Rim aperture of yeast autophagic membranes balances cargo inclusion with vesicle maturation.

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.

Biological and chemical properties of 2-in-1 calcium-chelating and antibacterial root canal irrigants.

OBJECTIVES: To evaluate the capacity of canal wall smear layer removal, precipitation caused by irrigant interaction, antibacterial activity and cytotoxicity of three 2-in-1 root canal irrigating solutions. METHODS: Forty single-rooted teeth were mechanically instrumented and irrigated with QMix, SmearOFF, Irritrol or 0.9% saline. Each tooth was evaluated for smear layer removal using scanning electron microscopy. Precipitation after interaction of the irrigating solutions with sodium hypochlorite (NaOCl) was evaluated with 1H nuclear magnetic resonance and mass spectroscopy. Confocal laser scanning microscopy was used to evaluate the antimicrobial activity of the irrigants against Enterococcus faecalis biofilms. Neutral red and clonogenic assays were performed on Chinese hamster V79 cells to evaluate the short-term and long-term cytotoxicity of the irrigants. RESULTS: There was no significant difference between QMix and SmearOFF in eliminating smear layers from the coronal-third and middle-third of the canal spaces. In the apical-third, SmearOFF removed smear layers effectively. Irritrol incompletely removed smear layers from all the canal-thirds. When mixed with NaOCl, precipitation was evident only with Irritrol. QMix demonstrated a higher E. faecalis cell death percentage and a smaller biovolume. SmearOFF exhibited a larger decrease in biovolume compared with Irritrol, although Irritrol had a higher death percentage. Irritrol was more cytotoxic than the other irrigants on a short-term interval. In terms of long-term cytotoxicity, both Irritrol and QMix were cytotoxic. CONCLUSION: QMix and SmearOFF performed better in smear layer removal and antimicrobial activity. QMix and Irritrol were cytotoxic when compared to SmearOFF. Irritrol was associated with precipitation after interacting with NaOCl. CLINICAL SIGNIFICANCE: Evaluation of the smear layer removal capability, antibacterial activity and cytotoxicity of 2-in-1 root canal irrigants is necessary to ensure that they are safe to use during root canal treatment.

Anisotropic long-range spin transport in canted antiferromagnetic orthoferrite YFeO3.

In antiferromagnets, the efficient transport of spin-waves has until now only been observed in the insulating antiferromagnet hematite, where circularly (or a superposition of pairs of linearly) polarized spin-waves diffuse over long distances. Here, we report long-distance spin-transport in the antiferromagnetic orthoferrite YFeO3, where a different transport mechanism is enabled by the combined presence of the Dzyaloshinskii-Moriya interaction and externally applied fields. The magnon decay length is shown to exceed hundreds of nanometers, in line with resonance measurements that highlight the low magnetic damping. We observe a strong anisotropy in the magnon decay lengths that we can attribute to the role of the magnon group velocity in the transport of spin-waves in antiferromagnets. This unique mode of transport identified in YFeO3 opens up the possibility of a large and technologically relevant class of materials, i.e., canted antiferromagnets, for long-distance spin transport.

Mangiferin, a naturally occurring polyphenol, mitigates oxidative stress induced premature senescence in human dermal fibroblast cells.

Chronic oxidative stress has been associated with several human ailments including the condition of aging. Extensive studies have shown the causal relationship between oxidative stress, aging, and cellular senescence. In this regard, forestalling or preventing senescence could delay the aging process as well as act as an intervention against premature aging. Hence, in the present study, we investigated the anti-senescence potential of Mangiferin (MGN) against Hydrogen peroxide (H2O2) induced premature senescence using human dermal fibroblast cells. Early passage human dermal fibroblasts cells were exposed to H2O2 (10 µM) for 15 days. In order to assess the anti-senescence property of MGN, cells were preconditioned with MGN (10 µM / 50 µM; 2 h) followed by addition of H2O2 (10 µM). H2O2 mediated induction of premature senescence was accompanied by elevated ROS, lowering of mitochondrial mass and membrane potential, changes in ATP content along with G0/G1 arrest and SA-ß-gal expression. While, conditioning the cells with MGN lowered oxidative burden, stabilized mitochondrial membrane potential / mass and protected the cells against cell cycle arrest, ultimately rendering protection against premature senescence. The present findings showed that MGN might act as a potential cytoprotective nutraceutical that can prolong the onset of chronic oxidative stress mediated premature senescence.

