Manual segmentation of the paraventricular nucleus of the hypothalamus and the dorsal and ventral bed nucleus of stria terminalis using multimodal 7 Tesla structural MRI: probabilistic atlases for a stress-control triad.

The paraventricular nucleus of the hypothalamus (PVN) is uniquely capable of proximal control over autonomic and neuroendocrine stress responses, and the bed nucleus of the stria terminalis (BNST) directly modulates PVN function, as well as playing an important role in stress control itself. The dorsal BNST (dBNST) is predominantly preautonomic, while the ventral BNST (vBNST) is predominantly viscerosensory, receiving dense noradrenergic signaling. Distinguishing the dBNST and vBNST, along with the PVN, may facilitate our understanding of dynamic interactions among these regions. T1-weighted MPRAGE and high resolution gradient echo (GRE) modalities were acquired at 7T. GRE was coregistered to MPRAGE and segmentations were performed in MRIcroGL based on their Atlas of the Human Brain depictions. The dBNST, vBNST and PVN were manually segmented in 25 participants; 10 images were rated by 2 raters. These segmentations were normalized and probabilistic atlases for each region were generated in MNI space, now available as resources for future research. We found moderate-high inter-rater reliability [n = 10; Mean Dice (SD); PVN = 0.69 (0.04); dBNST = 0.77 (0.04); vBNST = 0.62 (0.04)]. Probabilistic atlases were reverse normalized into native space for six additional participants that were segmented but not included in the original 25. We also found moderate to moderate-high reliability between the probabilistic atlases and manual segmentations [n = 6; Mean Dice (SD); PVN = 0.55 (0.12); dBNST = 0.60 (0.10); vBNST = 0.47 (0.12 SD)]. By isolating these hypothalamic and BNST subregions using ultra-high field MRI modalities, more specific delineations of these regions can facilitate greater understanding of mechanisms underlying stress-related function and psychopathology.

Efficient Delivery of Plasmid DNA Using Incorporated Nucleotides for Precise Conjugation of Targeted Nanoparticles.

Many obstacles restrict development of DNA plasmid-based therapeutic delivery, involving but not limited to poor cellular uptake, premature material dissociation, and inefficient response. Additionally, lack of precision loading of the plasmids on the carrier nanoparticle may affect the overall nonspecificity in terms of loading as well as the site of loading. Here we report a strategy using the incorporation of a biotin-modified nucleotide into a 4.7 kb plasmid sequence for the site-specific nanoparticle conjugation as an improvement on targeted DNA plasmid delivery. Initially, a designed 80-nucleotide sequence was elongated by incorporating biotin-16-aminoallyl-2'-dCTP that facilitated streptavidin binding as determined via polyacrylamide gel electrophoresis (PAGE). This modified sequence was ligated into a specific location of the EGFP plasmid to avoid possible interference with important functional elements and gene expression off of the plasmid. In parallel, a gold nanoparticle complex comprising of either a CD44 or mutant DNA conjugated aptamer, a PEGylated streptavidin, and a derivatized hyaluronic acid stabilizing polymer was synthesized. To delineate the ability of this nanoparticle-plasmid complex to exhibit an improved cellular delivery, MDA-MB-231 cells were treated with a set of plasmid and plasmid-nanoparticle complexes. Successful expression of EGFP was only observed in cells treated with the biotin-modified EGFP plasmid and a streptavidin-CD44 aptamer-nanoparticle. This demonstrated the need for the specific biotin-streptavidin binding to avoid nanoparticle-plasmid dissociation for improved efficacy. This proof-of-principle concept creates a flexible scaffold that can be assimilated into any plasmid and can produce small RNAs or encoding a therapeutic gene via an installation of a design that uses incorporated modified nucleotides as tethering points for nanoparticles which can play host to stabilizing ligands, additional therapeutic molecules and antibody conjugates among other possibilities. In our system, the nanoparticles are vehicles for the addition of targeting ligands that were essential for cell specificity and enhanced cellular uptake.