MnO2 nanoparticles as a minimalist multimode vaccine adjuvant/delivery system to regulate antigen presenting cells for tumor immunotherapy.

In the field of tumor immunotherapy, tumor vaccines have unique advantages including fewer side effects, tumor-specificity and immune memory, and hence attract more and more attention. In the development of tumor vaccines, a critical challenge lies in the exploitation of appropriate vaccine adjuvants/delivery systems that need to meet multiple requirements to achieve potent cellular immunity while simultaneously requiring single composition to simplify the clinical translation process. Among numerous materials, only manganese dioxide (MnO2) nanoparticles with rare physicochemical properties seem to meet the demanding criteria of simplicity and multifunctionality. However, the potential of MnO2 nanoparticles as vaccine adjuvants/delivery systems has not been well exploited, despite their widespread applications in the biomedical field. In this study, the mechanism and efficacy of single MnO2 nanoparticles as a minimalist multi-mode tumor vaccine adjuvant/delivery system were fully investigated by using a model antigen ovalbumin (OVA) to construct tumor vaccines OVA/MnO2. The obtained results show that MnO2 nanoparticles act as an ideal delivery system by multiple modes to deliver the antigen to the cytoplasm of dendritic cells to induce cellular immune response. Moreover, MnO2 nanoparticles also act as a superior adjuvant depot to sustainably release Mn2+ to enhance the immune response through a STING pathway in dendritic cells. Both the delivery function and the adjuvant effect of MnO2 nanoparticles contribute to improved cellular immunity and anti-tumor efficacy of tumor vaccines OVA/MnO2. From the results, MnO2 nanoparticles are found to be a promising minimalist multi-mode vaccine adjuvant/delivery system for the development of practical tumor vaccines.

Biodegradable flower-like manganese for synergistic photothermal and photodynamic therapy applications.

A flower-like nanostructured MnO2 with near-infrared (NIR) light-triggered high photothermal conversion capability of 30% and reactive oxygen species (ROS) generation ability was successfully developed. Different from the reported MnO2 nanomaterials those were used in the nanomedicine field for only relieving tumor hypoxia and/or imaging, the flower-like MnO2 inherently acts as a competent agent for simultaneously enhanced photothermal and photodynamic therapy. A flower-like nanostructured MnO2 with near-infrared (NIR) light triggered high photothermal conversion capability of 30% and reactive oxygen species (ROS) generation ability was successfully developed.

In Situ Visualizing Oxidase-Mimicking Activity of Single MnOOH Nanotubes with Mie Scattering-Based Absorption Microscopy.

Imaging the catalytic activity at the single-particle level can greatly promote the screening and rational design of highly efficient nanozymes, but conventional techniques are based on ensemble analysis. Here, we present a new absorption microscopy for in situ visualizing oxidase-mimicking activity of single MnOOH nanotubes. The particle with a size more than 700 nm roughly equally scatters all wavelengths of visible light via Mie scattering, and the scattering light is collected by dark-field optical microscopy. When the particles absorb a single color of the scattering light, each individual nanoparticle shows its complementary color, enabling a form of absorption microscopy that we name Mie scattering-based absorption microscopy. We find that MnOOH nanotubes can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate polyTMB nanowires at their tips. There are multiple active sites on the surface of the individual nanotube, and the nanozyme activity shows a large heterogeneity as well as pH-dependent characteristic.

A Smart Theranostic Nanocapsule for Spatiotemporally Programmable Photo-Gene Therapy.

The therapeutic performance of DNAzyme-involved gene silencing is significantly constrained by inefficient conditional activation and insufficient cofactor supply. Herein, a self-sufficient therapeutic nanosystem was realized through the delicate design of DNAzyme prodrugs and MnO2 into a biocompatible nanocapsule with tumor-specific recognition/activation features. The indocyanine green (ICG)-modified DNA prodrugs are designed by splitting the DNAzyme and then reconstituted into the exquisite catalyzed hairpin assembly (CHA) amplification circuit. Based on the photothermal activation of ICG, the nanocapsule was disassembled to expose the MnO2 ingredient which was immediately decomposed into Mn2+ ions to supplement an indispensable DNAzyme cofactor on-demand with a concomitant O2 generation for enhancing the auxiliary phototherapy. The endogenous microRNA catalyzes the amplified assembly of DNA prodrugs via an exquisite CHA principle, leading to the DNAzyme-mediated simultaneous silencing of two key tumor-involved mRNAs. This self-activated theranostic nanocapsule could substantially expand the toolbox for accurate diagnosis and programmable therapeutics.

