Structure Characterization of Silk Fibroin Crystalline Domain Polypeptides Expressed in Escherichia coli

The molecular conformations of four silk fibroin crystalline analogues [GAGAG-X] 16(G,Gly;A,Ala;X=Ala,Ser,Tyr or Val,designated eGA,eGS,eGY or eGV),carried out using molecular design and expressed by Escherichia coil(E.coli),were evaluated by Raman spectra analysis.The abilities of forming β-sheet structure were determined by thioflavin T(ThT) fluorescence spectra analysis.In terms of molecular conformation,except eGY that could not form significant typical molecular conformation,eGS and eGV were mainly composed of β-sheets while eGA tended to form β-turn.β-turn was also present in eGY and absent in eGS and eGV.In terms of β-sheet structure,eGS had the highest β-sheet content,followed by eGV,and eGA had the lowest content,furthermore,β-sheet structures were more stable in eGS and eGV than those in eGA and eGY.

Label-free fluorescent strategy for sensitive detection of tetracycline based on triple-helix molecular switch and G-quadruplex

In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T（Th T） as a signal transduction probe（STP）. In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to Th T which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

A Label-free and Functional Fluorescent Oligonucleotide Probe Based on a G-Quadruplex Molecular Beacon for the Detection of Kanamycin

A label-free and turn-off fluorescent method for the quantitative detection of kanamycin based on a funtional molecular beacon was developed. The molecular beacon consists of two hairpin structures with a split G-rich oligonucleotide in the middle. The kanamycin＇s aptamer formed the loops portion for recognizing kanamycin, and the G-quadruplex bound by Thioflavin T（ThT） was employed as the reporter. In the absence of target, the molecular beacon folded into double stem-loops and the splited G-rich oligonucleotid came close to form a G-quadruplex. When ThT bound to the G-quadruplex, the fluorescence intensity of the solution increased. Upon the addition of kanamycin, the function between kanamycin and aptamer unfolded the hairpin and disassembled the G-quadraplex structure, resulting in a significant decrease in the fluorescence intensity. A good linear relationship ranging from 0.7 nmol/L to 10 nmol/L was achieved and the limit of detection was 0.37 nmol/L. Besides, it could efficiently recognize kanamycin in real samples.

Phytomedicines as potential inhibitors of β amyloid aggregation: significance to Alzheimer's disease

Throughout the history of drug development, plants have been an important source for the discovery of novel therapeutically active compounds for many diseases. The ethnopharmacological approach has provided several leads to identify potential new drugs from plant sources, including those for memory disorders. For the treatment of Alzheimer's disease the drug discovery focus shifted from cholinesterase inhibitors, to other targets primarily based on two key neuropathological hallmarks, namely the hyperphosphorylation of the tau protein resulting in the formation of neurofibrillary tangles(NFTs), and the increased formation and aggregation of amyloid-beta peptide(Aβ) derived from amyloid precursor protein(APP). The present article aims to provide a comprehensive literature survey of plants and their constituents that have been tested for Aβ aggregation, thus possibly relieving several features of Alzheimer's disease(AD).

Single-molecule investigation of human telomeric G-quadruplex interactions with Thioflavin T

G-quadruplex ligands have been accepted as potential therapeutic agents for anticancer treatment. Thioflavin T （ThT）, a highly selective G-quadruplex ligand, can bind G-quadruplex with a fluorescent light-up signal change and high specificity against DNA duplex. However, there are still different opinions that ThT induces/stabilizes G-quadruplex foldings/topologies in human telomere sequence. Here, a sensitive single-molecule nanopore technology was utilized to analyze the interactions between human telomeric DNA （Tel DNA） and ThT. Both translocation time and current blockade were measured to investigate the translocation behaviors. Furthermore, the effects of metal ion （K~ and Na~） and pH on the translocation behaviors were studied. Based on the single-molecule level analysis, there are some conclusions： （1） In the electrolyte solution containing 50 mmol/L I（Cl and 450 mmol/L NaCl, ThT can bind strongly with Tel DNA but nearly does not change the G-quadruplex form; （2） in the presence of high concentration K~, the ThT binding induces the structural change of hybrid G-quadruplex into antiparallel topology with an enhanced structural stability; （3） In either alkaline or acidic buffer, G-quadruplex form will change in certain degree. All above conclusions are helpful to deeply understand the interaction behaviors between Tel DNA and ThT. This nanopore platform, investigating G-quadruplex ligands at the single-molecule level, has great potential for the design of new drugs and sensors.

Associations of cerebrospinal fluid amyloidogenic nanoplaques with cytokines in Alzheimer’s disease

Background:The aggregation of amyloidβ(Aβ)is central in the pathogenesis of Alzheimer’s disease(AD).Recently it has been shown that specifically,larger,Thioflavin T-binding Aβaggregates are associated with increased neuroinflammation and cytokine release.This study was aimed to quantify fibrillary amyloid aggregates,so-called nanoplaques,and investigate their relationship with cytokines in the cerebrospinal fluid(CSF).Methods:CSF was collected from 111 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic.The patients were grouped based on their amyloid status.The CSF nanoplaque concentration was quantified with the Thioflavin T-fluorescence correlation spectroscopy(ThT-FCS)assay.The levels of nine cytokines(eotaxin-1,granulocyte stimulating factor,interleukin[IL]-6,IL-7,IL-8,monocyte chemoattractant protein-1,gammainduced protein 10,macrophage inflammatory protein[MIP]-1α,and MIP-1β)were quantified with a magnetic bead-based multiplex assay and read on a Luminex IS 200 instrument.Results:There were 49 amyloid-negative and 62 amyloid-positive patients in the cohort;none of the cytokines differed significantly between the amyloid groups.The increased nanoplaque levels were associated with levels of MIP-1βbelow the lower limit of quantification,and with decreased levels of MIP-1αand IL-8.The associations remained significant when adjusted for age,sex,cognitive function,apolipoproteinε4 status and CSF core biomarker levels.Conclusion:The cytokine levels were not associated with amyloid status in this cohort.The nanoplaque levels were negatively associated with MIP-1β,MIP-1αand IL-8,which is in line with recent findings suggesting that the upregulation of some cytokine markers has a protective role and is negatively associated with AD progression.