Berberine, a natural alkaloid sensitizes human hepatocarcinoma to ionizing radiation by blocking autophagy and cell cycle arrest resulting in senescence.

OBJECTIVE: To study the radiosensitizing potential of Berberine and the underlying mechanism in human hepatocarcinoma (HepG2) cells. METHODS: HepG2 cells were challenged with X-rays in combination with Berberine treatment and several in vitro assays were performed. Alteration in cell viability was determined by MTT assay. Changes in intracellular ROS levels, mitochondrial membrane potential/mass, intracellular acidic vesicular organelles as well as cell cycle arrest and apoptotic cell death were analysed by flow cytometry. Induction of autophagy was assessed by staining the cells with Monodansylcadaverine/Lysotracker red dyes and immunoblotting for LC3I/II and p62 proteins. Phase-contrast/fluorescence microscopy was employed to study mitotic catastrophe and senescence. Cellular senescence was confirmed by immunoblotting for p21 levels and ELISA for Interleukin-6. KEY FINDINGS: X-rays + Berberine had a synergistic effect in reducing cell proliferation accompanied by a robust G2/M arrest. Berberine-mediated radiosensitization was associated with elevated levels of LC3II and p62 suggesting blocked autophagy that was followed by mitotic catastrophe and senescence. Treatment of cells with X-rays + Berberine resulted in increased oxidative stress, hyperpolarized mitochondria with increased mitochondrial mass and reduced ATP levels. CONCLUSIONS: The study expands the understanding of the pharmacological properties of Berberine and its applicability as a radiosensitizer towards treating liver cancer.

Stimulation of cytoprotective autophagy and components of mitochondrial biogenesis / proteostasis in response to ionizing radiation as a credible pro-survival strategy.

The present study illustrates mitochondria-mediated impact of ionizing radiation which is paralleled by activation of several pro-adaptive responses in normal human dermal fibroblast cells. Irradiation of cells with X-rays (5 Gy) led to an increase in fragmentation and mitochondrial mass. Distinct temporal changes in cytosolic and mitochondrial reactive oxygen species (ROS) were noted in response to radiation, which was associated with depletion in mitochondrial membrane potential followed by decrease in ATP levels. Long Amplicon-Polymerase Chain Reaction (LA-PCR) analysis showed time-dependent increase in mitochondrial DNA damage that preceded mitochondrial ROS generation. Irradiation of cells led to an initial G2/M arrest at 8 h that persisted till 16 h, with subsequent block at G0/G1 measured at 48 and 72 h time points. Interestingly, cells activated autophagy as a countermeasure against radiation-mediated cellular insults and aided in removal of damaged mitochondria. Blocking autophagy using 3-methyladenine led to cell death via activation of enhanced ROS, PARP-1 and caspase 3 cleavage. Upregulation of mitochondrial biogenesis factors Nrf1/PGC-1α, following irradiation was observed. Irradiated cells exhibited an increase in the phosphorylation of GCN2, PERK and eIF2α that might be responsible for the up-regulation of ATF4 and CHOP thereby regulating autophagy and components of integrated stress response. Apart from this, we measured accumulation of mitochondrial chaperones (HSP60/HSP10) and ATF5 which is a major molecule involved in mitochondrial stress. Taken together, mitochondria are one of the major cytoplasmic targets for ionizing radiation and possibly act as an early indicator of cellular insult. The findings also show that stressed mitochondria might influence endoplasmic reticulum (ER)-related signalling leading to the activation of adaptive mechanisms like cytoprotective autophagy, and molecules responsible for mitochondrial biogenesis and protein quality control in order to replenish mitochondrial pool and maintain cellular homeostasis.

