Motif-pattern dependence of biomolecular phase separation driven by specific interactions.

Eukaryotic cells partition a wide variety of important materials and processes into biomolecular condensates-phase-separated droplets that lack a membrane. In addition to nonspecific electrostatic or hydrophobic interactions, phase separation also depends on specific binding motifs that link together constituent molecules. Nevertheless, few rules have been established for how these ubiquitous specific, saturating, motif-motif interactions drive phase separation. By integrating Monte Carlo simulations of lattice-polymers with mean-field theory, we show that the sequence of heterotypic binding motifs strongly affects a polymer's ability to phase separate, influencing both phase boundaries and condensate properties (e.g. viscosity and polymer diffusion). We find that sequences with large blocks of single motifs typically form more inter-polymer bonds, which promotes phase separation. Notably, the sequence of binding motifs influences phase separation primarily by determining the conformational entropy of self-bonding by single polymers. This contrasts with systems where the molecular architecture primarily affects the energy of the dense phase, providing a new entropy-based mechanism for the biological control of phase separation.

Assessment of stunned and viable myocardium using manganese-enhanced MRI.

OBJECTIVE: In a proof-of-concept study, to quantify myocardial viability in patients with acute myocardial infarction using manganese-enhanced MRI (MEMRI), a measure of intracellular calcium handling. METHODS: Healthy volunteers (n=20) and patients with ST-elevation myocardial infarction (n=20) underwent late gadolinium enhancement (LGE) using gadobutrol and MEMRI using manganese dipyridoxyl diphosphate. Patients were scanned ≤7 days after reperfusion and rescanned after 3 months. Differential manganese uptake was described using a two-compartment model. RESULTS: After manganese administration, healthy control and remote non-infarcted myocardium showed a sustained 25% reduction in T1 values (mean reductions, 288±34 and 281±12 ms). Infarcted myocardium demonstrated less T1 shortening than healthy control or remote myocardium (1157±74 vs 859±36 and 835±28 ms; both p<0.0001) with intermediate T1 values (1007±31 ms) in peri-infarct regions. Compared with LGE, MEMRI was more sensitive in detecting dysfunctional myocardium (dysfunctional fraction 40.5±11.9 vs 34.9%±13.9%; p=0.02) and tracked more closely with abnormal wall motion (r2=0.72 vs 0.55; p<0.0001). Kinetic modelling showed reduced myocardial manganese influx between remote, peri-infarct and infarct regions, enabling absolute discrimination of infarcted myocardium. After 3 months, manganese uptake increased in peri-infarct regions (16.5±3.5 vs 22.8±3.5 mL/100 g/min, p<0.0001), but not the remote (23.3±2.8 vs 23.0±3.2 mL/100 g/min, p=0.8) or infarcted (11.5±3.7 vs 14.0±1.2 mL/100 g/min, p>0.1) myocardium. CONCLUSIONS: Through visualisation of intracellular calcium handling, MEMRI accurately differentiates infarcted, stunned and viable myocardium, and correlates with myocardial dysfunction better than LGE. MEMRI holds major promise in directly assessing myocardial viability, function and calcium handling across a range of cardiac diseases. TRIAL REGISTRATION NUMBERS: NCT03607669; EudraCT number 2016-003782-25.

Doing 'business as usual' comes with a cost: evaluating energy cost of maintaining plant intracellular K+ homeostasis under saline conditions.

Salinization of agricultural lands is a major threat to agriculture. Many different factors affect and determine plant salt tolerance. Nonetheless, there is a consensus on the relevance of maintaining an optimal cytosolic potassium : sodium ion (K+  : Na+ ) ratio for salinity tolerance in plants. This ratio depends on the operation of plasma membrane and tonoplast transporters. In the present review we focus on some aspects related to the energetic cost of maintaining that K+  : Na+ ratio. One of the factors that affect the cost of the first step of K+ acquisition - root K+ uptake through High Affinity K+ transporter and Arabidopsis K+ transport system 1 transport systems - is the value of the plasma membrane potential of root cells, a parameter that may differ amongst plant species. In addition to its role in nutrition, cytosolic K+ also is important for signalling, and K+ efflux through gated outward-rectifying K+ and nonselective cation channels can be regarded as a switch to redirect energy towards defence reactions. In maintaining cytosolic K+ , the great buffer capacity of the vacuole should be considered. The possible role of high-affinity K+ transporters (HKT)2s in mediating K+ uptake under saline conditions and the importance of cycling of K+ throughout the plant also are discussed.