In vivo and in vitro biocompatibility study of MnFe2O4 and Cr2Fe6O12 as photosensitizer for photodynamic therapy and drug delivery of anti-cancer drugs.

In The present project, a variety of MnFe2O4 (Mn) and Cr2Fe6O12 (Cr)-based nanocarriers (NCs) were synthesized as photosensitizer and NCs for delivery of chemotherapeutic curcumin (CUR) and provide a new structure for Photodynamic Therapy (PDT). For determining efficiency of NCs release study, MTT assay, lethal dose test and hemolysis assay were carried out. The release study showed the release of CUR from NCs was pH-dependent, but, every NCs had its own behavior for releasing the drug. The data acquired from the release study showed the CUR release from Mn can reach to over 90% at acidic media instead of 41% at neutral media. However, the CUR released from Cr were approximately equal as Cr had equal zeta potential at both media. Hemolysis activity and lethal dose test displayed the cytotoxicity of NCs was neglectable at both in vitro and in vivo study. Also, the results of anti-cancer activity assay (MTT assay) showed that both of Cr and Mn NCs are suitable systems for PDT. Therefore, the results demonstrated that Mn is suitable NCs for PDT and anticancer drugs delivery of therapeutic drugs.

Research Note: Evaluation of manganese hydroxychloride in 45-wk-old white leghorn layers using yolk and shell manganese content.

The objective of the current study was to evaluate increasing levels of manganese hydroxychloride (MHC) in 45-wk-old white leghorn laying hens, using yolk and shell manganese (Mn) content as a potential marker for Mn concentration. A total of 80, 45-wk-old white leghorns were assigned to 6 dietary treatments, each consisting of 14 individually caged laying hens, with the exception of the reference diet containing 10 individually caged laying hens. The experiment consisted of a reference diet that contained 70 ppm of supplemental inorganic Mn in the form of Mn oxide and 5 experimental treatments each containing 0, 15, 30, 60, and 90 ppm supplemental MHC. Experimental birds were subjected to a 21 D depletion phase in which no supplemental Mn was included in the diet; however, during this time reference fed birds were fed the control diet (70 ppm Mn). After the 21 D depletion phase, the depleted birds were fed experimental diets for a 35 D evaluation period. Yolk and shell Mn content were analyzed at the end of the depletion phase and during the experimental phase on day 5, 10, 15, 25, and 35. During the experimental phase, Mn was replenished in the yolk and shell in all experimental treatments containing supplemental Mn; however, dose and time impacted the rate of replenishment. The yolk tended to be more sensitive to variations in Mn level as increases in Mn inclusion significantly (P < 0.05) increased concentration. These data demonstrate the ability to deplete and replenish Mn, and the use of egg yolk Mn concentration as measurement for determining changes in dietary Mn. At the conclusion of the experiment at 35 D, 60 ppm of Mn hydroxychloride seemed to be adequate in replenishing Mn to the level of the reference.

Effect of manganese preconditioning and replacing inorganic manganese with organic manganese on performance of male broiler chicks.

The effects of manganese (Mn) preconditioning, 96 h post-hatch followed by the replacement of inorganic Mn with different levels of organic Mn (5 to 21 D), on growth, tissue excreta Mn content, gene expression, and enzyme activity were evaluated. A total of 420 day-old male Cobb 500 broilers were divided into 2 groups. One group was fed a corn-soybean meal basal diet containing 17 mg of Mn/kg (preconditioning diet, MnPD); the second group was fed the non-preconditioning diet (NPCD), which was the MnPD supplemented with 60 mg of Mn/kg from manganese sulfate (MnSO4). On day 5, each group was divided into 5 subgroups and were randomly assigned to dietary treatments consisting of MnPD alone or MnPD supplemented with 12 or 60 mg Mn/kg Mn as MnSO4 or Mn proteinate (6 replicate cages of 6 birds). Broiler chicks that were fed the MnPD had lower (P ≤ 0.05) body weight gain (BWG) and G:F ratio when compared to those that were fed the NPCD for 4 D. Birds that were fed MnPD (1 to 4 D) and switched to MnPD supplemented with 60 mg/kg Mn (5 to 21 D) had lower (P ≤ 0.05) BWG compared to those that were fed NPCD (1 to 4 D) and switched to MnPD supplemented with 60 mg/kg Mn for 21 D. Excreta, tibia ash, liver, and heart Mn levels were increased (P ≤ 0.05) by supplemental Mn. The expression of jejunum divalent metal transporter-1 mRNA levels, as well as activities of plasma total super oxide dismutase and liver alanine transaminase, was not affected by MnPD or Mn source and levels. These results confirmed that feeding marginally deficient Mn diets to broiler chicks post-hatch does affect growth rate and tissue Mn concentration.

