Spark Plasma Sintered HA-Fe3O4-Based Multifunctional Magnetic Biocomposites

Although HA is highly biocompatible, one of the major disadvantages of HA include the lack of antibacterial property. In an earlier study, we demonstrated the potential role of magnetic field stimulation on bactericidal property in vitro. Following this, it was hypothesized that antibacterial property can be realized if bacteria are grown on magnetic biocomposites in vitro. In addressing this issue, this study demonstrates the development of HA-Fe3O4-based magnetic substrate with multifunctional properties. For this purpose, HA-xFe(3)O(4) (x: 10, 20 and 40wt%) powder compositions were sintered using uniquely designed spark plasma sintering conditions (three stage sintering with final holding temperature of 1050 degrees C for 5min). A saturation magnetization of 24emu/g is measured with HA-40%Fe3O4. Importantly, all the HA-Fe3O4 composites demonstrated bactericidal property by rupturing the membrane of Escherichia coli bacteria, while supporting cell growth of metabolically active human fetal osteoblast cells over 8d culture. A systematic decrease in bacterial viability with Fe3O4 addition is consistent with a commensurate increase in reactive oxygen species (ROS).

Introduction of a catalytic triad increases the glutathione peroxidase-like activity of diaryl diselenides

Reactive oxygen species (ROS)-mediated diseased states are of major concern in modern day life. Under oxidative stress conditions, the cellular antioxidants deplete, leading to several biological disorders. Small molecule mimics of different antioxidant enzymes are found to be useful in supplementing the biological systems to detoxify ROS. In this study, we have synthesized a series of amine or amide-based diselenides containing an additional amino group as glutathione peroxidase (GPx) mimetics. These diselenides act as a catalytic triad model of the native GPx featuring two basic amino groups near the selenium centre. A comparison of the catalytic activities reveals that the additional amino group increases the activity significantly in the presence of aromatic thiols. Deprotonation of thiol by an additional amine either stabilizes the selenolate intermediate or facilitates the nucleophilic attack of thiol in other intermediates. The Se-77 NMR experiments and DFT calculations show that the amino group does not have any significant effect on the catalytic intermediates. Although the amino moiety increases the nucleophilicity of the thiol, it does not prevent the thiol exchange reactions that take place in the selenenyl sulfide intermediates.

Differential viability response of prokaryotes and eukaryotes to high strength pulsed magnetic stimuli

The present study examines the efficacy of a high strength pulsed magnetic field (PMF) towards bacterial inactivation in vitro, without compromising eukaryotic cell viability. The differential response of prokaryotes Staphylococcus aureus (MESA), Staphylococcus epidermidis, and Escherichia coli], and eukaryotes C2C12 mouse myoblasts and human mesenchymal stem cells, hMSCs] upon exposure to varying PMF stimuli (1-4 T, 30 pulses, 40 ms pulse duration) is investigated. Among the prokaryotes, similar to 60% and similar to 70% reduction was recorded in the survival of staphylococcal species and E. coli, respectively at 4 T PMF as evaluated by colony forming unit (CPU) analysis and flow cytometry. A 2-5 fold increase in intracellular ROS (reactive oxygen species) levels suggests oxidative stress as the key mediator in PMF induced bacterial death/injury. The 4 T PMF treated staphylococci also exhibited longer doubling times. Both TEM and fluorescence microscopy revealed compromised membranes of PMF exposed bacteria. Under similar PMF exposure conditions, no immediate cytotoxicity was recorded in C2C12 mouse myoblasts and hMSCs, which can be attributed to the robust resistance towards oxidative stress. The ion interference of iron containing bacterial proteins is invoked to analytically explain the PMF induced ROS accumulation in prokaryotes. Overall, this study establishes the potential of PMF as a bactericidal method without affecting eukaryotic viability. This non-invasive stimulation protocol coupled with antimicrobial agents can be integrated as a potential methodology for the localized treatment of prosthetic infections. (C) 2015 Elsevier B.V. All rights reserved.

Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation

Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.

A rapid method to assess reactive oxygen species in yeast using H2DCF-DA

A protocol to efficiently assess Reactive Oxygen Species (ROS) levels in yeast cells using H2DCF-DA is described here. This method employs lithium acetate to permeate the cell wall, and thus, augments the release of the fluorescent product, dichlorofluorescein from the cells. This protocol obviates the need for both physical and enzymatic lysis methods that are arduous and time consuming. This method is simple, less time consuming and reproducible, especially while dealing with a large sample size. The lithium acetate method gave significantly reproducible and linear results (P < 0.0001), as compared with direct measurement (P = 0.0005), sonication (P = 0.1466) and bead beating (P = 0.0028).

