Many OX PHOS and replication factor mRNAs target mitochondria through specific binding to the organelle surface, independent of cotranslational protein import.

A large number of nucleus-encoded messenger RNAs (mRNAs) encoding proteins involved in oxidative phosphorylation have been found to be associated with mitochondria in vivo, indicating organelle-specific mRNA targeting. However, the identification of mitochondrion-bound mRNA (Mtb-RNA) has traditionally relied on cumbersome isolations of polysomes from a large number of input cells and is therefore biased in favour of mRNAs associated through nascent targeting peptides emerging from the polysome during cotranslational import of their encoded proteins, and tends to ignore sequence-directed mRNA targeting. We have, therefore, sought to identify and quantify Mtb-RNAs rapidly in small numbers of cells, independently of their polysomal status. We isolated Mtb-RNAs from tissue-cultured cells under different conditions and assayed them by endpoint or real-time polymerase chain reaction (RT-PCR). We observed that (i) different Mtb-RNAs are differentially affected by cycloheximide-induced polysome arrest, indicating possible artifacts of the use of this translation elongation inhibitor; (ii) several Mtb-RNAs have direct affinity for the mitochondrial surface in vitro, indicating the possibility of targeting through mRNA recognition by surface-bound RNA-binding proteins (RBP); and (iii) mRNA-mitochondrion interactions are stabilized by formaldehyde crosslinking. Our results reveal the importance of sequence-directed targeting of mRNAs to mitochondria.

OXPHOS deficiency induces mitochondrial DNA synthesis through non-canonical AMPK-dependent mRNA compartmentalization.

Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is dynamically regulated by mitochondrial biogenesis and mitophagy processes. However, the conditions regulating mtDNA replication, an essential component of biogenesis, are unknown. We observed that short-term (2 h) treatment of rat myoblasts with oligomycin, a specific inhibitor of the mitochondrial F1F0 ATP synthase, resulted in stimulation of mtDNA synthesis from the OH replication origin. This effect was abrogated by Compound C, an antagonist of the AMP-dependent protein kinase (AMPK), a universal intracellular energy sensor, and in AMPK-knockdown cells, indicating that mtDNA replication is regulated by AMPK under oxidative phosphorylation (OXPHOS)- deficient conditions. Using antibody decoration, enzymatically active AMPK, phosphorylated at T172 of the α1 subunit, was found to be located on the mitochondrial surface. Furthermore, oligomycin induced the compartmentalization of several mRNAs encoding OXPHOS components and mtDNA replication factors to mitochondria. Compartmentalization of mRNAs was inhibited by Compound C. We infer that AMPK is locally activated by inhibition of the F1F0 ATP synthase to stimulate association of mtDNA replication factor mRNAs, leading to stimulation of mtDNA synthesis. The findings have implications for the clonal expansion of OXPHOS-deficient mtDNA mutant mitochondria in human patients, with clinical consequences.

Nanonization of a chemically synthesized flavone HMDF (3-hydroxy-3',4'-methylenedioxyflavone) by entrapping within calcium phosphate nanoparticles and exploring its antioxidant role on neural cellsin vitroand zebrafishin vivo.

The chemical synthesis of 3-hydroxy-3',4'-methylenedioxyflavone (HMDF) was reported to generate a modified flavone of potent antioxidant activity with significant neuropharmacological properties. In this study, HMDF was nanonized by entrapping within calcium phosphate nanoparticles (CPNPs). HMDF-CPNPs were of (i) size 25 nm, (ii) zeta potential (-) [22 ± 3] mV and (iii) entrapment efficiency 67%. HMDF-CPNPs, but not HMDF alone, inhibited thein vitroactivity of acetylcholinesterase enzymes to break down the major neurotransmitter compound acetylcholine. Moreover, nanonized HMDF had more antioxidant activity than bulk HMDF, as observed from its ability to protect mouse neural (N2A) cells from oxidative damage caused by H2O2exposure at the levels of cell viability, intracellular reactive oxygen species, mitochondrial membrane potential, cell cycle stages, nuclear integrity and neural connectivity. Anin vivostudy on zebrafish larvae (Denio rerio) also demonstrated that H2O2-mediated larval death was checked by HMDF-CPNP treatment. These results, therefore, suggest that HMDF-CPNPs may be developed as a potential antioxidant, particularly as a neuroprotectant.