A colorimetric assay for acetylcholinesterase activity and inhibitor screening based on the thiocholine-induced inhibition of the oxidative power of MnO2 nanosheets on 3,3',5,5'-tetramethylbenzidine.

The authors describe a colorimetric method for the determination of the activity of acetylcholinesterase (AChE). Manganese dioxide (MnO2) nanosheets directly reacts with 3,3',5,5'-tetramethylbenzidine (TMB) in the absence of hydrogen peroxide (H2O2). This leads to the formation of a blue product (oxTMB) with an absorption peak at 652 nm. If AChE hydrolyzes its substrate acetylthiocholine chloride, thiocholine is formed which blocks the oxidative power of the MnO2 nanosheets. Hence, oxTMB will not be formed. The decreased absorbance is directly related to the AChE activity in the 0.01-1.0 mU·mL-1 range. The detection limit is 0.01 mU·mL-1 and the relative standard deviation is 1.2% (for n = 11 at 0.5 mU·mL-1). The method was also applied to screen for inhibitors of AChE. Graphical abstract Based on the oxidizing properties of manganese dioxide nanosheets (MnO2 nanosheets), we report a colorimetric method for determining acetylcholinesterase activity with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB).

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

Evaluation of Manganese Chloride's Effect on Biosynthetic Properties of In Vitro Cultures of Eschscholzia californica Cham.

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4'-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

Dietary manganese supplementation affects mammillary knobs of eggshell ultrastructure in laying hens.

This study evaluated the mechanism by which dietary manganese (Mn) supplementation-in either an organic or inorganic form-affects mammillary knobs of the eggshell ultrastructure in laying hens. A total of 225 54-week-old Hy-Line Brown laying hens were fed a basal diet containing 27.5 mg Mn/kg feed for 2 wk, after which they were randomly allocated into 3 groups and fed a basal diet (control) or a basal diet supplemented with 120 mg Mn/kg feed from monohydrate Mn sulfate (an inorganic source of Mn) or with 80 mg Mn/kg feed from an amino acid-Mn complex (an organic source of Mn) for 10 wk. For each group, 5 replicates of 15 hens each were used with 1 hen per cage. Compared with the control, dietary Mn supplementation increased the mammillary-knob density of eggs at 9.5 h post-oviposition (P < 0.05). The Mn content in both blood and eggshell gland was increased with the supplementation of Mn in inorganic and organic forms (P < 0.05), but the blood Mn content was higher after inorganic-Mn supplementation as compared with organic-Mn supplementation (P < 0.05). RNA sequencing and quantitative real-time PCR analysis of the eggshell gland showed that dietary Mn supplementation increased the expression of genes encoding some proteoglycans, glycoproteins, and calcium-binding proteins in the eggshell gland (P < 0.05), and involved in the process of the protein glycosylation and glycan metabolism in the eggshell gland (P < 0.05). Overall, dietary Mn supplementation can involve in the process of protein glycosylation and glycan metabolism and improve the expression of genes encoding proteoglycans and glycoproteins in the eggshell gland, thus increasing the mammillary-knob density during the initial deposition stage of shell formation.

Dietary manganese supplementation modulated mechanical and ultrastructural changes during eggshell formation in laying hens.

