Detecting In Vivo Free Radicals in Various Disease Models

In vivo free radical imaging in pre-clinical models of disease is now possible. Free radicals have traditionally been characterized by ESR or EPR spin trapping spectroscopy. The disadvantage of the ESR/EPR approach is that spin adducts are short-lived due to biological reductive and/or oxidative processes. Immuno-spin trapping (IST) involves the use of an antibody that recognizes macromolecular DMPO-spin adducts (anti-DMPO antibody), regardless of the oxidative/reductive state of trapped radical adducts. The IST approach has been extended to an in vivo application that combines IST with molecular magnetic resonance imaging (mMRI). This combined IST-mMRI approach involves the use of a spin trapping agent, DMPO, to trap free radicals in disease models, and administration of a mMRI probe, an anti-DMPO probe, that combines an antibody against DMPO-radical adducts and a MRI contrast agent, resulting in targeted free radical adduct detection. The combined IST-mMRI approach has been used in several rodent disease models, including diabetes, ALS, gliomas, and septic encephalopathy. The advantage of this approach is that heterogeneous levels of trapped free radicals can be detected directly in vivo and in situ to pin-point where free radicals are formed in different tissues. The approach can also be used to assess therapeutic agents that are either free radical scavengers or generate free radicals. The focus of this review will be on the different applications that have been studied, advantages and limitations, and future directions

Region-specific dendritic simplification induced by Aβ, mediated by tau via dysregulation of microtubule dynamics: a mechanistic distinct event from other neurodegenerative processes.

BackgroundDendritic simplification, a key feature of the neurodegenerative triad of Alzheimer's disease (AD) in addition to spine changes and neuron loss, occurs in a region-specific manner. However, it is unknown how changes in dendritic complexity are mediated and how they relate to spine changes and neuron loss.ResultsTo investigate the mechanisms of dendritic simplification in an authentic CNS environment we employed an ex vivo model, based on targeted expression of enhanced green fluorescent protein (EGFP)-tagged constructs in organotypic hippocampal slices of mice. Algorithm-based 3D reconstruction of whole neuron morphology in different hippocampal regions was performed on slices from APPSDL-transgenic and control animals. We demonstrate that induction of dendritic simplification requires the combined action of amyloid beta (Aβ) and human tau. Simplification is restricted to principal neurons of the CA1 region, recapitulating the region specificity in AD patients, and occurs at sites of Schaffer collateral input. We report that γ-secretase inhibition and treatment with the NMDA-receptor antagonist, CPP, counteract dendritic simplification. The microtubule-stabilizing drug epothilone D (EpoD) induces simplification in control cultures per se. Similar morphological changes were induced by a phosphoblocking tau construct, which also increases microtubule stability. In fact, low nanomolar concentrations of naturally secreted Aβ decreased phosphorylation at S262 in a cellular model, a site which is known to directly modulate tau-microtubule interactions.ConclusionsThe data provide evidence that dendritic simplification is mechanistically distinct from other neurodegenerative events and involves microtubule stabilization by dendritic tau, which becomes dephosphorylated at certain sites. They imply that treatments leading to an overall decrease of tau phosphorylation might have a negative impact on neuronal connectivity

Discovery of Sex-Specific Regions in a Salamander Genome

Biological Aspects: Salamander (Ambystoma mexicanum) has a gigantic genome: ~32,000,000,000 bases (10X of size of human genome) Sex is determined by a pair of morphologically identical chromosomes: ZZ in male ZW in female Object: Find (if there are any) genomic differences between chromosomes W and Z Workflow: Sequencing and de novo assembly of the reference salamander genome Alignment of short sequences from male and female genomes to the reference Coverage analysi

Miniscule Differences Between Sex Chromosomes in the Giant Genome of a Salamander

In the Mexican axolotl (Ambystoma mexicanum), sex is determined by a single Mendelian factor, yet its sex chromosomes do not exhibit morphological differentiation typical of many vertebrate taxa that possess a single sex-determining locus. As sex chromosomes are theorized to differentiate rapidly, species with undifferentiated sex chromosomes provide the opportunity to reconstruct early events in sex chromosome evolution. Whole genome sequencing of 48 salamanders, targeted chromosome sequencing and in situ hybridization were used to identify the homomorphic sex chromosome that carries an A. mexicanum sex-determining factor and sequences that are present only on the W chromosome. Altogether, these sequences cover ~300 kb of validated female-specific (W chromosome) sequence, representing ~1/100,000th of the 32 Gb genome. Notably, a recent duplication of ATRX, a gene associated with mammalian sex-determining pathways, is one of few functional (non-repetitive) genes identified among these W-specific sequences. This duplicated gene (ATRW) was used to develop highly predictive markers for diagnosing sex and represents a strong candidate for a recently-acquired sex determining locus (or sexually antagonistic gene) in A. mexicanum