Combining in vitro digestion model with cell culture model: Assessment of encapsulation and delivery of curcumin in milled starch particle stabilized Pickering emulsions.

To investigate the encapsulation and oral delivery efficiency of milled starch particles stabilized Pickering emulsions for lipophilic bioactive compounds, in vitro digestion model coupled with Caco-2 cells models were used. Physicochemical and biological properties of curcumin encapsulated Pickering emulsions were analyzed regarding to emulsion structure, curcumin retention, in vitro digestion, in vitro anti-proliferate ability and cellular uptake. Milled starch particles stabilized Pickering emulsion system was able to protect curcumin against harsh gastric conditions. Around 80% of the encapsulated curcumin was retained after 2 h of simulated gastric digestion. By being encapsulated in Pickering emulsion, the bioaccessibility of curcumin was increased from 11% for curcumin in bulk oil phase to 28% under simulated intestinal digestion process. The resulting curcumin-loaded micelle phase from digested emulsion exhibited significant anti-cancer ability and enhanced cellular uptake. This research provides an exploratory study on the possible future application of milled starch particles stabilized Pickering emulsions as nutraceutical delivery vehicles in the creation of novel functional foods.

Doppler fluctuation spectroscopy of intracellular dynamics in living tissue.

Intracellular dynamics in living tissue are dominated by active transport driven by bioenergetic processes far from thermal equilibrium. Intracellular constituents typically execute persistent walks. In the limit of long mean free paths, the persistent walks are ballistic, exhibiting a "Doppler edge" in light scattering fluctuation spectra. At shorter transport lengths, the fluctuations are described by lifetime-broadened Doppler spectra. Dynamic light scattering from transport in the ballistic, diffusive, or the crossover regimes is derived analytically, including the derivation of autocorrelation functions through a driven damped harmonic oscillator analog for light scattering from persistent walks. The theory is validated through Monte Carlo simulations. Experimental evidence for the Doppler edge in three-dimensional (3D) living tissue is obtained using biodynamic imaging based on low-coherence interferometry and digital holography.

Novel Glycosylated Naphthalimide-Based Activatable Fluorescent Probe: A Tool for the Assessment of Hexosaminidase Activity and Intracellular Hexosaminidase Imaging.

The development of effective detection methods for hexosaminidase is of great importance for the rapid screening of potential inhibitors in vitro and for the early diagnosis of related diseases ex vivo. In this study, the activatable fluorescent probes that are based on naphthalimide decorated with ethylene glycol units were synthesized using N-acetyl-ß-d-glucosaminide as a hexosaminidase-responsive group. When exposed to this enzyme, the glucoside-linked naphthalimide moiety of 1c can be cleaved quickly with significant changes in both color (from colorless to yellow) and fluorescence (from blue to green). Probe 1c shows better water-solubility and fluorescence properties than common substrate 4-methylumbelliferyl N-acetyl-ß-d-glucosaminide. Furthermore, the response mechanism of 1c to hexosaminidase was evaluated using HPLC analysis and TD-DFT calculations. Molecular docking was performed to investigate the interaction mode. In addition, 1c has successfully achieved the straightforward rapid discovery of effective hexosaminidase inhibitors. Fluorescence imaging experiments indicate that 1c has good cell safety and can be employed as a useful tool for detecting intracellular hexosaminidase activity.

Tissue-Specific Subcellular Localization Prediction Using Multi-Label Markov Random Fields.