This study evaluated the mechanical and ultrastructural changes during eggshell formation in laying hens by using the optimal levels of organic and inorganic manganese (Mn). A total of 270 62-wk-old Hy-Line Brown laying hens were fed a basal diet containing 25.1 mg Mn per kg feed for 2 wks, after which they were randomly allocated into 3 groups and fed the basal diet (control) or the basal diet supplemented with 120 mg Mn per kg feed from monohydrate Mn sulfate (an inorganic source of Mn), or 80 mg Mn per kg feed from an amino acid-Mn complex (an organic source of Mn) for 12 wks. For each group, 6 replicates of 15 hens were used with one hen per cage. Dietary Mn supplementation significantly increased eggshell-breaking strength and thickness in laying hens (P < 0.05). In neither was the elasticity of their eggshell membranes, measured during the nucleation and mammillary knob formation stages, affected by dietary Mn compared with the control (P > 0.05), whereas the breaking strength of the eggshells was greater at the linear and terminate deposition stages compared with the control (P < 0.05). Dietary Mn supplementation decreased the width of the mammillary knobs and the proportion of mammillary thickness, and increased the proportion of effective thickness of the whole eggshells (P < 0.05). Ultrastructural changes during the eggshell formation indicated that dietary Mn supplementation increased the nucleation site and mammillary knob densities, decreased the mammillary thickness, and increased the proportion of effective thickness and total thickness of the eggshells compared with the control (P < 0.05). Therefore, dietary Mn supplementation can improve the breaking strength and ultrastructure of the eggshells during their formation, and the mammillary and palisade layers are both crucial structures affected by Mn.

Effect of dietary supplementation of organic or inorganic manganese on eggshell quality, ultrastructure, and components in laying hens.

This study evaluated the effects of dietary supplemental levels and sources of manganese (Mn) on performance, eggshell quality, ultrastructure, and components in laying hens. A total of 1,080 46-wk-old Jing Brown hens were fed a basal diet (Mn, 32.7 mg/kg) for 2 wks laying and then randomly allocated to 9 groups that were fed a basal diet (control) or the basal diet supplemented with inorganic (MnSO4·H2O) or organic (amino-acid-Mn, 8.78%) Mn at 40, 80, 120, or 160 mg per kg of feed for 8 wks. Each group had 8 replicates of 15 hens. The results showed that dietary Mn supplementation did not affect the performance of hens (P > 0.05). Dietary Mn supplementation resulted in linear and quadratic increases of breaking strength and thickness in both inorganic and organic forms (P < 0.05), but fracture toughness increased quadratically only in organic groups (P < 0.05). Linear and quadratic effects on effective and mammillary thickness were observed with Mn supplementation from inorganic and organic sources (P < 0.05), and lower mammillary thickness was observed in organic groups (P < 0.05). However, the width of mammillary knobs decreased quadratically only with the supplementation of organic Mn (P < 0.05). Dietary Mn supplementation had a quadratic effect on the shell Mn content in both inorganic and organic forms (P < 0.05). Linear and quadratic effects on the content of sulfated glycosaminoglycans (GAGs) were observed only in calcified eggshell with inorganic Mn supplementation (P < 0.05), while the supplementation of organic Mn had a quadratic effect on sulfated GAGs content in both calcified eggshell and membranes (P < 0.05). Overall, dietary Mn supplementation, regardless of the source, could increase breaking strength and thickness by improving the ultrastructure, which partly results from increased sulfated GAGs content in the eggshell. Moreover, the supplementation of organic Mn could increase fracture toughness by decreasing the width of mammillary knobs, which is partially due to increased sulfated GAGs content in the membranes.

Biological synthesis of manganese dioxide nanoparticles by Kalopanax pictus plant extract.

Manganese dioxide (MnO2) nanoparticles were synthesised by the reduction of potassium permanganate (KMnO4) using Kalopanax pictus leaf extract at room temperature. A transparent dark-brown colour appeared after the addition of K. pictus leaf extract to the solution of permanganate. The time course of the reduction of KMnO4and synthesis of MnO2 nanoparticles was monitored by means of UV-Vis spectra. The reduction of KMnO4occurred after addition of plant extract with disappearance of KMnO4specific peaks and emergence of peak specific for MnO2nanoparticles. MnO2nanoparticles showed absorption maxima at 404 nm. The electron dispersive X-ray spectroscopy analyses confirmed the presence of Mn and O in the sample. X-ray photoelectron spectroscopy revealed characteristic binding energies for MnO2nanoparticles. Transmission electron microscopy micrographs revealed presence of uniformly dispersed spherical shaped particles with average size of 19.2 nm. The selected area electron diffraction patterns revealed the crystalline nature of MnO2nanoparticles. Fourier transform-infrared spectroscopy spectra of pure MnO2show the occurrence of O-Mn-O vibrational mode at around 518 cm⁻¹. The phyto-synthesised MnO2nanoparticles showed degradation ability of dyes (congo red and safranin O) similar to chemically synthesised MnO2nanoparticles. This study shows simple and eco-friendly synthesis of MnO2nanoparticles by plant extract and their utilisation for dye degradation for the first time.