A simple robust method of synthesis of copper-silver core-shell nano-particle: evaluation of its structural and chemical properties with anticancer potency.

A simple method of synthesis of a stable bimetallic copper-silver nano-particle (CuAg-NP) was developed by successive reduction of Cu(NO3)2 and AgNO3, using hydrazine hydrate as the reducing agent and gelatin and poly-vinyl pyrrolidone (PVP) as the capping agents. The round-shaped particles were of a core-shell structure with a core of Cu0 atoms surrounded by a shell of Ag0 atoms. The size and the mol. wt. of the NPs were (100 ± 10) nm and (820 ± 157) Kd, respectively; the particles were crystalline in nature and 90% of the precursors Cu(NO3)2 and AgNO3 were converted to the NPs. The particles were more toxic to cancer cells than normal cells; the dose of the NPs (4-5 µg ml-1), that killed about 75% of the different human cancer cell lines viz, HepG2 (liver cancer), A549 (lung cancer) and AGS (stomach cancer), killed only about 22.5% of the normal cell lines viz, WRL68 (liver) and WI38 (lung). Therefore, the NP may be developed as a potent anticancer drug in future. The more detailed study on the cytotoxicity of the CuAg-NP on the HepG2 cell line revealed that the particles caused cell cycle arrest in a G2/M phase, depolarization of mitochondrial membrane potential, translocation of phosphatidylserine residues from inner to outer leaflets of cell membrane and DNA degradation; these phenomena confirmed that the NP-induced cell death was apoptotic in nature.

Calcium phosphate-quercetin nanocomposite (CPQN): A multi-functional nanoparticle having pH indicating, highly fluorescent and anti-oxidant properties.

Calcium phosphate quercetin nanocomposite (CPQN) i.e., quercetin entrapped in calcium phosphate nanoparticle was synthesized by a precipitation method at 80°C, taking ammonium hydrogen phosphate, calcium nitrate and quercetin as precursors and sodium citrate as stabilizer. The nanocomposite suspension had different color at different pH values, a property that could render the nanoparticle a pH indicator. Besides color, the particles also had different size, shape, stability and quercetin content with change of pH. In addition, the CPQN was highly fluorescent having two sharp emission peaks at 460 and 497nm, when excited at 370nm; by this property it behaved as an effective fluorophore to label biological cell. Moreover, the nanocomposite had potential anti-oxidant property, for which mortality of mouse neuroblastoma cell N2A, by H2O2-induced oxidative stress, was found to be lowered by the pre-treatment of the cells with CPQN.

Green synthesized cerium oxide nanoparticle: A prospective drug against oxidative harm.

Cerium oxide nanoparticle (CeONP) of size 2-3nm was synthesized by a new, simple and green method at ambient temperature, using cerium nitrate as prime precursor and Aloe vera leaf extract as stabilizing agent. Of the two oxidation states (+3) and (+4) of cerium, it was dominantly present in (+3) state in CeONP and cyclic conversion of Ce(III)O→Ce(IV)O→Ce(III)O by reaction with H2O2 implied uninterrupted antioxidant property of CeONP. Moreover, the higher oxygen defect in the crystal lattice produced particles with higher antioxidant activity. CeONP was found to neutralize the deleterious effects of H2O2 viz., cell death, generation of intracellular reactive oxygen species and loss of connectivity in mouse neural cells. Therefore, CeONP might have potential use in future as an anti-oxidant drug.

Tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP): Rejuvenation of an obsolete antibiotic to further action.