The understanding of subcellular localization (SCL) of proteins and proteome variation in the different tissues and organs of the human body are two crucial aspects for increasing our knowledge of the dynamic rules of proteins, the cell biology, and the mechanism of diseases. Although there have been tremendous contributions to these two fields independently, the lack of knowledge of the variation of spatial distribution of proteins in the different tissues still exists. Here, we proposed an approach that allows predicting protein SCL on tissue specificity through the use of tissue-specific functional associations and physical protein-protein interactions (PPIs). We applied our previously developed Bayesian collective Markov random fields (BCMRFs) on tissue-specific protein-protein interaction network (PPI network) for nine types of tissues focusing on eight high-level SCL. The evaluated results demonstrate the strength of our approach in predicting tissue-specific SCL. We identified 1,314 proteins that their SCL were previously proven cell line dependent. We predicted 549 novel tissue-specific localized candidate proteins while some of them were validated via text-mining.

Low Variance Couplings for Stochastic Models of Intracellular Processes with Time-Dependent Rate Functions.

A number of coupling strategies are presented for stochastically modeled biochemical processes with time-dependent parameters. In particular, the stacked coupling is introduced and is shown via a number of examples to provide an exceptionally low variance between the generated paths. This coupling will be useful in the numerical computation of parametric sensitivities and the fast estimation of expectations via multilevel Monte Carlo methods. We provide the requisite estimators in both cases.

Pyruvate dehydrogenase complex plays a central role in brown adipocyte energy expenditure and fuel utilization during short-term beta-adrenergic activation.

Activation of brown adipose tissue (BAT) contributes to total body energy expenditure through energy dissipation as heat. Activated BAT increases the clearance of lipids and glucose from the circulation, but how BAT accommodates large influx of multiple substrates is not well defined. The purpose of this work was to assess the metabolic fluxes in brown adipocytes during ß3-adrenergic receptor (ß3-AR) activation.T37i murine preadipocytes were differentiated into brown adipocytes and we used Seahorse respirometry employing a set of specific substrate inhibitors in the presence or absence of ß3-AR agonist CL316,243. The main substrate used by these brown adipocytes were fatty acids, which were oxidized equally during activation as well as during resting condition. [U-13C]-glucose tracer-based metabolomics revealed that the flux through the TCA cycle was enhanced and regulated by pyruvate dehydrogenase (PDH) activity. Based on 13C-tracer incorporation in lipids, it appeared that most glucose was oxidized via TCA cycle activity, while some was utilized for glycerol-3-phosphate synthesis to replenish the triglyceride pool. Collectively, we show that while fatty acids are the main substrates for oxidation, glucose is also oxidized to meet the increased energy demand during short term ß3-AR activation. PDH plays an important role in directing glucose carbons towards oxidation.

The COOLER Code: A Novel Analytical Approach to Calculate Subcellular Energy Deposition by Internal Electron Emitters.

COmputation Of Local Electron Release (COOLER), a software program has been designed for dosimetry assessment at the cellular/subcellular scale, with a given distribution of administered low-energy electron-emitting radionuclides in cellular compartments, which remains a critical step in risk/benefit analysis for advancements in internal radiotherapy. The software is intended to overcome the main limitations of the medical internal radiation dose (MIRD) formalism for calculations of cellular S-values (i.e., dose to a target region in the cell per decay in a given source region), namely, the use of the continuous slowing down approximation (CSDA) and the assumption of a spherical cell geometry. To this aim, we developed an analytical approach, entrusted to a MATLAB-based program, using as input simulated data for electron spatial energy deposition directly derived from full Monte Carlo track structure calculations with PARTRAC. Results from PARTRAC calculations on electron range, stopping power and residual energy versus traveled distance curves are presented and, when useful for implementation in COOLER, analytical fit functions are given. Example configurations for cells in different culture conditions (V79 cells in suspension or adherent culture) with realistic geometrical parameters are implemented for use in the tool. Finally, cellular S-value predictions by the newly developed code are presented for different cellular geometries and activity distributions (uniform activity in the nucleus, in the entire cell or on the cell surface), validated against full Monte Carlo calculations with PARTRAC, and compared to MIRD standards, as well as results based on different track structure calculations (Geant4-DNA). The largest discrepancies between COOLER and MIRD predictions were generally found for electrons between 25 and 30 keV, where the magnitude of disagreement in S-values can vary from 50 to 100%, depending on the activity distribution. In calculations for activity distribution on the cell surface, MIRD predictions appeared to fail the most. The proposed method is suitable for Auger-cascade electrons, but can be extended to any energy of interest and to beta spectra; as an example, the 3H case is also discussed. COOLER is intended to be accessible to everyone (preclinical and clinical researchers included), and may provide important information for the selection of radionuclides, the interpretation of radiobiological or preclinical results, and the general establishment of doses in any scenario, e.g., with cultured cells in the laboratory or with therapeutic or diagnostic applications. The software will be made available for download from the DTU-Nutech website: http://www.nutech.dtu.dk/ .