Identification of Mn(II)-oxidizing bacteria from a low-pH contaminated former uranium mine.

Biological Mn oxidation is responsible for producing highly reactive and abundant Mn oxide phases in the environment that can mitigate metal contamination. However, little is known about Mn oxidation in low-pH environments, where metal contamination often is a problem as the result of mining activities. We isolated two Mn(II)-oxidizing bacteria (MOB) at pH 5.5 (Duganella isolate AB_14 and Albidiferax isolate TB-2) and nine strains at pH 7 from a former uranium mining site. Isolate TB-2 may contribute to Mn oxidation in the acidic Mn-rich subsoil, as a closely related clone represented 16% of the total community. All isolates oxidized Mn over a small pH range, and isolates from low-pH samples only oxidized Mn below pH 6. Two strains with different pH optima differed in their Fe requirements for Mn oxidation, suggesting that Mn oxidation by the strain found at neutral pH was linked to Fe oxidation. Isolates tolerated Ni, Cu, and Cd and produced Mn oxides with similarities to todorokite and birnessite, with the latter being present in subsurface layers where metal enrichment was associated with Mn oxides. This demonstrates that MOB can be involved in the formation of biogenic Mn oxides in both moderately acidic and neutral pH environments.

Effects of diets supplemented with zinc and manganese on performance and related parameters in laying hens.

Iron is often found to be of excessive concentrations in laying hens' diets, which may cause antagonistic interactions with other minerals. This study was conducted to investigate how to supplement Zn and Mn in the diets without Fe supplementation. In experiment 1, 420 18-week Lohmann Brown layers were fed a basal diet or a basal diet supplemented with 30-0, 65-30 and 100-60 mg/kg of Zn and Mn, respectively. In experiment 2, 360 40-week Lohmann Brown layers were fed a basal diet or a basal diet supplemented with 15-0, 35-0 and 55-15 mg/kg of Mn and Zn, respectively. Minerals were supplemented in the form of sulfate. Egg production was improved by supplementing 30 mg/kg Zn or 65 mg/kg Zn in combination with 30 mg/kg Mn in experiment one. In experiment two, a significant reduction of egg performance occurred with 35 mg/kg Mn supplementation. Mn and/or Zn supplementation increased eggshell thickness in experiment one, and decreased yolk cholesterol in both experiments. Mn and/or Zn supplementation increased Zn and Mn excretion in both experiments. Serum growth hormone (GH), thyroxine (T(4) ), and insulin levels, or alkaline phosphatase (AP) activity were not affected by treatments; serum estrogen (E(2) ) and triiodothyronine (T(3) ) were different but there was no consistency by dietary treatments. This study demonstrates that 30 mg/kg supplemental Zn is necessary to obtain maximal egg production, and there seems to be no need to supply Mn in this type of diet.

Roles of siderophore in manganese-oxide reduction by Shewanella oneidensis MR-1.

Dissimilatory metal-reducing bacteria (DMRB), such as Shewanella oneidensis MR-1, are of great interest for their importance in the biogeochemical cycling of metals and utility in biotechnological processes, such as bioremediation and microbial fuel cells. To identify genes necessary for metal reduction, this study constructed a random transposon-insertion mutant library of MR-1 and screened it for isolating mutants that were deficient in metal reduction. Examination of approximately 5000 mutants on lactate minimal-medium plates containing MnO(2) resulted in the isolation of one mutant, strain N22-7, that showed a decreased MnO(2)-reduction activity. Determination of a transposon-insertion site in N22-7 followed by deletion and complementation experiments revealed that the disruption of SO3030, a siderophore biosynthesis gene, was responsible for the decreased MnO(2)-reduction activity. In ΔSO3030 cells, iron and cytochrome contents were decreased to approximately 50% of those in the wild-type cells, when they were incubated under MnO(2)-reduction conditions. In addition, the transcription of genes encoding outer-membrane cytochromes necessary for metal reduction was repressed in ΔSO3030 under MnO(2)-reduction conditions, while their transcription was upregulated after supplementation of culture media with ferrous iron. These results suggest that siderophore is important for S. oneidensis MR-1 to respire MnO(2), because iron availability influences the expression of cytochromes necessary for metal reduction.

