Selenium supplementation enhanced the expression of selenoproteins in hippocampus and played a neuroprotective role in LPS-induced neuroinflammation.

Selenium (Se) is obtained from organic and inorganic selenium food content, which mainly depends on the regional soil selenium content. Selenium deficiency and decreased selenoprotein functions have been shown to associate with the progression of cognitive decline and neurodegenerations including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Selenoproteins are well recognized for their anti-oxidative activities. Given the high oxygen consumption, mammalian brains preferent@ially supplied with Se. Here, we propose a beneficiary role for dietary supplementation of sodium selenite (300 ng per gram of body weight) in ameliorating neuroinflammation induced by bilateral intracerebroventricular injection of 1 µL LPS (1 µg/µL), evidenced by the significantly reduced oxidative stress, downregulated pro-inflammatory cytokines expression, improved integrity of blood-brain barrier, as well as suppressed glial activation and shifted microglial MI/M2 polarization in Se-sup mouse brain. Se intake also reduced neural cell death and significantly improved the cognition in Se-sup mice following LPS challenge. The neuroprotective role for supplementary Se is likely to be ascribed to the overall elevated expression of selenoproteins, especially Selenoprotein P (SELENOP) and Glutathione peroxidase 4 (GPX4), ranking on top of the change in selenoprotein expression hierarchy. The regional hierarchy of Se induced elevation of SELENOP expression was further characterized. The SELENOP expression in the mediodorsal thalamic nucleus, dendric gyrus (DG) and cornu ammonis 3 (CA3) of hippocampus and lateral habenular nucleus was highly sensitive to dietary Se intake.

miR-203, fine-tunning neuroinflammation by juggling different components of NF-κB signaling.

BACKGROUND: miR-203 was first indicated in maintaining skin homeostasis and innate immunity. Aberrant expression of miR-203 was found associated with pathological progressions of immune disorders, cancers, as well as neurodegenerations. Recently, increasing data on miR-203 in regulating neuroinflammation and neuronal apoptosis has raised extensive concern about the biological function of this microRNA. METHODS: Mouse model with ectopic miR-203 expression in the hippocampus was constructed by stereotactic injection of lentiviral expression vector of pre-miR-203. Association of miR-203 and mRNA of Akirin2, as well as the competition for miR-203 targeting between Akirin2 3'UTR and another recently characterized miR-203 target, 14-3-3θ, was verified using Dual-Luciferase Reporter Gene Assay and western blot. Microglia activation and pro-inflammatory cytokines expression in the hippocampus of mice overexpressing miR-203 was evaluated using immunohistochemistry analysis and western blot. Neuronal cell death was monitored using anti-caspase 8 in immunohistochemistry as well as TUNEL assay. Cognition of mice was assessed with a behavior test battery consisting of nesting behavior test, Barnes maze and fear conditioning test. RESULTS: Akirin2, an activator of NF-κB signaling, was identified as a direct target of miR-203. By also targeting 14-3-3θ, a negative regulator of NF-κB signaling, miR-203 displayed an overall pro-inflammatory role both in vitro and in vivo. Promoted nuclear translocation of NF-κB and increased expression of proinflammatory cytokines were observed in cultured BV2 cells transfected with miR-203 mimics. Microglia activation and upregulation of NF-κB, IL-1ß and IL-6 were observed in mouse hippocampus with overexpression of miR-203. In addition, promoted neuronal cell death in the hippocampus and impaired neuronal activities resulted in cognitive dysfunction of mice with ectopic miR-203 expression in the hippocampus. CONCLUSION: A pro-inflammatory and neurodisruptive role of miR-203 was addressed based on our data in this study. Given the identification of Akirin2 as a direct target of miR-203 and the competition with 14-3-3θ for miR-203 targeting, together with the findings of other signaling molecules in NF-κB pathway as targets of miR-203, we proposed that miR-203 was a master modulator, fine-tunning neuroinflammation by juggling different components of NF-κB signaling.

Identification of the transcription factor, AFF4, as a new target of miR-203 in CNS.

