Interplay of Aryl Hydrocarbon Receptor (AHR) and Wnt/β-Catenin Pathways in Kidney Development Under the Influence of Environmental Pollutants.

Understanding the intricate molecular mechanisms governing aryl hydrocarbon receptor (AHR) and Wnt/β-Catenin pathways crosstalk is of paramount importance for elucidating normal development. We investigated the repercussions of aberrant activation of these signaling pathways on kidney development. HEK-293 cells were subjected to AHR and Wnt activators and inhibitors for 3 and 24h. Subsequently, pregnant adult female BALB/c mice were administered treatments at gestation day 9 (GD-9), and embryos were analyzed at GD-18 using a combination of cellular, molecular, stereological, and histopathological techniques. Our results demonstrated a noteworthy escalation in oxidative stress and gene expression endpoints associated with apoptosis. Moreover, stereological analyses exhibited alterations in cortex, proximal tubule, and kidney tissue vessels volumes. Remarkably, co-treatment with 6-formylindolo [3,2-b] carbazole (FICZ) and cadmium (Cd) resulted in a significant reduction in glomerulus volume, while elevating the volumes of distal tubule, Henle loop, and connective tissue, compared to the control group. Histopathological investigations further confirmed structural changes in the loop of Henle and proximal tubule, alongside a decline in glomerular volume. Additionally, the expression levels of AHR and Ctnnb1 genes significantly increased in the Cd-treated group compared to the control group. Enhanced expression of apoptosis-related genes, including Bcl-x, Bax, and Caspase3, along with alterations in mitochondrial membrane potential and cytochrome C release, was observed. In contrast, Gsk3 gene expression was significantly decreased. Our findings robustly establish that chemical pollutants, such as Cd, disrupt the AHR and Wnt/β-Catenin physiological roles during developmental stages by inhibiting the metabolic degradation of FICZ.

Toxicity of superparamagnetic iron oxide nanoparticles on retinoblastoma mitochondria

Purpose Retinoblastoma (RB) is one of the most important cancers in children with a higher rate of prevalence in developing countries. Despite different approaches to the treatment of RB, it seems necessary to discover a new approach to its treatment. Today, mitochondria are recognised as an important target in the treatment of cancer. Superparamagnetic iron oxide nanoparticles (SPIONs) have been studied by researchers due to their important biological effects. Methods In this study, the effects of SPIONs on mitochondria isolated from Y79 retinoblastoma cells were investigated. Results The results showed that SPIONs were able to increase the reactive oxygen species (ROS) level and subsequently damage the mitochondrial membrane and release cytochrome c a as one of the important pro-apoptotic proteins of RB mitochondria. Furthermore, the results indicated a decrease in cell viability and an increase in caspase-3 activity in Y79 retinoblastoma cells. Conclusions These events can lead to the killing of cancerous mitochondria. Our results suggest that SPIONs can cause mitochondrial dysfunction and death in RB mitochondria.

Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation.

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from - 150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool. This article is protected by copyright. All rights reserved.

Enhancement of microbial current production by riboflavin requires the reduced heme centers in outer membrane cytochromes in Shewanella oneidensis MR-1

Enhancement of microbial current production by riboflavin requires the reduced heme centers in outer membrane cytochromes in Shewanella oneidensis MR-1

Biochar promoted microbial iron reduction in competition with methanogenesis in anaerobic digestion

Microbial Fe (III) reduction generally could outcompete methanogenesis due to its thermodynamic advantage, while the low bioavailability of Fe (III) compounds limits this process in the anaerobic digestion system, which could result in the low recovery of vivianite. Therefore, this study investigated the competition between Fe (III) reduction and methanogenesis in the presence of different biochar (pyrochar and hydrochar). The results showed that pyrochar obtained at 500 °C (P5) resulted in the highest Fe (III) reduction (80.3%) compared to the control experiment (29.1%). P5 also decreased methane production by 9.4%. Both conductivity and surface oxygen-containing functional groups contributed to the promotion of direct electron transfer for Fe (III) reduction. Genomic-centric metatranscriptomics analysis showed that P5 led to the highest enrichment of Geobacter soli A19 and induced the significant expression of out membrane cytochrome c and pilA in Geobacter soli A19, which was related to higher Fe (III) reduction.

Cytochrome gene expression shifts in Geobacter sulfurreducens to maximize energy conservation in response to changes in redox conditions

Previous studies have identified that Geobacter sulfurreducens has three different electron transfer pathways for respiration, and it switches between these pathways to adapt to the redox potential of its electron acceptor. However, only a small fraction of the electron carriers from each pathway have been identified. In this study, we combined electrochemical and gene expression data to identify electron carriers in the inner membrane, periplasm, outer membrane, and exterior of the cell that may be induced by the use of the three different electron transfer pathways. Cyclic voltammetry was performed on thin biofilms grown on anodes poised at different redox potentials, providing a quantitative assessment of the relative use of three electron-transfer pathways in each condition (catalytic midpoint potentials (EKAs) of −0.227 V [Low], −0.15 V [Medium], −0.1 V [High] vs. SHE). Transcriptomic analyses as a function of electrochemical signals or fumarate utilization showed differential induction in inner membrane (Medium: cbcL), periplasmic (Low: ppcB/ppcE, Medium: ppcA), outer membrane (Low: extA/extC, Medium: extJ/extK, Fumarate: extF/extG), and extracellular (Medium: omcZ, High/Fumarate: omcS/omcT) cytochromes, suggesting the pathway signals are associated with complex transcriptomic responses in genes across the electron transfer pathway. Our method combining electrochemical modeling and transcriptomics could be adapted to better understand electron transport in other electroactive organisms with complex metabolisms.