Bioaccumulation of heavy metals by Phragmites australis cultivated in synthesized substrates.

Accumulation of heavy metals from various oxides with adsorbed cadmium by wetland plant Phragmites australis was studied to evaluate the fate of heavy metals in the sediment of constructed wetlands. Hoagland solution was used as nutrition supply, and single metal oxide with adsorbed cadmium was applied as contaminant to study the accumulation characteristics of cadmium and the substrate metals by P. australis. After 45-d treatment, the bioaccumulation degree in root followed the order: Al(OH)3 > Al2O3 > Fe3O4 > MnO2 > FeOOH. Heavy metals absorbed by P. australis were largely immobilized by the roots with little translocation to aboveground parts.

Iron requirement for Mn(II) oxidation by Leptothrix discophora SS-1.

A common form of biocatalysis of Mn(II) oxidation results in the formation of biogenic Mn(III, IV) oxides and is a key reaction in the geochemical cycling of Mn. In this study, we grew the model Mn(II)-oxidizing bacterium Leptothrix discophora SS-1 in media with limited iron (0.1 microM iron/5.8 mM pyruvate) and sufficient iron (0.2 microM iron/5.8 mM pyruvate). The influence of iron on the rate of extracellular Mn(II) oxidation was evaluated. Cultures in which cell growth was limited by iron exhibited reduced abilities to oxidize Mn(II) compared to cultures in medium with sufficient iron. While the extracellular Mn(II)-oxidizing factor (MOF) is thought to be a putative multicopper oxidase, Mn(II) oxidation in the presence of zero added Cu(II) was detected and the decrease in the observed Mn(II) oxidation rate in iron-limited cultures was not relieved when the medium was supplemented with Cu(II). The decline of Mn(II) oxidation under iron-limited conditions was not accompanied by siderophore production and is unlikely to be an artifact of siderophore complex formation with Mn(III). The temporal variations in mofA gene transcript levels under conditions of limited and abundant iron were similar, indicating that iron limitation did not interfere with the transcription of the mofA gene. Our quantitative PCR results provide a step forward in understanding the regulation of Mn(II) oxidation. The mechanistic role of iron in Mn(II) oxidation is uncertain; the data are consistent with a direct requirement for iron as a component of the MOF or an indirect effect of iron resulting from the limitation of one of many cellular functions requiring iron.

Using thermal energy produced by irradiation of Mn-Zn ferrite magnetic nanoparticles (MZF-NPs) for heat-inducible gene expression.

One of the main advantages of gene therapy over traditional therapy is the potential to target the expression of therapeutic genes in desired cells or tissues. To achieve targeted gene expression, we developed a novel heat-inducible gene expression system in which thermal energy generated by Mn-Zn ferrite magnetic nanoparticles (MZF-NPs) under an alternating magnetic field (AMF) was used to activate gene expression. MZF-NPs, obtained by co-precipitation method, were firstly surface modified with cation poly(ethylenimine) (PEI). Then thermodynamic test of various doses of MZF-NPs was preformed in vivo and in vitro. PEI-MZF-NPs showed good DNA binding ability and high transfection efficiency. In AMF, they could rise to a steady temperature. To analyze the heat-induced gene expression under an AMF, we combined P1730OR vector transfection with hyperthermia produced by irradiation of MZF-NPs. By using LacZ gene as a reporter gene and Hsp70 as a promoter, it was demonstrated that expression of a heterogeneous gene could be elevated to 10 to 500-fold over background by moderate hyperthermia (added 12.24 or 25.81 mg MZF-NPs to growth medium) in tissue cultured cells. When injected with 2.6 or 4.6 mg MZF-NPs, the temperature of tumor-bearing nude mice could rise to 39.5 or 42.8 degrees C, respectively, and the beta-gal concentration could increase up to 3.8 or 8.1 mU/mg proteins accordingly 1 day after hyperthermia treatment. Our results therefore supported hyperthermia produced by irradiation of MZF-NPs under an AMF as a feasible approach for targeted heat-induced gene expression. This novel system made use of the relative low Curie point of MZF-NPs to control the in vivo hyperthermia temperature and therefore acquired safe and effective heat-inducible transgene expression.