Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

A framework for analysis of real-time nucleic acid amplification data using novel multidimensional standard curves

ABSTRACTResearch into improving methods for absolute quantification of nucleic acids using standard curves has plateaued despite its positive, far-reaching impact on biomedical applications and clinical diagnostics. Currently, the mathematics involved in this mature area is restricted by the simplicity of conventional standard curves such as the gold standard cycle-threshold (Ct) method. Here, we propose a novel framework that expands current methods into multidimensional space and opens the door for more complex mathematical techniques, signal processing and machine learning to be implemented. The heart of this work revolves around two new concepts: the multidimensional standard curve and its home - the feature space. This work has been validated using phage lambda DNA and standard qPCR instruments. We show that the capabilities of standard curves can be extended in order to simultaneously: enhance absolute quantification, detect outliers and provide insights into the intersection between molecular biology and amplification data. This work and its vision aims to maximise the information extracted from amplification data using current instruments without increasing the cost or complexity of existing diagnostic settings.

A New Subcritical Nanostructure of Graphene—Crinkle-Ruga Structure and Its Novel Properties

Here, we report an experimental characterization of a new subcritical graphene nanostructure termed a crinkle ruga. Multilayer graphene forms crinkles as a periodic mode of buckling if the ratio of periodic buckling span to thickness is smaller than a critical value. Otherwise, it forms wrinkles. The crinkles have sawtooth-shaped profiles with their faces perfectly flat and the tips of the peaks and valleys highly curved. Our AFM measurements show that the width of the curvature focusing band at the tip is very narrow, e.g. smaller than 16 nm for a 6o crinkle, indicating a strong influence of flexoelectric coupling in crinkle formation. We also found that concavity or convexity of crinkle tips, i.e. parity of the crinkle, can be controlled. Due to the flexoelectric coupling, the concave tip at the crinkle valley is positively charged, and the convex tip at the crinkle peak negatively charged. In addition, here, we demonstrate that the charges at the crinkle tips can attract macromolecules in adsorption experiments. We show linearly-aligned adsorption of C60 along crinkle valleys on an HOPG surface. In another experiment, we exhibit period-doubled adsorption of lambda DNA on an HOPG surface, possibly caused by ion kinetics involved in the DNA adsorption along the crinkle valleys.