Design of Nucleic Acid Biosensors Based on CRISPR/Cas Systems and Reporter Split Proteins.

Highly sensitive, specific, rapid, and easy-to-use diagnostic methods for the detection of nucleic acids of pathogens are required for the diagnosis of many human, animal, and plant diseases and environmental monitoring. The approaches based on the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with a high specificity under isothermal conditions are an alternative to the polymerase chain reaction method, which requires expensive laboratory equipment. The development of the methods for signal registration with the formation of a DNA/RNA/Cas9 protein complex is a separate bioengineering task. In this work, a design was developed and the applicability of a biosensor system based on the binding of two dCas9 proteins with target DNA sequences (without their cutting) and detection of their colocalization using reporter systems based on split enzymes was studied. Using the methods of molecular modeling, possible mutual positions of two dCas9 proteins at a detectable locus of genomic DNA, allowing the split enzyme domains attached to them to interact in an optimal way, were determined. The optimal distances on DNA between binding sites of dCas9 proteins in different orientations were determined, and the dependence of the complex structure on the distance between the binding sites of dCas9 proteins was modeled. Using the methods of bioinformatics, the genomes of a number of viruses (including SARS-CoV-2) were analyzed, and the presence of genomic loci unique to the species, allowing the possibility of landing pairs of dCas9 proteins in optimal positions, was demonstrated. The possibility of a combined use of dCas9 proteins from different bacteria to expand the spectrum of detected loci was analyzed. The results of the work indicate a fundamental possibility of the creation of highly specific nucleic acid biosensors based on a combination of CRISPR/Cas9 technologies and split enzymes.

Correlating results from high resolution EBSD with TEM- and ECCI-based dislocation microscopy: Approaching single dislocation sensitivity via noise reduction.

High resolution electron backscatter diffraction (HREBSD), an SEM-based diffraction technique, may be used to measure the lattice distortion of a crystalline material and to infer the geometrically necessary dislocation content. Uncertainty in the image correlation process used to compare diffraction patterns leads to an uneven distribution of measurement noise in terms of the lattice distortion, which results in erroneous identification of dislocation type and density. This work presents a method of reducing noise in HREBSD dislocation measurements by removing the effect of the most problematic components of the measured distortion. The method is then validated by comparing with TEM analysis of dislocation pile-ups near a twin boundary in austenitic stainless steel and with ECCI analysis near a nano-indentation on a tantalum oligocrystal. The HREBSD dislocation microscopy technique is able to resolve individual dislocations visible in TEM and ECCI and correctly identify their Burgers vectors.

Highly mobile ferroelastic domain walls in compositionally graded ferroelectric thin films.

Domains and domain walls are critical in determining the response of ferroelectrics, and the ability to controllably create, annihilate, or move domains is essential to enable a range of next-generation devices. Whereas electric-field control has been demonstrated for ferroelectric 180° domain walls, similar control of ferroelastic domains has not been achieved. Here, using controlled composition and strain gradients, we demonstrate deterministic control of ferroelastic domains that are rendered highly mobile in a controlled and reversible manner. Through a combination of thin-film growth, transmission-electron-microscopy-based nanobeam diffraction and nanoscale band-excitation switching spectroscopy, we show that strain gradients in compositionally graded PbZr1-xTixO3 heterostructures stabilize needle-like ferroelastic domains that terminate inside the film. These needle-like domains are highly labile in the out-of-plane direction under applied electric fields, producing a locally enhanced piezoresponse. This work demonstrates the efficacy of novel modes of epitaxy in providing new modalities of domain engineering and potential for as-yet-unrealized nanoscale functional devices.

Electronic Origins of Anomalous Twin Boundary Energies in Hexagonal Close Packed Transition Metals.

Density-functional-theory calculations of twin-boundary energies in hexagonal close packed metals reveal anomalously low values for elemental Tc and Re, which can be lowered further by alloying with solutes that reduce the electron per atom ratio. The anomalous behavior is linked to atomic geometries in the interface similar to those observed in bulk tetrahedrally close packed phases. The results establish a link between twin-boundary energetics and the theory of bulk structural stability in transition metals that may prove useful in controlling mechanical behavior in alloy design.

In situ and tomographic observations of defect free channel formation in ion irradiated stainless steels.

The effects of heavy-ion irradiation on dislocation processes in stainless steels were investigated using in situ irradiation and deformation in the transmission electron microscope as well as post mortem electron tomography to retrieve information on the three-dimensional dislocation state. Irradiation-induced defects were found to pose a strong collective barrier to dislocation motion, leading to dislocation pileups forming in grain interiors and at grain boundaries. The passage of multiple dislocations along the same slip plane removes the irradiation defects and leads to the eventual formation of a defect-free channel. These channels are composed of densely tangled dislocation networks which line the channel-matrix walls as well as residual dislocation debris in the channel interiors. The structures of the dislocation tangles were found to be similar to those encountered in later stages of deformation in unirradiated materials, with the exception that they developed earlier in the deformation process and were confined to the defect free channels. Also, defect free channels were found to widen through both source widening as well as complex cross-slip mechanisms.

miR-21 and miR-155 are associated with mitotic activity and lesion depth of borderline melanocytic lesions.

BACKGROUND: Expression of microRNAs (miRs) has been shown to be altered in many solid tumours and is being explored in melanoma. The malignant potential of some melanocytic lesions is difficult to predict. We hypothesised that characterisation of miR expression in borderline melanocytic proliferations would lead to the identification of a molecular profile that could be used with known prognostic factors to differentiate lesions with high malignant potential. METHODS: The miR expression profile of melanocytic lesions (benign naevi, malignant melanoma and borderline melanocytic tumours) was evaluated by real-time PCR. RESULTS: PCR analysis revealed primary cutaneous melanomas had an 8.6-fold overexpression of miR-21 and a 7.5-fold overexpression of miR-155 compared with benign naevi (P<0.0001). In situ hybridisation confirmed these results. miR-21 and miR-155 were significantly overexpressed within borderline lesions (P=0.0011 and P=0.0048, respectively). When borderline lesions were categorised by mitotic activity and Breslow thickness, miR-21 was associated with mitotic activity and miR-155 was associated with thickness (P<0.025). Among 14 patients with borderline lesions who underwent sentinel lymph node biopsy (SLNB), positive SLNB was associated with increased miR-21 and miR-155 in the primary lesion compared with lesions with a negative SLNB. CONCLUSION: MicroRNA expression profiles can be used to characterise atypical melanocytic lesions.