MiR-203 was identified as a hub of a potential regulatory miRNA network in central nervous system. Overexpressing of miR-203 in the frontal cortex of C57BL/6J wild type mouse induced neurodegeneration by increasing the apoptotic pathway and neuron death. AFF4, a transcription factor, was identified as a new bona fida protein target of miR-203 in CNS. The miRNA:mRNA interaction of miR-203 and AFF4 was verified using Dural-luciferase assay. Down-regulated expression of AFF4 was induced by overexpressing miR-203 both in vitro and in vivo. Open field test, Y maze and Morris water maze test were conducted for the behavioral assessment of the mice with stereotactic injection of lentiviral vector overexpressing miR-203 in the hippocampus. No anxiety-like behavior or impaired cognition was noticed in these mice. Consistent with the results of the behavioral assessment, the electron micrograph and Nissl staining revealed no significant change in the synaptic density and no neuron injuries in the hippocampus of mice overexpressing miR-203, respectively. Our results indicated that instead of promoting neurodegenerative phenotype, a more profound function should be ascribed to miR-203 in regulating neuron behavioral activities and cognition. Neuron-type specific functions of miR-203 are likely to be executed via its various downstream protein interactors.

Electrochemical and spectroscopic characteristics of cytochrome P450 55A3 and its interaction with nitric oxide.

Cytochrome P450 55A3 (CYP55A3) is an enzyme with the catalytic activity of nitric oxide (NO) to nitrous oxide using NADH or NADPH as the electron donor. Herein CYP55A3 has been expressed in E. coli and purified by His-tag columns. The electrochemical and spectroscopic characteristic of CYP55A3 and its interaction with NO has been studied. The direct electrochemistry of Fe3+/Fe2+ redox peaks in CYP55A3 was realized on the pyrolitic graphite electrode with the redox potential of -475 mV in pH 7.0 phosphate buffer. With the addition of NO a ferric nitroxyl complex (Fe3+-NO) formed with a new reduction peak at -0.78 V. The reduction peak current increased with the concentration of NO and showed typical Michaelis-Menten kinetic characteristics with the apparent Michaelis constant Kmapp 9.78 µM. The binding constant K calculated to be 3.93 × 104 M by UV-vis method. The fluorescence emission spectra of iron porphyrin in CYP55A3 showed with the peak wavelength 633 nm, and its fluorescence intensity increased after binding with NO. The fluorescence analysis demonstrated that NADH can relay electrons to iron porphyrin and reduce NO. The reductive product of NO released and the iron porphyrin in CYP55A3 turned back to the original form.

Chemical nature of electrochemical activation of carbon electrodes.

Electrochemical activation of carbonaceous electrodes is a common step in the preparation of high-performance electrochemical (bio-)sensors. The underlying mechanism has long been discussed but is, however, not yet fully understood. Here, we propose that electrochemical activation can produce graphene derivatives that largely enhance the electrochemical performance of carbonaceous electrodes using a carbon paste electrode or a carbon rod electrode as samples. Based on morphological, chemical and electrochemical evidence, we conclude that in the electrochemical activation of carbonaceous electrodes, graphene oxide or reduced graphene oxide could be generated in situ, which is the main reason for the enhancement of the electrode performance. Moreover, chemical and biological sensors made from electrochemically activated carbonaceous electrodes resemble their counterparts made from chemically prepared graphene-derivative-modified carbonaceous electrodes and even exhibit better performance. These findings could enable us to establish an efficient and predictable solution for graphene-related electrochemistry and surface chemistry.

Direct electrochemistry of bacterial surface displayed cytokinin oxidase and its application in the sensitive electrochemical detection of cytokinins.

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E0') of -0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0 µM and a lower limit of detection of 0.7 µM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins.

Rapid and simple detection of endospore counts in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker.