BACKGROUND: Increasing resistance in bacteria towards antibiotics has made it imperative to research on their revitalization to combat infectious diseases. This study dealt with synthesis of a nano-form of the antibiotic tetracycline, its characterization and potency of killing different multi-drug resistant diarrhea-causing bacteria. METHODS: Nano-formulation was done by loading tetracycline within biocompatible calcium phosphate nanoparticle. The synthesized tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP) was characterized by the techniques like TEM, DLS, EDS, FTIR, spectrofluorimetry and dialysis. Bactericidal activity of nano-particulate tetracycline was investigated by agar plating, spectrophotometry, phase contrast-fluorescence-atomic force microscopy and flow cytometry techniques. RESULTS: The Tet-CPNPs were 8±5nm in size and nearly spherical in shape, efficiency of tetracycline loading in CPNP was about 20% and the release of antibiotic from Tet-CPNPs was sustainable during 7days. Minimum inhibitory concentration (MIC) of Tet-CPNP on multiple antibiotic (including tetracycline) resistant bacteria like Escherichia coli, Salmonella kentuckey and Shigella flexneri was in the range of 20-40µg/ml, whereas MIC of free tetracycline was in the range of 150-180µg/ml. NP-mediated cell filamentation and cell membrane disintegration caused cell killing. Moreover, death of Shigella-infected Zebra fish larvae was stalled by Tet-CPNP treatment. CPNP itself had no toxic effect on bacteria as well as on Zebra fish. CONCLUSION: Our nano-formulation of tetracycline might reclaim a nearly obsolete antibiotic to further potential function. GENERAL SIGNIFICANCE: Such a study on revival of an old, cheap, broad-spectrum antibiotic to further action is highly beneficial to developing countries with limited health care budgets.

In vitro holdase activity of E. coli small heat-shock proteins IbpA, IbpB and IbpAB: a biophysical study with some unconventional techniques.

E. coli small heat shock proteins IbpA and IbpB (inclusion body binding proteins A and B) are known to act as holding chaperones on denaturing, aggregate-prone proteins. But, there is no clear understanding about which of the IbpA and IbpB has more holdase activity and how the holdase activity of one was influenced by the presence of the other. This study was conducted to resolve the questions, using some uncommon physical techniques like dynamic light scattering, micro-viscometry and atomic force microscopy in addition to the common techniques of spectrophotometry and spectrofluorimetry. The holdase activity was investigated on the heat-denatured L-lactate dehydrogenase (LDH) of rabbit muscle. LDH was found to be deactivated completely without any aggregation at 52°C and with transient aggregation at 60°C; molecular dynamics simulation also revealed that at 52°C, denaturation occurred only at the active site of LDH. When LDH was allowed to be deactivated in the presence of IbpA, IbpB or (IbpA + IbpB), partial inhibition of i) denaturation at 52°C and ii) aggregation at 60°C were observed. The results further demonstrated that the holdase activity of IbpB was higher than that of IbpA and their combined effect was higher than their individual one.

Calcium phosphate nanoparticles: a study of their synthesis, characterization and mode of interaction with salmon testis DNA.

Calcium phosphate nanoparticles (CPNPs) are presently emerging as a second generation vector for efficient delivery and stabilization of nucleic acids inside cells, although the detailed mode of interaction between CPNPs and DNA is still obscure. This study discloses some features of the interaction. For this study, we synthesized CPNPs by a modified co-precipitation method and characterized the particles by different techniques such as dynamic light scattering, X-ray diffraction, electron dispersive spectroscopy, Fourier transform infra-red spectroscopy, differential thermal and thermo-gravimetric analysis, and atomic force, scanning and transmission electron microscopy. The characterization studies showed that the nanoparticles were spherical in shape, about 45 nm in size and were composed of the hydroxyapatite form of calcium phosphate; almost 90% of the starting materials were converted to nanoparticles (NPs). The different aspects of the interaction between CPNPs and salmon testis DNA were investigated using techniques such as UV-Vis spectrophotometry, circular dichroism, Fourier transform infra-red spectrometry, thermal denaturation, microviscometry, agarose gel electrophoresis, cyclic voltammetry and atomic force microscopy. The results revealed that CPNPs interacted with DNA with ~1 : 3.3 stoichiometry with a binding constant of the order of 10(4) M(-1) through groove-interacting mode and a single nanoparticle covered about 6.2 base pairs of the DNA chain. Moreover, the binding interaction was spontaneous, cooperative, exothermic and enthalpy-driven and some electrostatic nature of the binding was also evident; however, the non-polyelectrolyte contribution was dominant. The binding interaction finally caused an increase in the melting temperature of DNA from 70.8 °C to 75 °C and alteration of its secondary structure from the naturally occurring B-form to C-form.