Architecture of the human interactome defines protein communities and disease networks.

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.

Markov modeling reveals novel intracellular modulation of the human TREK-2 selectivity filter.

Two-pore domain potassium (K2P) channel ion conductance is regulated by diverse stimuli that directly or indirectly gate the channel selectivity filter (SF). Recent crystal structures for the TREK-2 member of the K2P family reveal distinct "up" and "down" states assumed during activation via mechanical stretch. We performed 195 µs of all-atom, unbiased molecular dynamics simulations of the TREK-2 channel to probe how membrane stretch regulates the SF gate. Markov modeling reveals a novel "pinched" SF configuration that stretch activation rapidly destabilizes. Free-energy barrier heights calculated for critical steps in the conduction pathway indicate that this pinched state impairs ion conduction. Our simulations predict that this low-conductance state is accessed exclusively in the compressed, "down" conformation in which the intracellular helix arrangement allosterically pinches the SF. By explicitly relating structure to function, we contribute a critical piece of understanding to the evolving K2P puzzle.

Innovative Technologies in Nanomedicines: From Passive Targeting to Active Targeting/From Controlled Pharmacokinetics to Controlled Intracellular Pharmacokinetics.

Nanomedicines promise to extend drug therapy from small molecular compounds to proteins/nucleic acids/genes. Multifunctional envelope-type nanodevices (MENDs) have been developed for delivering such molecules to the site of action. The YSK-MEND contains new types of pH-responsive cationic lipids to efficiently deliver siRNA to hepatocytes via receptor-mediated endocytosis and use in treating hepatitis C and B in model mice. The RGD ligand is introduced to target tumor endothelial cells (TEC) and RGD-MEND is able to send siRNA to TEC to regulate the function of tumor microenvironments. The MITO-Porter is also developed to target mitochondria via membrane fusion. Antisense oligo RNA in the MITO-Porter permits the knock down of mitochondrial function. Finally, the ssPalms is designed based on a new concept of pH-dependent protonation in endosomes and cleavage of SS bonds in the reducing conditions in cytosol. These new technologies promise to stimulate the use of Nanomedicines in the future.

Two for the Price of One: PAMAM-Dendrimers with Mixed Phosphoryl Choline and Oligomeric Poly(Caprolactone) Surfaces.

The application of dendrimers for biological and medical purposes is highly dependent on the type of surface group in relation to cytotoxicity. Since amine terminated PAMAM dendrimers have been shown to have toxic properties and thereby limited applications in the medical field, the discovery of a new nontoxic surface coating is of great interest. In the present work, amine terminated DAB-PAMAM dendrimers from generation zero to four have been coated with statistical surface functionalization giving a dendrimer surface consisting of an approximately 1:1 mixture of zwitterionic phosphoryl choline hexanamide and 6-((6-hydroxyhexanoyl)oxy)hexanamide. The cytotoxic properties of generation two to four were tested on three different human cancer cell lines, SKBR3 human breast cancer cells, HeLa human cervical cancer cells, and Hep G2 human hepatocellular liver carcinoma cells and compared to the toxicity of amine terminated PAMAM dendrimers. In addition to lower cytotoxicity than observed for amine terminated dendrimers, the coated dendrimers showed minor cytotoxicity against all three human cell lines, negligible influence on ROS generation and mitochondrial membrane potential. These observations support the conclusion that the analyzed group of phosphorylcholine dendrimers may be suitable for medical applications.

