Surface Display of Bacterial Metallothioneins and a Chitin Binding Domain on Escherichia coli Increase Cadmium Adsorption and Cell Immobilization

To increase the level of adsorption of cadmium ions to the surface of Escherichia coli, we fused cyanobacterial metallothioneins, SmtA (from Synechococcus elongatus PCC 3601) and MtnA (from Synechococcus vulcanus) to the E. coli cell surface using a Lpp??-OmpA-based display system. E. coli strains expressing Lpp??-OmpA?CSmtA-linker-ChBD (chitin-binding domain from Bacillus pumillus SG2 chitinase S; chiS) and Lpp??-OmpA?CMtnA-linker-ChBD on their surface adsorbed more cadmium compared to the E. coli cells expressing only the Lpp??-OmpA-linker-ChBD hybrid. These constructs also were bound to chitin through their chitin-binding domain, allowing them to be immobilized on a chitin matrix. We assessed surface presentation of Lpp??-OmpA?CSmtA-linker-ChBD, Lpp??-OmpA?CMtnA-linker-ChBD, and Lpp??-OmpA-linker-ChBD using immunostaining. The Lpp??-OmpA?CSmtA-linker-ChBD chimera adsorbed metal and was bound to chitin with the highest efficiency compared to the other chimeras, suggesting that it is an effective bioadsorbent. This is the first example of coupling metal adsorption with cell immobilization using a whole-cell bioadsorbent.     

Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.     

Active vibration control using optimized modified acceleration feedback with Adaptive Line Enhancer for frequency tracking

Modified acceleration feedback (MAF) control, an active vibration control method that uses collocated piezoelectric actuators and accelerometer is developed and its gains optimized using an optimal controller. The control system consists of two main parts: (1) frequency adaptation that uses Adaptive Line Enhancer (ALE) and (2) an optimized controller. Frequency adaptation method tracks the frequency of vibrations using ALE. The obtained frequency is then fed to MAF compensators. This provides a unique feature for MAF, by extending its domain of capabilities from controlling a certain mode of vibrations to any excited mode. The optimized MAF controller can provide optimal sets of gains for a wide range of frequencies, based on the characteristics of the system. The experimental results show that the frequency tracking method works quite well and fast enough to be used in a real-time controller. ALE parameters are numerically and experimentally investigated and tuned for optimized frequency tracking. The results also indicate that the MAF can provide significant vibration reduction using the optimized controller. The control power varies for vibration suppression at different resonance frequencies; however, it is always optimized.     

Tuning the primary resonances of a micro resonator,using piezoelectric actuation

Microbeam dynamics is important in MEMS filters and resonators. In this research, the effect of piezoelectric actuation on the resonance frequencies of a piezoelectrically actuated capacitive clamped-clamped microbeam is studied. The microbeam is sandwiched with piezoelectric layers throughout its entire length. The lower piezoelectric layer is exposed to a combination of a DC and a harmonic excitation voltage. The DC electrostatic voltage is applied to prevent the doubling of the excitation frequency. The traditional resonators are tuned using DC electrostatic actuation, which tunes the resonance frequency only in backward direction on the frequency domain. The proposed model enables tuning the resonance frequencies in both forward and backward directions. For small amplitudes of harmonic excitation and high enough quality factor, the frequency response curves obtained by the shooting method are validated with those of the multiple time scales technique. Unlike the perturbation technique, which imposes limitation on both the amplitude of the harmonic excitation and the quality factor to be applicable, the shooting method can be applied to capture the periodic attractors regardless of how big the amplitude of harmonic excitation and the quality factor are.     

Electromagnetic fields with 217 Hz and 0.2 mT as hazardous factors for tubulin structure and assembly (in vitro study)

Nowadays exposure to extremely low frequency electromagnetic fields (ELF-EMFs) is an unavoidable fact in human societies. In spite of destructive effects of ELF-EMF such as impression on short-term memory and cognitive performances, neuroprotective and neurogeneration effects of ELF-EMF have been reported. On the other hand, microtubule (MT) proteins, the dynamic cytoskeleton proteins that are mostly located in axons, are able to produce local EMFs and possess key role in memory formation and consciousness. Therefore, MTs have strong potential to be affected by exogenous ELF-EMFs. In the present study, decline of MT polymerization was shown by transmission electron microscopy images and turbidimetry method after exposure to ELF-EMFs with 50, 100, and 217 Hz frequencies at 0.2 mT intensity. Circular dichroism and 8-anilino-1-naphthalenesulfonate (ANS) fluorometry were used to demonstrate the MT secondary and third structural changes. Decrement of MT polymerization through disruption of tubulin native structure, and increasing the β-sheets and random coils were observed in all exposure conditions. Therefore, ELF-EMFs with 50, 100, and specially 217 Hz frequencies at 0.2 mT could lead to disruption of MT functions in the neural cells.