Sensing of mycobacterial arabinogalactan by galectin‐9 exacerbates mycobacterial infection

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.     

Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: application to the diagnosis of Japanese encephalitis virus

A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.     

Carbon nanotube powders as electrode modifier to enhance the activity of anodic biofilm in microbial fuel cells

Carbon nanotube (CNT) is a promising electrode material and has been used as an anode modifier in microbial fuel cells (MFCs). In this study, a new method of simultaneously adding CNT powders and Geobacter sulfurreducens into the anode chamber of a MFC was used, aiming to form a composite biofilm on the anode. The performance of MFCs such as startup time and steady-state power generation was investigated under conditions of different CNT powders dosages. Results showed that both the startup time and the anodic resistance were reduced. The optimal dosage of CNT powders pre-treated by acid was 4 mg/mL for the anode chamber with an effective volume of 25 mL. The anodic resistance and output voltage of the MFC with CNT powders addition were maintained around 180 Ω and 650 mV during 40 days operation, while those of the MFC without CNT powders addition increased from 250 Ω to 540 Ω and decreased from 630 mV to 540 mV, respectively, demonstrating that adding CNT powders helped stabilize the anodic resistance, thus the internal resistance and power generation during long-term operation. Based on cyclic voltammogram, the electrochemical activity of anodic biofilm was enhanced by adding CNT powders, though no significant increase of the biomass in anodic biofilm was detected by phospholipids analysis. There was no remarkable change of ohmic resistance with an addition of CNT powders revealed by current interrupt method, which indicated that the rate of mass transfer might be promoted by the presence of CNT powders.     

Nickel oxide microfibers immobilized onto electrode by electrospinning and calcination for nonenzymatic glucose sensor and effect of calcination temperature on the performance

Nickel oxide microfibers (NiO-MFs) were directly immobilized onto the surface of fluorine tin oxide (FTO) electrode by electrospinning and calcination without using any immobilization matrix for nonenzymatic glucose sensor. Morphology and structure of NiO-MFs were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction pattern (XRD). The electrochemical and electrocatalytic performances of the NiO-MFs modified electrodes prepared at different calcination temperatures ranging from 300 to 500°C were evaluated by cyclic voltammetry (CV). The CV results have demonstrated that NiO-MFs modified electrode prepared at 300°C displayed distinct increase in electrocatalytic activity toward the oxidation of glucose, which is explored to develop an amperometric nonenzymatic glucose sensor. The NiO-MFs prepared at 300°C based amperometric nonenzymatic glucose sensor has ultrasensitive current (1785.41 μA mM(-1) cm(-2)) response and low detection limit of 3.3×10(-8) M (signal/noise ratio (S/N)=3), which are among the best values reported in literature. Additionally, excellent selectivity and stability have also been obtained.     

Phylogenetic diversity and characterization of 2-haloacid degrading bacteria from the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis>

A total of 139 2-haloacid degrading bacteria strains were isolated from the marine sponge Hymeniacidon perlevis using a modified enrichment medium and a pH indicator method. After screening on indicator agar and 2-chloropropionic acid (2-CPA) liquid medium, 11 isolates with high degrading activities were characterized and initially identified. Seven of the 11 isolates were able to degrade 2-CPA at 8% salt, and four isolates (DEH 66, DEH 99, DEH125 and DEH138) degraded 2-CPA at 15% salt. Eight of the 11 isolates utilized all four types of organohalogen compounds used in this study. The DEH99 and DEH138 isolates exhibited the best enantioselectivity towards (S)-2-chloropropionic acid (S-CPA) and (R)-2-chloropropionic acid (R-CPA), respectively. The dehalogenase activities of DEH84 against racemic CPA, DEH99 against S-CPA, DEH138 against R-CPA and DEH130 against racemic CPA were 0.16U/mg, 0.06U/mg, 0.12U/mg and 0.19U/mg, respectively. Based on 16S rRNA sequence analysis, the 11 isolates were clustered into the Rhodobacteraceae family of α-proteobacteria and the Pseudomonadaceae family of γ-proteobacteria. To our knowledge, this is the first report detailing the isolation of organisms of Pseudomonas stuzeri sp. and the Rhodobacteraceae family with 2-haloacid dehalogenase activity from marine sponges.     

Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds

Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation.     

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.     

Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin

Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 μg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.     

A fungal enzyme with the ability of aflatoxin B₁ conversion: purification and ESI-MS/MS identification

Armillariella tabescens, a Chinese edible and medicinal fungus, whose multienzyme exist ability of AFB₁-converting, and ADTZ (aflatoxin-detoxizyme) had previously purified from the A. tabescens multienzyme monitored by AFB₁ conversion_. However, the enzyme now confirmed an oxidase and renamed aflatoxin-oxidase (AFO). In this paper, AFO was purified by an economical and practical three-step procedure monitored by AFB₁ conversion. And ESI-MS/MS analysis was done for identification of AFO. The following database searching (Protein Blast on NCBI) results did not show any homologous oxidase protein, which implied that AFO was mostly a new oxidase differing from other reported aflatoxin-converting enzymes such as fungal laccase and horse radish peroxidase. HPTLC analysis of the purified AFO activity suggested that the enzyme reacted at the bisfuran ring of AFB₁ which was the key toxic structure. Therefore, all these investigations implied a new choice for biodegradation of aflatoxin in foods and feeds with the practical application of AFO.