Evaluation of developmental toxicity and apoptotic induction of the aqueous extract of Millettia pachycarpa using zebrafish as model organism

Extensive and indiscriminate use of synthetic compounds and natural compounds obtained from plant sources have resulted in serious threats to the aquatic ecosystem and human health. Aqueous extract of the root of the plant, Milletia pachycarpa Benth, is currently used for killing fish in the state of Manipur, India. Moreover, this plant is also used as traditional medicine in this region. Although it is widely used in traditional medicine, there is limited information available regarding the adverse effects and mechanism underlying its toxicity. This study examined the effects of exposure to aqueous extract of M. pachycarpa (AEMP) on early embryonic development of zebrafish embryos and mechanisms underlying toxicity. Zebrafish embryos treated with different concentrations of the AEMP produced embryonic lethality and developmental defects. The 96-hr-LC50 of AEMP was found to be 4.276 mu g/mL. Further, multiple developmental abnormalities such as pericardial edema, yolk sac edema, spinal curvature, swim bladder deflation, decreased heart rate, and delayed hatching were also observed in a dose-dependent manner. Zebrafish embryo showing moderate-to-severe developmental defects following AEMP exposure cannot swim properly. Further, this study examined oxidative stress and apoptosis in embryos exposed to AEMP. Enhanced production of ROS and apoptosis was found in brain, trunk, and tail of zebrafish embryos treated with AEMP. Data suggest that oxidative stress and apoptosis are associated with AEMP-induced embryonic lethality and developmental toxicity in zebrafish embryos.

Visible light-induced cytotoxicity of a dinuclear iron(III) complex of curcumin with low-micromolar IC50 value in cancer cells

Photoactive metal complexes have emerged as potential candidates in the photodynamic therapy (PDT) of cancer. We present here the synthesis, characterization and visible light-triggered anticancer activity of two novel mixed-ligand oxo-bridged iron(III) complexes, viz., {Fe(L)(acac)}(2)(mu-O)](ClO4)(2) (1) and {Fe (L)(cur)}(2)(mu-O)](ClO4)(2) (2) where L is bis-(2-pyridylmethyl)-benzylamine, acac is acetylacetonate and cur is the monoanion of curcumin (bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione). The crystal structure of complex 1 (as PF6 salt, 1a) shows distorted octahedral geometry of each iron(III) centre formed by the FeN3O3 core. The 1: 2 electrolytic complexes are stable in solution and retain their oxo-bridged identity in aqueous medium. Complex 2 has a strong absorption band in the visible region and shows promising photocytotoxicity in HeLa and MCF-7 cancer cells in visible light giving respective IC50 values of 3.1 +/- 0.4 lM and 4.9 +/- 0.5 lM while remains non-toxic in the dark (IC50 > 50 lM). The control complex 1 is inactive both in the light and dark. Complex 2 accumulates in cytoplasm of HeLa and MCF-7 cells as evidenced from fluorescence microscopy and triggers apoptotic cell death via light-assisted generation of reactive oxygen species (ROS). Taken together, complex 2 with its promising photocytotoxicity but negligible dark toxicity in cancer cells has significant photochemotherapeutic potential for applications in PDT. (C) 2015 Elsevier B.V. All rights reserved.

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein, Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.

Mitochondria-Targeting Iron(III) Catecholates for Photoactivated Anticancer Activity under Red Light

Iron(III) catecholates Fe(R-bpa)(R-dopa)Cl] (1, 2) with a triphenylphosphonium (TPP) moiety, where R-bpa is 2-(TPP-N,N-bis((pyridin-2-yl)methyl)ethanamine) chloride (TPPbpa) and R-dopa is 4-{2-(anthracen-9-yl)methylamino]ethyl}benzene-1,2-diol (andopa, 1) or 4-{2-(pyren-1-yl)-methylamino]ethyl}benzene-1,2-diol (pydopa, 2), were synthesized and their photocytotoxicity studied. Complexes 3 and 4 with phenyl-N,N-bis(pyridin-2-yl)methyl]methanamine (phbpa) were used as controls. The catecholate complexes showed an absorption band near 720 nm. The 5e(-) paramagnetic complexes showed a Fe-III/Fe-II irreversible response near -0.45 V and a quasi-reversible catechol/semiquinone couple near 0.5 V versus saturated calomel electrode (SCE) in DMF/0.1 M tetrabutylammonium perchlorate. They showed photocytotoxicity in red/visible light in HeLa, HaCaT, MCF-7, and A549 cells. Complexes 1 and 2 displayed mitochondrial localization, reactive oxygen species (ROS) generation under red light, and apoptotic cell death. Control complexes 3 and 4 exhibited uniform distribution throughout the cell. The complexes showed DNA photocleavage under red light (785 nm), forming hydroxyl radicals as the ROS.

