Acquisition and Analysis of Excised Neocortex from Pediatric Patients with Focal Cortical Dysplasia Using Mesoscale Diffusion MRI.

Non-invasive classification of focal cortical dysplasia (FCD) subtypes remains challenging from a radiology perspective. Quantitative imaging biomarkers (QIBs) have the potential to distinguish subtypes that lack pathognomonic features and might help in defining the extent of abnormal connectivity associated with each FCD subtype. A key motivation of diagnostic imaging is to improve the localization of a "lesion" that can guide the surgical resection of affected tissue, which is thought to cause seizures. Conversely, surgical resections to eliminate or reduce seizures provided unique opportunities to develop magnetic resonance imaging (MRI)-based QIBs by affording long scan times to evaluate multiple contrast mechanisms at the mesoscale (0.5 mm isotropic voxel dimensions). Using ex vivo hybrid diffusion tensor imaging on a 9.4 T MRI scanner, the grey to white matter ratio of scalar indices was lower in the resected middle temporal gyrus (MTG) of two neuropathologically confirmed cases of FCD compared to non-diseased control postmortem fixed temporal lobes. In contrast, fractional anisotropy was increased within FCD and also adjacent white matter tracts. Connectivity (streamlines/mm3) in the MTG was higher in FCD, suggesting that an altered connectivity at the lesion locus can potentially provide a tangible QIB to distinguish and characterize FCD abnormalities. However, as illustrated here, a major challenge for a robust tractographical comparison lies in the considerable differences in the ex vivo processing of bioptic and postmortem samples. Mesoscale diffusion MRI has the potential to better define and characterize epileptic tissues obtained from surgical resection to advance our understanding of disease etiology and treatment.

E. coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA.

Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.

Stretching DNA to quantify nonspecific protein binding.

Nonspecific binding of regulatory proteins to DNA can be an important mechanism for target search and storage. This seems to be the case for the lambda repressor protein (CI), which maintains lysogeny after infection of E. coli. CI binds specifically at two distant regions along the viral genome and induces the formation of a repressive DNA loop. However, single-molecule imaging as well as thermodynamic and kinetic measurements of CI-mediated looping show that CI also binds to DNA nonspecifically and that this mode of binding may play an important role in maintaining lysogeny. This paper presents a robust phenomenological approach using a recently developed method based on the partition function, which allows calculation of the number of proteins bound nonspecific to DNA from measurements of the DNA extension as a function of applied force. This approach was used to analyze several cycles of extension and relaxation of λ DNA performed at several CI concentrations to measure the dissociation constant for nonspecific binding of CI (~100 nM), and to obtain a measurement of the induced DNA compaction (~10%) by CI.