Structural and functional study of a simple,rapid, and label‐free DNAzyme‐based DNA biosensor for optimization activity

Peroxidase‐mimicking DNAzyme has a potential to self‐assemble into a G‐quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase‐mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non‐labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV–Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G‐quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg²⁺. Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.     

Ultra‐sensitive,rapid gold nanoparticle‐quantum dot plexcitonic self‐assembled aptamer‐based nanobiosensor for the detection of human cardiac troponin I

Acute myocardial infarction (AMI) is one of the leading causes of death throughout the world. Usual methods for detecting AMI are expensive, time‐consuming and using blood samples as biological samples. Therefore, creating an ultra‐fast, sensitive and non‐invasive diagnostic test is necessary. Herein, a novel ultra‐sensitive, fluorescent, plasmon‐exciton coupling hybrid of a gold nanoparticle‐quantum dot (PQ)‐based aptamer nanobiosensor is presented for the detection of human cardiac troponin I (cTnI), the golden biomarker of AMI, and a preclinical test is performed within saliva. The binding of the cTnI protein to aptamer leads to a fluorescence enhancement of the plexcitonic hybrid system. The response range of this nanobiosensor is 0.4–2500 fM and the limit of detection is 0.3 fM. It seems that this novel design of nanobiosensor in the form of the PQ plexcitonic hybrid system can presents new opportunities for nanobiosensor progress.     

Binding of Tris to Bacillus licheniformis alpha-amylase can affect its starch hydrolysis activity

Bacillus licheniformis alpha-amylase (BLA) is routinely used as a model thermostable amylase in biochemical studies. Its starch hydrolysis activity has recently been studied in Tris buffer. Here, we address the question that whether the application of Tris buffer may influence the results of BLA activity analyses. Based on the inhibition studies and docking simulations, we suggest that Tris molecule is a competitive inhibitor of starch-hydrolyzing activity of BLA, and it has a high tendency to bind the enzyme active site. Hence, it is critically important to consider such effect when interpreting the results of activity studies of this enzyme in Tris buffer.     

Selecting the signals for a brain-machine interface

Brain-machine interfaces are being developed to assist paralyzed patients by enabling them to operate machines with recordings of their own neural activity. Recent studies show that motor parameters, such as hand trajectory, and cognitive parameters, such as the goal and predicted value of an action, can be decoded from the recorded activity to provide control signals. Neural prosthetics that use simultaneously a variety of cognitive and motor signals can maximize the ability of patients to communicate and interact with the outside world. Although most studies have recorded electroencephalograms or spike activity, recent research shows that local field potentials (LFPs) offer a promising additional signal. The decode performances of LFPs and spike signals are comparable and, because LFP recordings are more long lasting, they might help to increase the lifetime of the prosthetics.     

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.     

Ketamine/xylazine anesthesia for radiologic imaging of neurologically impaired rats: dose response, respiratory depression, and management of complications

In vivo imaging of rats represents an important tool for outcome evaluation in research on stroke, brain trauma, and other neurologic diseases. Since sedation of animals is necessary to avoid artifacts, a mixture of ketamine and xylazine is frequently used for anesthesia. We assessed the suitable dosage of narcotics and its correlation to severe respiratory adverse events in 269 cases of ketamine/xylazine anesthesia in male Wistar rats for performance of magnetic resonance imaging after middle cerebral artery occlusion (MCAO) or sham surgery. Anesthesia depth was not measured. Anesthesia was efficacious in avoiding movement artifacts during imaging. Necessary dosage was lower if rodents were subjected to MCAO instead of sham surgery, if body weight was below baseline, and if time since surgery was short. If anesthesia was induced during the first 2 days after surgery in animals with body weight loss, necessary dose rates were 27% below doses required for rats more than 10 days post-surgery with body weight above baseline (91.4/8.3 versus 125.1/11.3 mg of ketamine/xylazine/kg). A dose adaptation scale for the prediction of necessary dose rates was developed. Apnea developed in 3.3% of all animals. Use of ketamine/xylazine anesthesia for imaging procedures is feasible and safe, though it is associated with a small risk of respiratory arrest. In case of apnea, inspiration can be provoked by a puff of air into the rat's pelt. If unsuccessful, endotracheal intubation and mechanical ventilation are needed until spontaneous breathing is restored or xylazine effects are antagonized.     

A three-dimensional model of the human transglutaminase 1: insights into the understanding of lamellar ichthyosis

The stratum corneum, the outer layer of the epidermis, serves as a protective barrier to isolate the skin from the external environment. Keratinocyte transglutaminase 1 (TGase 1) catalyzes amide crosslinking between glutamine and lysine residues on precursor proteins forming the impermeable layers of the epidermal cell envelopes (CE), the highly insoluble membranous structures of the stratum corneum. Patients with the autosomal recessive skin disorder lamellar ichthyosis (LI) appear to have deficient cross-linking of the cell envelope due to mutations identified in TGase 1, linking this enzyme to LI. In the absence of a crystal structure, molecular modeling was used to generate the structure of TGase 1. We have mapped the known mutations of TGase 1 from our survey obtained from a search of PubMed and successfully predicted the impact of these mutations on LI. Furthermore, we have identified Ca²⁺ binding sites and propose that Ca²⁺ induces a cis to trans isomerization in residues near the active site as part of the enzyme transamidation activation. Docking experiments suggest that substrate binding subsequently induces the reverse cis to trans isomerization, which may be a significant part of the catalytic process. These results give an interpretation at the molecular level of previously reported mutations and lead to further insights into the structural model of TGase 1, providing a new basis for understanding LI. Figure Ribbon image of the model of the human TGase 1 structure. The side chains of residues reported to be mutated in patients with LI (34 amino acid mutation sites) are shown as spheres. This paper is dedicated to the memory of Dr. Peter M. Steinert (April 7, 2003).