A novel photoelectrochemical immunosensor by integration of nanobody and TiO2 nanotubes for sensitive detection of serum cystatin C.

Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO2 nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO2 nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM-3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO2 nanotubes.

Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae.

Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative to the wild-type strain. Our results indicated that different types of histone mutation resulted in different phenotypes and a distinct reorganization of nucleosomes. Nucleosome occupancy was altered at telomeres, but not at centromeres. The first nucleosomes upstream (-1) and downstream (+1) of the transcription start site (TSS) were more dynamic than other nucleosomes. Mutations in histones affected the nucleosome array downstream of the TSS. Highly expressed genes, such as ribosome genes and genes involved in glycolysis, showed increased nucleosome occupancy in many types of histone mutant. In particular, the H3K56A mutant exhibited a high percentage of dynamic genomic regions, decreased nucleosome occupancy at telomeres, increased occupancy at the +1 and -1 nucleosomes, and a slow growth phenotype under stress conditions. Our findings provide insight into the influence of histone mutations on nucleosome dynamics.

Characterization and applications of Nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library.

BACKGROUND: Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). Their unique advantageous properties make Nbs highly attractive in various applications. The general approach to obtain Nbs is to isolate them from immune libraries by phage display technology. However, it is unfeasible when the antigens are toxic, lethal, transmissible or of low immunogenicity. Naïve libraries could be an alternative way to solve the above problems. RESULTS: We constructed a large camel naïve phage display Nanobody (Nb) library with great diversity. The generated library contains to 6.86 × 10(11) clones and to our best of knowledge, this is the biggest naïve phage display Nb library. Then Nbs against human procalcitonin (PCT) were isolated from this library. These Nbs showed comparable affinity and antigen-binding thermostability at 37°C and 60°C compared to the PCT Nbs from an immune phage-displayed library. Furthermore, two PCT Nbs that recognize unique epitopes on PCT have been successfully applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect PCT, which showed a linear working range from 10-1000 ng/mL of PCT. CONCLUSION: We have constructed a large and diverse naïve phage display Nb library, which potentially functioning as a good resource for selecting antigen-binders with high quality. Moreover, functional Nbs against PCT were successfully characterized and applied, providing great values on medical application.

Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin.

The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.

Elevated CO2 alters the feeding behaviour of the pea aphid by modifying the physical and chemical resistance of Medicago truncatula.

Elevated CO(2) compromises the resistance of leguminous plants against chewing insects, but little is known about whether elevated CO(2) modifies the resistance against phloem-sucking insects or whether it has contrasting effects on the resistance of legumes that differ in biological nitrogen fixation. We tested the hypothesis that the physical and chemical resistance against aphids would be increased in Jemalong (a wild type of Medicago truncatula) but would be decreased in dnf1 (a mutant without biological nitrogen fixation) by elevated CO(2). The non-glandular and glandular trichome density of Jemalong plants increased under elevated CO(2), resulting in prolonged aphid probing. In contrast, dnf1 plants tended to decrease foliar trichome density under elevated CO(2), resulting in less surface and epidermal resistance to aphids. Elevated CO(2) enhanced the ineffective salicylic acid-dependent defence pathway but decreased the effective jasmonic acid/ethylene-dependent defence pathway in aphid-infested Jemalong plants. Therefore, aphid probing time decreased and the duration of phloem sap ingestion increased on Jemalong under elevated CO(2), which, in turn, increased aphid growth rate. Overall, our results suggest that elevated CO(2) decreases the chemical resistance of wild-type M. truncatula against aphids, and that the host's biological nitrogen fixation ability is central to this effect.

Antennal morphology and sensilla ultrastructure of three Tomicus species (Coleoptera: Curculionidae, Scolytinae).

The antennal morphology and sensilla ultrastructure of Tomicus yunnanensis, T. minor, and T. brevipilosus were studied by scanning electron microscopy and transmission electron microscopy. Eight common sensilla types were recorded: (1) sensilla trichodea (S.tr.) types 1 and 2 were located on the club and were innervated by five and eight dendrites, respectively; (2) sensilla chaetica (S.ch.) types 1 and 2 had no dendrites in the sensilla lymph lumen; (3) sensilla basiconica (S.b.), top-protuberated S.b. and fluted cones (Fl.c.) occurred on the club; and Böhm bristles (B.b.) occurred on the funicle. S.b. were the most abundant and were innervated by 10-14 dendritic branches. Top-protuberated S.b., a new sensilla type, were innervated by one dendrite. Fl.c. were innervated by five dendrites. Only three sensilla furcatea were visible on the antennae of female T. yunnanensis. The possible functions of these sensilla are discussed in relation to their morphology and ultrastructure. No statistical differences between sexes were found in the size and numbers of each sensilla type. Although the three species had similar antennal morphology and sensilla type, sensilla on sub-segments 3 and 4 of the antennal club of T. minor were much sparser than those of T. yunnanensis or T. brevipilosus. Concerning the antennae of T. yunnanensis, there were more S.tr. type 2, S.ch. type 2 and S.b. and the size of S.tr. type 1 and S.b. were significantly greater than those of T. minor.