Photoelectrochemical Assay Based on SnO2/BiOBr p-n Heterojunction for Ultrasensitive DNA Detection.

Herein, a photoelectrochemical (PEC) assay was designed for a highly sensitive DNA determination relying upon the SnO2/BiOBr p-n heterojunction as a photoactive material and SiO2 as a signal quencher. Compared with most traditional heterojunctions, the SnO2/BiOBr p-n heterostructure not only lessened the recombination of the photogenerated electron-hole pairs but also promoted the light-harvesting in the ultraviolet-visible (UV-vis) region, leading to further enhanced photoelectric conversion efficiency and photocurrent, which demonstrated 12.1-fold and 6.4-fold increments versus those of pure SnO2 and BiOBr, respectively. Additionally, the limited quantity of target DNA (a fragment of p53 gene) could be transformed into abundant output DNA-SiO2 by employing the Nt·BstNBI enzyme-assisted signal amplification procedure, leading to a highly improved detection sensitivity of the biosensor. Then, output DNA-SiO2 hybridized with the capture DNA anchored on the modified electrode surface, remarkably diminishing the PEC signal and thus achieving sensitive DNA determination. The elaborated PEC biosensor demonstrated outstanding performance within the linear range between 0.5 fM and 5 nM and a low limit of detection down to 0.18 fM, paving a new way for fabricating heterojunction with exceptional photoactive performance and demonstrating the enormous potential for detecting multitudinous biomarkers in bioanalysis and clinical therapy.

Fullerenol as a photoelectrochemical nanoprobe for discrimination and ultrasensitive detection of amplification-free single-stranded DNA.

Traditional approaches for nucleic acids detection require prior amplification of target genes, while nanomaterials-aided DNA biosensors are very magnificent but still suffer from the nanomaterial acquirement and limited sensitivity (above picomolar level). Herein, fullerenol C60(OH)25, a representative fullerene derivative, was employed as a photoelectrochemical (PEC) nanoprobe to achieve discrimination and ultrasensitive detection of amplification-free single-stranded DNA (ssDNA) down to sub-femtomolar level. The bonded hydroxyl groups with intense density endowed fullerenol to directly recognize and capture ssDNA-AuNPs via the hydrogen bonding interactions (H-bonds), leading to a sharply decreased photocurrent with quenching efficiency up to 85%, which could be attributed to the photo-generated electrons on the conduction band of fullerenol (-4.66 eV) preferentially migrating to the Fermi level of AuNPs (-5.1 eV) rather than the electrode. In the presence of target gene (mutant human p53 gene fragment), the H-bonds between fullerenol and ssDNA were competitively depleted during the base pairing process of complete hybridization between ssDNA and target, making double-stranded DNA-AuNPs (dsDNA-AuNPs) depart so that the photocurrent powerfully recovered. On basis of the photocurrent variation before and after target introduction, this proposed simple, rapid and ultrasensitive PEC biosensor for amplification-free target gene detection illustrated a wide liner ranged from 1 fM to 100 pM and a detection limit of 0.338 fM. This work presented an ingenious strategy for the discrimination and ultrasensitive detection of nucleic acids, and the well-designed PEC biosensor was further conducive to the impetus of clinic diagnostics.

Crystal Structures of Cystathionine ß-Synthase from Saccharomyces cerevisiae: One Enzymatic Step at a Time.

Cystathionine ß-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H2S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation.

In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical "molecular switch" to promote differentiation should expand the utility of this method in basic research and clinical applications.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

Polarization-sensitive optical frequency domain imaging based on unpolarized light.

Polarization-sensitive optical coherence tomography (PS-OCT) is an augmented form of OCT, providing 3D images of both tissue structure and polarization properties. We developed a new method of polarization-sensitive optical frequency domain imaging (PS-OFDI), which is based on a wavelength-swept source. In this method the sample was illuminated with unpolarized light, which was composed of two orthogonal polarization states (i.e., separated by 180° in the Poincaré sphere) that are uncorrelated to each other. Reflection of these polarization states from within the sample was detected simultaneously and independently using a frequency multiplexing scheme. This simultaneous sample probing with two polarization states enabled determination of the depth-resolved Jones matrices of the sample. Polarization properties of the sample were obtained by analyzing the sample Jones matrices through eigenvector decomposition. The new PS-OFDI system ran at 31K wavelength-scans/s with 3072 pixels per wavelength-scan, and was tested by imaging a polarizer and several birefringent tissues such as chicken muscle and human skin. Lastly the new PS-OFDI was applied to imaging two cancer animal models: a mouse model by injecting cancer cells and a hamster cheek pouch model. These animal model studies demonstrated the significant differences in tissue polarization properties between cancer and normal tissues in vivo.

Zinc coordination geometry and ligand binding affinity: the structural and kinetic analysis of the second-shell serine 228 residue and the methionine 180 residue of the aminopeptidase from Vibrio proteolyticus.

The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure-function studies were carried out on the dizinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.

Differential regulation by magnesium of the two MsbB paralogs of Shigella flexneri.

Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.