A fast and specific method to screen for intracellular amyloid inhibitors using bacterial model systems.

The aggregation of a large variety of amyloidogenic proteins is linked to the onset of devastating human disorders. Therefore, there is an urgent need for effective molecules able to modulate the aggregative properties of these polypeptides in their natural environment, in order to prevent, delay or halt the progression of such diseases. On the one hand, the complexity and cost of animal models make them inefficient at early stages of drug discovery, where large chemical libraries are usually screened. On the other hand, in vitro aggregation assays in aqueous solutions hardly reproduce (patho)physiological conditions. In this context, because the formation of insoluble aggregates in bacteria shares mechanistic and functional properties with amyloid self-assembly in higher organisms, they have emerged as a promising system to model aggregation in the cell. Here we show that bacteria provide a powerful and cost-effective system to screen for amyloid inhibitors using fluorescence spectroscopy and flow cytometry, thanks to the ability of the novel red fluorescent ProteoStat dye to detect specifically intracellular amyloid-like aggregates. We validated the approach using the Alzheimer's linked Aß40 and Aß42 peptides and tacrine- and huprine-based aggregation inhibitors. Overall, the present method bears the potential to replace classical in vitro anti-aggregation assays.

A Lattice-Boltzmann scheme for the simulation of diffusion in intracellular crowded systems.

BACKGROUND: The intracellular environment is a complex and crowded medium where the diffusion of proteins, metabolites and other molecules can be decreased. One of the most popular methodologies for the simulation of diffusion in crowding systems is the Monte Carlo algorithm (MC) which tracks the movement of each particle. This can, however, be computationally expensive for a system comprising a large number of molecules. On the other hand, the Lattice Boltzmann Method (LBM) tracks the movement of collections of molecules, which represents significant savings in computational time. Nevertheless in the classical manifestation of such scheme the crowding conditions are neglected. METHODS: In this paper we use Scaled Particle Theory (SPT) to approximate the probability to find free space for the displacement of hard-disk molecules and in this way to incorporate the crowding effect to the LBM. This new methodology which couples SPT and LBM is validated using a kinetic Monte Carlo (kMC) algorithm, which is used here as our "computational experiment". RESULTS: The results indicate that LBM over-predicts the diffusion in 2D crowded systems, while the proposed coupled SPT-LBM predicts the same behaviour as the kinetic Monte Carlo (kMC) algorithm but with a significantly reduced computational effort. Despite the fact that small deviations between the two methods were observed, in part due to the mesoscopic and microscopic nature of each method, respectively, the agreement was satisfactory both from a qualitative and a quantitative point of view. CONCLUSIONS: A crowding-adaptation to LBM has been developed using SPT, allowing fast simulations of diffusion-systems of different size hard-disk molecules in two-dimensional space. This methodology takes into account crowding conditions; not only the space fraction occupied by the crowder molecules but also the influence of the size of the crowder which can affect the displacement of molecules across the lattice system.

Assessment of biological effectiveness of boron neutron capture therapy in primary and metastatic melanoma cell lines.