Mitochondrial selectivity and remarkable photocytotoxicity of a ferrocenyl neodymium(III) complex of terpyridine and curcumin in cancer cells

A series of four novel neodymium(III) complexes of the formulation Nd(R-tpy)(O-O)(NO3)(2)] (1-4), where R-tpy is 4'-phenyl-2,2': 6', 2''-terpyridine (Ph-tpy; 1, 2) and 4'-ferrocenyl-2,2': 6', 2''-terpyridine (Fc-tpy; 3, 4); O-O is the conjugate base of acetylacetone (Hacac; 1, 3) or curcumin (Hcurc; 2, 4), are synthesized and characterized. The single crystal structure of 1 shows that the complex is a discrete mononuclear species with the Nd(III) centre in a nine coordinate environment provided by a set of O6N3 donor atoms. Complexes 1 and 3 having the simple acac ligand are prepared as control compounds. Complex 4, possessing an appended ferrocenyl (Fc) and the curcumin moiety, is remarkably photocytotoxic to HeLa and MCF-7 cancer cells in visible light giving respective IC50 values of 0.7 mu M and 2.1 mu M while being significantly less toxic to MCF-10A normal cells (IC50 = 34 mu M) and in the dark (IC50 > 50 mu M). The phenyl appended complex 2, lacking a ferrocenyl moiety, is significantly less toxic to both the cell lines when compared with 4. Complexes 1 and 3, lacking the photoactive curcumin moiety, do not show any apparent toxicity both in light and in the dark. The cell death is apoptotic in nature and is mediated by the light-induced formation of reactive oxygen species (ROS). Fluorescence imaging experiment with HeLa cells reveals mitochondrial accumulation of complex 4 within 4 h of incubation. The complexes bind to calf thymus (ct) DNA with moderate affinity giving K-b values in the range of 10(4)-10(5) M-1. The curcumin complexes 2 and 4 cleave plasmid supercoiled DNA to its nicked circular form in visible light via O-1(2) and (OH)-O-center dot pathways. The presence of the ferrocenyl moiety is likely to be responsible for the enhanced cellular uptake and photocytotoxicity of complex 4. Thus, the mitochondria targeting complex 4, being remarkably cytotoxic in light but non-toxic in the dark and to normal cells, is a potential candidate for photochemotherapeutic applications.

Unimpeded permeation of water through biocidal graphene oxide sheets anchored on to 3D porous polyolefinic membranes

3D porous membranes were developed by etching one of the phases (here PEO, polyethylene oxide) from melt-mixed PE/PEO binary blends. Herein, we have systematically discussed the development of these membranes using X-ray micro-computed tomography. The 3D tomograms of the extruded strands and hot-pressed samples revealed a clear picture as to how the morphology develops and coarsens over a function of time during post-processing operations like compression molding. The coarsening of PE/PEO blends was traced using X-ray micro-computed tomography and scanning electron microscopy (SEM) of annealed blends at different times. It is now understood from X-ray micro-computed tomography that by the addition of a compatibilizer (here lightly maleated PE), a stable morphology can be visualized in 3D. In order to anchor biocidal graphene oxide sheets onto these 3D porous membranes, the PE membranes were chemically modified with acid/ethylene diamine treatment to anchor the GO sheets which were further confirmed by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and surface Raman mapping. The transport properties through the membrane clearly reveal unimpeded permeation of water which suggests that anchoring GO on to the membranes does not clog the pores. Antibacterial studies through the direct contact of bacteria with GO anchored PE membranes resulted in 99% of bacterial inactivation. The possible bacterial inactivation through physical disruption of the bacterial cell wall and/or reactive oxygen species (ROS) is discussed herein. Thus this study opens new avenues in designing polyolefin based antibacterial 3D porous membranes for water purification.