Neuroprotective role of naringenin against methylmercury induced cognitive impairment and mitochondrial damage in a mouse model.

Human exposure to organomercurials like methylmercury (MeHg) may occur by consumption of contaminated seafood, affecting various vital organs especially, brain contributing to neuro disorders. The citrus flavanone, naringenin (NAR) has shown strong antioxidant and anti-inflammatory effects and therefore may exert cytoprotective effect against xenobiotic agents. Herein, we investigated the neuroprotective role of NAR against MeHg induced functional changes in mitochondria, neuronal cell death and cognitive impairment in a mouse model. A neurotoxic dose of MeHg (4 mg/kg.b.wt.) was administered orally to mice for 15 days. This resulted in the reduction of GSH and GST, an increase in mitochondrial DNA damage and memory impairment. On the contrary, NAR pre-treatment (100 mg/kg.b.wt.), helped in lowering the oxidative burden which in turn maintained mitochondrial function and prevented induced neuronal cell death, ultimately improving the cognitive impairment. As MeHg intoxication occurs chronically, consumption of the dietary components rich in NAR may have its positive human health impact, ultimately improving the quality of life.

Switching of multi-state magnetic structures via domain wall propagation triggered by spin-orbit torques.

Spin-orbit torques emerge as a promising method for manipulating magnetic configurations of spintronic devices. Here, we show that these torques can induce a magnetization reversal via domain wall propagation which may open new ways in developing novel spintronic devices and in particular in realizing high-density multi-level magnetic memory. Our devices are bi-layer heterostructures of Ni0.8Fe0.2 on top of ß-Ta patterned in the form of two or three crossing ellipses which exhibit in the crossing area shape-induced biaxial and triaxial magnetic anisotropy, respectively. We demonstrate field-free switching between discrete remanent magnetic states of the structures by spin-orbit torques induced by flowing electrical current through one of the ellipses. We note switchings induced by the coupling between the ellipses where current flowing in one ellipse triggers a reversal in a neighboring ellipse which propagates from the center outwards. Numerical tools successfully simulate the observed coupling-induced switching using experimentally extracted parameters.

Magnetization switching of multi-state magnetic structures with current-induced torques.

Spintronic devices often require the ability to locally change the magnetic configuration of ferromagnetic structures on a sub-micron scale. A promising route for achieving this goal is the use of heavy metal/ferromagnetic heterostructures where current flowing through the heavy metal layer generates field-like and anti-damping like torques on the magnetic layer. Commonly, such torques are used to switch magnets with a uniaxial anisotropy between two uniformly magnetized states. Here, we use such torques to switch magnetization in Ta/Ni0.80Fe0.20 heterostructures with uniaxial and biaxial anisotropy, where in the latter the magnetization is non-uniform. The anisotropies are induced by shape and the magnetic state is monitored using the planar Hall effect. As structures with several easy axes induced by shape can be part of a magnetic memory element, the results pave the way for multi-level magnetic memory with spin-orbit torque switching.

Kondo effect with tunable spin-orbit interaction in LaTiO3/CeTiO3/SrTiO3 heterostructure.

We have fabricated epitaxial films of CeTiO3 (CTO) on (0 0 1) oriented SrTiO3 (STO) substrates, which exhibit highly insulating and diamagnetic properties. X-ray photoelectron spectroscopy was used to establish the 3+ valence state of the Ce and Ti ions. Furthermore, we have also fabricated δ (CTO) doped LaTiO3 (LTO)/SrTiO3 thin films which exhibit variety of interesting properties including Kondo effect and spin-orbit interaction (SOI) at low temperatures. The SOI shows a non-monotonic behaviour as the thickness of the CTO layer is increased and is reflected in the value of characteristic SOI field ([Formula: see text]) obtained from weak anti-localization fitting. The maximum value of [Formula: see text] is 1.00 T for δ layer thickness of 6 u.c. This non-monotonic behaviour of SOI is attributed to the strong screening of the confining potential at the interface. The screening effect is enhanced by the CTO layer thickness and the dielectric constant of STO which increases at low temperatures. Due to the strong screening, electrons confined at the interface are spread deeper into the STO bulk where it starts to populate the Ti [Formula: see text] subbands; consequently the Fermi level crosses over from [Formula: see text] to the [Formula: see text] subbands. At the crossover region of [Formula: see text] where there is orbital mixing, SOI goes through a maximum.