PURPOSE: In order to optimize the effectiveness of Boron Neutron Capture Therapy (BNCT), Relative Biological Effectiveness (RBE) and Compound Biological Effectiveness (CBE) were determined in two human melanoma cell lines, M8 and Mel-J cells, using the amino acid p-boronophenylalanine (BPA) as boron carrier. MATERIALS AND METHODS: The effects of BNCT on the primary amelanotic cell line M8 and on the metastatic pigmented melanoma cell line Mel-J were studied using colony formation assay. The RBE values were determined using both a gamma ray source, and the neutron beam from the Nuclear Reactor of the National Atomic Energy Commission (RA-3). For the determination of the RBE, cells were irradiated with increasing doses of both sources, between 1 and 8 Gy; and for the determination of CBE factors, the cells were pre-incubated with BPA before irradiation. Afterwards, the cell surviving fraction (SF) was determined for each treatment. RESULTS: Marked differences were observed between both cell lines. Mel-J cells were more radioresistant than the M8 cell line. The clonogenic assays showed that for a SF of 1%, the RBE values were 1.3 for M8 cells and 1.5 for Mel-J cells. Similarly, the CBE values for a 1% SF were 2.1 for M8 and 3 for Mel-J cell lines. For the endpoint of 0.1% of SF the RBE values obtained were 1.2 for M8 and 1.4 for Mel-J cells. Finally, CBE values calculated for a 0.1% were 2 and 2.6 for M8 and Mel-J cell lines respectively. In order to estimate the uptake of the non-radioactive isotope Boron 10 ((10)B), a neutron induced autoradiographic technique was performed showing discrepancies in (10)B uptake between both cell lines. CONCLUSIONS: These obtained in vitro results are the first effectiveness factors determined for human melanoma at the RA-3 nuclear reactor and show that BNCT dosimetry planning for patients could be successfully performed using these new factors.

In vitro toxicity assessment of silver nanoparticles in the presence of phenolic compounds--preventive agents against the harmful effect?

The increasing commercial use of silver nanoparticles (Ag-NPs) will inevitably lead to elevated silver exposure and thus to potential human health complications. In this study the acute toxicity of Ag-NPs <20 nm alone and upon co-administration with food matrix component phenolic compounds (PCs) on the cell-based models of the gastrointestinal tract was investigated. An improved co-culture model of Caco-2 and RajiB cells was applied for more precise in vitro simulation of the gastrointestinal tract. The involvement of two major factors contributing to the toxicity of Ag-NPs, i.e. the release of Ag(+) and the induction of oxidative stress, was investigated. Ag-NPs were cytotoxic for Caco-2 cells with an EC50 of ca. 40 µg/ml. Ag-NPs led to oxidative stress starting from ca. 45 µg/ml. The epithelial barrier integrity disruption by Ag-NPs on Caco-2 cell mono- and co-cultures was established by decreased transepithelial electrical resistances and increased passages of Lucifer Yellow, a paracellular marker. Immunofluorescence staining demonstrated that Ag-NPs affect occludin and zonula occludens 1 distributions, suggesting the opening of tight junctions. Ag(+), corresponding to the release from Ag-NPs, demonstrated a partial contribution in the toxic parameters, induced by Ag-NPs. Two PCs, quercetin and kaempferol, partially protected the Caco-2 cells from Ag-NP-induced toxicity and maintained the epithelial barrier integrity, disrupted by NPs. No protective effect was observed for resveratrol. The protective effect could be beneficial and decrease the potential toxicity of ingested Ag-NPs. However, the precise mechanisms of barrier-integrity-destabilising action of Ag-NPs/Ag(+) and protective effect of PCs still require further elucidation.

Dynamic neuroanatomy at subcellular resolution in the zebrafish.

Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

Synthesis and characterization of a novel o-tolyltelluronic trimethyltin ester and its cytotoxic assessment in vitro.

A new arenetelluronic triorganotin ester, namely (Me3Sn)4[o-Me-PhTe(µ-O)(OH)O2)]2 (1) has been prepared by the reaction of o-tolyltelluronic acid and Me3SnCl in the presence of potassium hydroxide. The complex was fully characterized by elemental analysis, FT-IR, NMR ((1)H, (13)C, (119)Sn) spectroscopy and X-ray crystallography. Structure analysis revealed that the complex crystallized as Sn4Te2 units and a 1D linear chain was formed by intermolecular C-HO interactions. Cytotoxic assessments showed that the complex can induce apoptotic cell death via accumulation of ROS, collapse of the MMP and activating caspase-3. The results indicated that ROS is crucial to the cytotoxicity induced by the complex.