Spore counting in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker was studied for developing a rapid and simple detection method. The newly developed method is based on the fluorescence enhancement by a new chelating agent, which forms a complex with EuCl3 and DPA. The results showed that 1,2-cyclohexanediamine-N,N,N'N'-tetraacetic acid (CyDTA) greatly enhanced the fluorescence intensity in all selected chelating agents. The optimal composition of the fluorescence complex DPA-Eu-CyDTA had a detection limit of 0.3 nM of DPA. Metal ions in high concentrations, including Cu2+, Fe2+, Fe3+, Al3+, and Zn2+, might lower the detection sensitivity, which could be eliminated by diluting the sample with the metal ions below 10 µM. The maximum release of DPA was achieved by heating treatments at 121 °C for at least 10 min for two types of Bacillus endospores. The spore concentrations and corresponding released DPA fluorescence intensities were linearly associated (coefficient R2 = 0.9993 and 0.9995 for Bacillus subtilis MA139 and Bacillus licheniformis BL20386, respectively). The detection limit for both strains reached approximately 6800 spores/mL. The verification results showed that the DPA fluorimetry assay developed in the present study was fully consistent with the plate-counting assay. The study shows that the fluorescence complex DPA-Eu-CyDTA can be reliably used for the detection of endospores in Bacillus fermentation for the production of probiotics.

Expression and Purification of Cytochrome P450 55B1 from Chlamydomonas reinhardtii and Its Application in Nitric Oxide Biosensing.

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 µmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0.70 µmol/g of the total protein, which showed a single band on a SDS-PAGE and Western blot. The direct electrochemistry of CYP55B1 in dihexadecylphosphate (DHP) film was realized with an electric potential at -0.47 V at the scan rate of 1 V s-1. We studied the in vitro interaction between P450 55B1 and NO by the fluorescence spectrometric method. The results show that the fluorescence intensity of iron-porphyrin in P450 55B1 changes gradually with the addition of NO. The fluorescence intensity change values against NO concentrations were plotted, and it showed a linear range of NO from 0 to 22.5 µM with a sensitivity of 0.15 µM/AU and a detection limit of 0.15 µM.

Enhancing growth of Chlamydomonas reinhardtii and nutrient removal in diluted primary piggery wastewater by elevated CO2 supply.

The coupling of primary piggery wastewater as a culture medium with elevated CO2 aeration is thought to be an economically feasible option for the cultivation of microalgae. However, little information is available regarding the photosynthetic characteristics of microalgae and nutrient removal from wastewater at different CO2 concentrations. It was found that elevated CO2 aeration provided sustained growth at CO2 concentrations ranging from 5% to 15% and performed best with 5% CO2 aeration in primary piggery wastewater for Chlamydomonas reinhardtii growth. Photosynthesis, respiration, and nutrient uptake (total nitrogen and total phosphorus) were stimulated in response to CO2 enrichment, thus increasing nutrient uptake in primary piggery wastewater, particularly total nitrogen and total phosphorus. A study of carbon-concentrating mechanism-related gene expression revealed that the levels of mRNAs, such as CAH1, LCIB and HLA3, were significantly downregulated. This represents a possible method for the reconciliation of CO2-stimulated growth with mixotrophic cultivation of C. reinhardtii in diluted primary piggery wastewater.

Application of an M13 bacteriophage displaying tyrosine on the surface for detection of Fe(3+) and Fe(2+) ions.

Ferric and ferrous ion plays critical roles in bioprocesses, their influences in many fields have not been fully explored due to the lack of methods for quantification of ferric and ferrous ions in biological system or complex matrix. In this study, an M13 bacteriophage (phage) was engineered for use as a sensor for ferric and ferrous ions via the display of a tyrosine residue on the P8 coat protein. The interaction between the specific phenol group of tyrosine and Fe(3+) / Fe(2+) was used as the sensor. Transmission electron microscopy showed aggregation of the tyrosine-displaying phages after incubation with Fe(3+) and Fe(2+). The aggregated phages infected the host bacterium inefficiently. This phenomenon could be utilized for detection of ferric and ferrous ions. For ferric ions, a calibration curve ranging from 200 nmol/L to 8 µmol/L with a detection limit of 58 nmol/L was acquired. For ferrous ions, a calibration curve ranging from 800 nmol/L to 8 µmol/L with a detection limit of 641.7 nmol/L was acquired. The assay was specific for Fe(3+) and Fe(2+) when tested against Ni(2+), Pb(2+), Zn(2+), Mn(2+), Co(2+), Ca(2+), Cu(2+), Cr(3+), Ba(2+), and K(+). The tyrosine displaying phage to Fe(3+) and Fe(2+) interaction would have plenty of room in application to biomaterials and bionanotechnology.