Naringin abates adverse effects of cadmium-mediated hepatotoxicity: An experimental study using HepG2 cells.

This study investigated the protective potential of Naringin (NIN) against cadmium chloride (CdCl2 ) mediated hepatotoxicity using human hepatocellular carcinoma (HepG2) cells. An optimal concentration of NIN (5 µM) was potent enough to confer cytoprotection against CdCl2 (50 µM) as was observed by MTT assay. Preconditioning with NIN maintained redox homeostasis, mitochondrial membrane potential, and reduced apoptosis as marked by decrease in the percentage sub-G0 /G1 and Annexin V-FITC/propidium iodide positive cells (apoptotic). NIN pretreatment maintained the levels of protein thiol along with endogenous activities of Superoxide dismutase, Glutathione S-transferase, and Catalase and lowered lipid peroxidation. Decreased Bax/Bcl2 ratio along with reduced Caspase 3 cleavage and Cytochrome c release indicated that NIN conditioning blocked mitochondrial-mediated apoptosis. Increased Nrf2 and metallothionein (MT) acted as adaptive response in the presence of cadmium. Thus, the protective mechanism of NIN is attributed to its antioxidant potential which aids in redox homeostasis and prevents CdCl2 mediated cytotoxicity.

Efficient T3P® mediated synthesis, differential cytotoxicity and apoptosis induction by indolo-triazolo-thiadiazoles in human breast adenocarcinoma cells.

The limited efficacy of marketed anticancer agents demands the design of novel target-specific hybrid molecules incorporating multiple bioactive pharmacores to combat cancer. In the present study, a one-pot simple and efficient T3P® mediated procedure for the preparation of twelve new 3-(substituted- [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazolo)-1H-indoles with short reaction times, easy workup procedure, good yields, and purity of products is described. Cytotoxicity assay (MTT), flow-cytometric univariate cell cycle analysis, Annexin V-FITC staining and DNA fragmentation for cell death mechanism suggested that compound 3d with chloro-substituted phenyl ring induced enhanced cytotoxicity by an apoptotic pathway with high differential toxicity to breast adenocarcinoma cells (MCF-7) when compared with normal human dermal fibroblast cells. Additionally, the interaction between the BH3 domain of anti-apoptotic proteins Bcl-2 and Bcl-xL with the pharmacophore 3d was examined by molecular docking simulations to assess its potential to induce apoptosis. The docking solutions were proposed to explain the observed selectivity of 3d to Bcl-xL protein. From the present findings, the lead compound, 3d exhibited better anticancer activity when related to the other synthesized molecules with specific action on MCF-7 cells and hence can be considered as a plausible candidate chemo-therapeutic agent, although this warrants further experimentation.

Harmonization of Mangiferin on methylmercury engendered mitochondrial dysfunction.