Mechanisms of magnetic sensing and regulating extracellular electron transfer of electroactive bacteria under magnetic fields

Electroactive bacteria can display notable plasticity in their response to magnetic field (MF), which prompted bioelectrochemical system as promising candidates for magnetic sensor applications. In this study, we explored the sensing and stimulatory effect of MF on current generation by Geobacter sulfurreducens, and elucidated the related molecular mechanism at the transcriptomic level. MF treatment significantly enhanced electricity generation and overall energy efficiency of G. sulfurreducens by 50 % and 22 %, respectively. The response of current to MFs was instantaneous and reversible. Cyclic voltammetry analysis of the anode biofilm revealed that the redox couples changed from −0.31 to −0.39 V (vs. Ag/AgCl), suggesting that MFs could alter electron transfer related components. Differential gene expression analysis further verified this hypothesis, genes associated with electron transfer were upregulated in G. sulfurreducens under MF treatment relative to the control group, specifically, genes encoding periplasmic c-type cytochromes (ppcA and ppcD), outer membrane cytochrome (omcF, omcZ, omcB), pili (pilA-C, pilM, and pilV2), and ribosome. The enhanced bacterial extracellular electron transfer process was also linked to the overexpression of the NADH dehydrogenase I subunit, the ABC transporter, transcriptional regulation, and ATP synthase. Overall, our findings shed light on the molecular mechanism underlying the effects of magnetic field stimuli on EAB and provide a theoretical basis for its further application in magnetic sensors and other biological system.

The Outer Membrane Cytochrome OmcA Is Essential for Infection of Shewanella oneidensis by a Zebrafish-Associated Bacteriophage.

The microbiota-the mixture of microorganisms in the intestinal tract of animals-plays an important role in host biology. Bacteriophages are a prominent, though often overlooked, component of the microbiota. The mechanisms that phage use to infect susceptible cells associated with animal hosts, and the broader role they could play in determining the substituents of the microbiota, are poorly understood. In this study, we isolated a zebrafish-associated bacteriophage, which we named Shewanella phage FishSpeaker. This phage infects Shewanella oneidensis strain MR-1, which cannot colonize zebrafish, but it is unable to infect Shewanella xiamenensis strain FH-1, a strain isolated from the zebrafish gut. Our data suggest that FishSpeaker uses the outer membrane decaheme cytochrome OmcA, which is an accessory component of the extracellular electron transfer (EET) pathway in S. oneidensis, as well as the flagellum to recognize and infect susceptible cells. In a zebrafish colony that lacks detectable FishSpeaker, we found that most Shewanella spp. are sensitive to infection and that some strains are resistant to infection. Our results suggest that phage could act as a selectivity filter for zebrafish-associated Shewanella and show that the EET machinery can be targeted by phage in the environment. IMPORTANCE Phage exert selective pressure on bacteria that influences and shapes the composition of microbial populations. However, there is a lack of native, experimentally tractable systems for studying how phage influence microbial population dynamics in complex communities. Here, we show that a zebrafish-associated phage requires both the outer membrane-associated extracellular electron transfer protein OmcA and the flagellum to infect Shewanella oneidensis strain MR-1. Our results suggest that the newly discovered phage-FishSpeaker-could exert selective pressure that restricts which Shewanella spp. colonize zebrafish. Moreover, the requirement of OmcA for infection by FishSpeaker suggests that the phage preferentially infects cells that are oxygen limited, a condition required for OmcA expression and an ecological feature of the zebrafish gut.

Agaritine derived from Agaricus blazei Murrill induces apoptosis via mitochondrial membrane depolarization in hematological tumor cell lines.

Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50μM AGT for 24h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

Synthesis, characterization, anticancer efficacy evaluation of ruthenium(II) and iridium(III) polypyridyl complexes toward A549 cells.

A new ligand DFIP (2-(dibenzo[b,d]furan-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its two complexes iridium(III) [Ir(ppy)2(DFIP)](PF6) (ppy = 2-phenylpyridine, Ir1) and ruthenium(II) [Ru(bpy)2(DFIP)](PF6)2 (bpy = 2,2'-bipyridine, Ru1) were synthesized and characterized. The anticancer effects of the two complexes on A549, BEL-7402, HepG2, SGC-7901, HCT116 and normal LO2 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complex Ir1 shows high cytotoxic activity on A549, BEL-7402, SGC-7901 and HepG2, Ru1 exhibits moderate anticancer activity toward A549, BEL-7402 and SGC-7901 cells. The IC50 values of Ir1 and Ru1 toward A549 are 7.2 ± 0.1 and 22.6 ± 1.4μM, respectively. The localization of complexes Ir1 and Ru1 in the mitochondrial, intracellular accumulation of reactive oxygen species (ROS) levels, and the changes of mitochondrial membrane potential (MMP) and cytochrome c (cyto-c) were investigated. Apoptosis and cell cycle were detected by flow cytometry. Immunogenic cell death (ICD) was used to detect the effects of Ir1 and Ru1 on the A549 using a confocal laser scanning microscope. The expression of apoptosis-related proteins was detected by western blotting. Ir1 and Ru1 can increase the intracellular ROS levels and release cyto-c, reduce the MMP, leading to the apoptosis of A549 cells and blocking the A549 cells at the G0/G1 phase. Additionally, the complexes caused a decrease of the expression of polyADP-ribose polymerase (PARP), caspase 3, Bcl-2 (B-cell lymphoma-2), PI3K (phosphoinositide-3 kinase) and upregulated the expression of Bax. All these findings indicated that the complexes exert anticancer efficacy to induce cell death through immunogenic cell death, apoptosis, and autophagy.