Encapsulation of gold nanoparticles by simian virus 40 capsids.

Viral capsid-nanoparticle hybrid structures constitute a new type of nanoarchitecture that can be used for various applications. We previously constructed a hybrid structure comprising quantum dots encapsulated by simian virus 40 (SV40) capsids for imaging viral infection pathways. Here, gold nanoparticles (AuNPs) are encapsulated into SV40 capsids and the effect of particle size and surface ligands (i.e. mPEG and DNA) on AuNP encapsulation is studied. Particle size and surface decoration play complex roles in AuNP encapsulation by SV40 capsids. AuNPs ≥15 nm (when coated with mPEG750 rather than mPEG2000), or ≥10 nm (when coated with 10T or 50T DNA) can be encapsulated. Encapsulation efficiency increased as the size of the AuNPs increased from 10 to 30 nm. In addition, the electrostatic interactions derived from negatively charged DNA ligands on the AuNP surfaces promote encapsulation when the AuNPs have a small diameter (i.e. 10 nm and 15 nm). Moreover, the SV40 capsid is able to carry mPEG750-modified 15-nm AuNPs into living Vero cells, whereas the mPEG750-modified 15-nm AuNPs alone cannot enter cells. These results will improve our understanding of the mechanisms underlying nanoparticle encapsulation in SV40 capsids and enable the construction of new functional hybrid nanostructures for cargo delivery.

Colorimetric detection of melamine in complex matrices based on cysteamine-modified gold nanoparticles.

A sensitive assay for melamine in complex matrices is built using cysteamine-modified gold nanoparticles (AuNPs) and an effective sample pretreatment protocol. Citrate-stabilized AuNPs were modified by cysteamine in order to weaken the electrostatic repulsion force between the gold nanoparticles. Detection sensitivity gained through this modification increased about 100 fold compared with the result using the unmodified AuNPs. Direct colorimetric visualizations of melamine in milk products, eggs and feeds was successfully demonstrated within the linear ranges of 1-200 mg L(-1) and detection limits below 1 mg L(-1). The proposed scheme could be an alternative means for onsite detection of melamine without costly instruments.

Construction of bifunctional phage display for biological analysis and immunoassay.

A phage display-based bifunctional display system was developed for simple and sensitive immunoassay. The resulting bifunctional phage could simultaneously display a few single-chain variable fragment (ScFv) and many copies of the gold-binding peptide on its surface, thereby mediating antigen recognition and signal amplification. As a demonstration study, it was possible for bifunctional phage-based immunoassay to identify Bacillus anthracis spores from other Bacillus strains with detection sensitivity 10-fold higher than that of conventional phage enzyme-linked immunosorbent assay (ELISA). This protocol may be applied to build other bifunctional phage clones for broad applications (e.g., immunoassay kits, affinity biosensors, biorecognition assays).

Seeding-induced self-assembling protein nanowires dramatically increase the sensitivity of immunoassays.

Aiming to build a supersensitive and easily operable immunoassay, bifunctional protein nanowires were generated by seeding-induced self-assembling of the yeast amyloid protein Sup35p that genetically fused with protein G and an enzyme (methyl-parathion hydrolase, MPH), respectively. The protein nanowires possessed a high ratio of enzyme molecules to protein G, allowing a dramatic increase of the enzymatic signal when protein G was bound to an antibody target. As a result, a 100-fold enhancement of the sensitivity was obtained when applied in the detection of the Yersinia pestis F1 antigen.