Mangiferin (MGN), a C-glucosylxanthone abundantly found in mango plants, was studied for its potential to ameliorate methylmercury (MeHg) induced mitochondrial damage in HepG2 (human hepatocarcinoma) cell line. Cell viability experiments performed using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) showed protective property of MGN in annulling MeHg-induced cytotoxicity. Conditioning the cells with optimal dose of MGN (50 µM) lowered MeHg-induced oxidative stress, calcium influx/efflux, depletion of mitochondrial trans-membrane potential and prevented mitochondrial fission as observed by decrease in Mitotracker red fluorescence, expression of pDRP1 (serine 616), and DRP1 levels. MGN pre-treated cells demonstrated elevation in the activities of glutathione (GSH), Glutathione-S-transferase (GST), Glutathione peroxidase (GPx), Glutathione reductase (GR), reduced levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) and mitochondrial electron transport chain (ETC) enzyme complexes. In addition, the anti-apoptotic effect of MGN was clearly indicated by the reduction in MeHg-induced apoptotic cells analyzed by flowcytometric analysis after Annexin V-FITC/propidium iodide staining. In conclusion, the present work demonstrates the ability of a dietary polyphenol, MGN to ameliorate MeHg-mediated mitochondrial dysfunction in human hepatic cells in vitro. This hepatoprotective potential may be attributed predominantly to the free radical scavenging/antioxidant property of MGN, by facilitating the balancing of cellular Ca2+ ions, maintenance of redox homeostasis and intracellular antioxidant activities, ultimately preserving the mitochondrial function and cell viability after MeHg intoxication. As MeHg intoxication occurs over a period of time, continuous consumption of such dietary compounds may prove to be very useful in promoting human health. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 630-644, 2017.

Hydroxytyrosol, a dietary phenolic compound forestalls the toxic effects of methylmercury-induced toxicity in IMR-32 human neuroblastoma cells.

This study demonstrates the protective potential of hydroxytyrosol (HT), an olive oil phenol, against methylmercury (MeHg)-induced neurotoxicity using IMR-32 human neuroblastoma cell line. HT inhibited MeHg-induced cytotoxicity and genotoxicity as confirmed by MTT, micronucleus, and comet assays. Cells preconditioned with HT showed reduction of MeHg-induced cellular oxidative stress along with the maintenance of glutathione, superoxide dismutase, glutathione-S-tranferase, and catalase. Fluorescence microscopy and DNA ladder assays indicated the inhibitory effect of HT against MeHg-induced apoptosis, which was further established by Western blotting. An effective concentration of 5 µM HT caused downregulation of p53, bax, cytochrome c, and caspase 3 and upregulation of prosurvival proteins including nuclear factor erythroid 2-related factor 2 (Nrf2) and metallothionein. This work indicates the cytoprotective potential of HT against MeHg-induced toxicity primarily by the lowering of oxidative stress, which may be endorsed to its antigenotoxic and antiapoptotic potential, in addition to its free radical scavenging ability. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1264-1275, 2016.

Some new indole-coumarin hybrids; Synthesis, anticancer and Bcl-2 docking studies.

Hybrid molecules have attracted attention for their improved biological activity, selectivity and lesser side effects profile, distinct from their individual components. In the quest for novel anticancer drug entities, three series of indole-coumarin hybrids - 3-(1-benzyl-1H-indol-2-yl)-2H-chromen-2-ones, 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehydes and 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acids were synthesized. All the synthesized compounds were characterized by spectral techniques like IR, (1)H NMR, (13)C NMR, mass spectrometry and elemental analysis. In silico docking studies of synthesized molecules with apoptosis related gene Bcl-2 that is recognized to play an important role in tumerogenesis were carried out. Dose-dependent cytotoxic effect of the compounds in human breast adenocarcinoma (MCF-7) and normal cell lines were assessed using MTT assay and compared with that of the standard marketed drug, Vincristine. Compound 4c had a highly lipophilic bromine substituent capable of forming halogen bond and was identified as a potent molecule both in docking as well as cytotoxicity studies. Flow cytometric cell cycle analysis of 4c exhibited apoptotic mode of cell death due to cell cycle arrest in G2/M phase. Structure activity relationship of these hybrid molecules was also studied to determine the effect of steric and electronic properties of the substituents on cell viability.

Radio-modifying potential of Saraca indica against ionizing radiation: an in vitro study using Chinese hamster lung fibroblast (V79) cells.

This study demonstrated the radioprotective efficacy of extracts prepared from stem bark of Saraca indica (SI) against X-rays induced cellular damage, which was evaluated by a battery of cytotoxicity, genotoxicity, apoptotic, and biochemical assays using Chinese hamster fibroblast (V79) cells. Cell viability and surviving fraction were increased significantly when V79 cells were preconditioned with optimal concentration of hydroalcoholic extract (HE; 50 µg/mL) of SI for 2 h prior exposure to X-rays. Radiation induced cellular damage was correlated with a significant elevation in intracellular ROS and increased mitochondrial depolarization and loss of intracellular antioxidant enzymes. However, cells preconditioned with 50 µg/mL of HE reversed this effect. Pretreatment of HE resulted in inhibition of radiation induced GSH, GST, SOD, catalase levels, and lipid peroxidation to that of radiation-alone treated group. Also, a significant decrease in radiation induced DNA damage, apoptotic and necrotic cell death was observed in case of cells preconditioned with HE. Supporting to this HPLC analysis indicated the presence of ellagic acid as one of the major phytochemical present in HE. Thus, the maintenance of cellular redox status by pretreatment with HE, conferred protection of cellular DNA, oxidative stress by neutralizing free radicals generated by the cellular irradiation and resulted in increased in cell survival may be attributed to the presence of ellagic acid indicating SI's radioprotective potential.

Thymol, a monoterpene phenolic derivative of cymene, abrogates mercury-induced oxidative stress resultant cytotoxicity and genotoxicity in hepatocarcinoma cells.

Thymol (TOH) was investigated for its ability to protect against mercuric chloride (HgCl2 )-induced cytotoxicity and genotoxicity using human hepatocarcinoma (HepG2) cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay confirmed the efficacy of TOH pretreatment in attenuating HgCl2 -induced cytotoxicity. Pretreatment with TOH inhibited HgCl2 -induced genotoxicity, depolarization of mitochondrial membrane, oxidative stress, and mitochondrial superoxide levels. Interestingly, TOH (100 µM) alone elevated the intracellular basal glutathione S-transferase (GST) levels and TOH pretreatment abrogated the decrease in glutathione, GST, superoxide dismutase, and catalase levels even after HgCl2 intoxication. Furthermore, TOH was also capable of inhibiting HgCl2 -induced apoptotic as well as necrotic cell death analyzed by flowcytometric analysis of cells dual stained with Annexin-FITC/propidium iodide. The present findings clearly indicate the cytoprotective potential of TOH against HgCl2 -induced toxicity, which may be attributed to its free radical scavenging ability which facilitated in reducing oxidative stress and mitochondrial damage thereby inhibiting cell death.

Mangiferin attenuates methylmercury induced cytotoxicity against IMR-32, human neuroblastoma cells by the inhibition of oxidative stress and free radical scavenging potential.

Mangiferin (MGN), a C-glucosylxanthone was investigated for its ability to protect against methylmercury (MeHg) induced neurotoxicity by employing IMR-32 (human neuroblastoma) cell line. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and clonogenic cell survival assays confirmed the efficacy of MGN supplementation in attenuating MeHg-induced cytotoxicity. Pre-treatment with MGN significantly (p<0.01) inhibited MeHg-induced DNA damage (micronuclei, olive tail moment and % tail DNA) thereby demonstrating MGN's antigenotoxic potential. Also, pre-treatment with MGN significantly reduced MeHg-induced oxidative stress, intra-cellular Ca(2+) influx and inhibited depolarization of mitochondrial membrane. MGN pre-treated cells demonstrated a significant (p<0.05) increase in the GSH and GST levels followed by a significant (p<0.05) decrease in malondialdehyde (MDA) formation. In addition, inhibition of MeHg induced apoptotic cell death by MGN was demonstrated by microscopic, Annexin-V FITC and DNA fragmentation assays and further confirmed by western blot analysis. The present findings indicated the protective effect of MGN against MeHg induced toxicity, which may be attributed to its anti-genotoxic, anti-apoptotic and anti-lipid peroxidative potential plausibly because of its free radical scavenging ability, which reduced the oxidative stress and in turn facilitated the down-regulation of mitochondrial apoptotic signalling pathways.