In Vitro Phase-Contrast Magnetic Resonance Investigation on Development of Human Carotid Sinus in Young Age

Figure 1 . Three dimensional reconstruction process of morphologically averaged human carotid artery bifurcations using the measurements of arterial dimensions from two orthogonal (AP and LAT) projections

A Chromosome-Scale Assembly of the Axolotl Genome

The axolotl (Ambystoma mexicanum) provides critical models for studying regeneration, evolution, and development. However, its large genome (∼32 Gb) presents a formidable barrier to genetic analyses. Recent efforts have yielded genome assemblies consisting of thousands of unordered scaffolds that resolve gene structures, but do not yet permit large-scale analyses of genome structure and function. We adapted an established mapping approach to leverage dense SNP typing information and for the first time assemble the axolotl genome into 14 chromosomes. Moreover, we used fluorescence in situ hybridization to verify the structure of these 14 scaffolds and assign each to its corresponding physical chromosome. This new assembly covers 27.3 Gb and encompasses 94% of annotated gene models on chromosomal scaffolds. We show the assembly\u27s utility by resolving genome-wide orthologies between the axolotl and other vertebrates, identifying the footprints of historical introgression events that occurred during the development of axolotl genetic stocks, and precisely mapping several phenotypes including a large deletion underlying the cardiac mutant. This chromosome-scale assembly will greatly facilitate studies of the axolotl in biological research

Skin biopsy analysis of concurrent keloidal morphoea and systemic sclerosis confirms overlapping pathogenic pathways

OBJECTIVES: Although localised forms of scleroderma (morphoea) have very different clinical features and outcomes from systemic sclerosis the two conditions can occur together in some patients. In this study we have explored skin gene expression in a series of patients with keloidal morphoea, a distinct clinical variant, concurrently with systemic sclerosis. METHODS: We have compared skin gene expression from the keloidal lesions with that from skin elsewhere. We have also examined a series of patients with diffuse or limited cutaneous SSc without morphoea and some healthy control skin biopsies. RESULTS: Keloidal morphoea has a distinct gene expression signature that is mainly driven by differential expression of fibroblast related genes compared with other cell types. Indeed, the signature reflects a profibrotic pattern seen in diffuse cutaneous SSc but is much more extreme. We propose that keloidal morphoea skin provides a unique insight into the profibrotic population of cells driving dcSSc. CONCLUSIONS: Understanding the biology of keloidal morphoea may give valuable insight into the molecular and cellular pathology of systemic sclerosis. The discrete nature of keloidal lesions raises the possibility of haematogenous spread and we suggest that the driving cells could represent blood derived cells derived from circulating progenitors

Initial Characterization of the Large Genome of the Salamander \u3cem\u3eAmbystoma mexicanum\u3c/em\u3e Using Shotgun and Laser Capture Chromosome Sequencing

Vertebrates exhibit substantial diversity in genome size, and some of the largest genomes exist in species that uniquely inform diverse areas of basic and biomedical research. For example, the salamander Ambystoma mexicanum (the Mexican axolotl) is a model organism for studies of regeneration, development and genome evolution, yet its genome is ~10× larger than the human genome. As part of a hierarchical approach toward improving genome resources for the species, we generated 600 Gb of shotgun sequence data and developed methods for sequencing individual laser-captured chromosomes. Based on these data, we estimate that the A. mexicanum genome is ~32 Gb. Notably, as much as 19 Gb of the A. mexicanum genome can potentially be considered single copy, which presumably reflects the evolutionary diversification of mobile elements that accumulated during an ancient episode of genome expansion. Chromosome-targeted sequencing permitted the development of assemblies within the constraints of modern computational platforms, allowed us to place 2062 genes on the two smallest A. mexicanum chromosomes and resolves key events in the history of vertebrate genome evolution. Our analyses show that the capture and sequencing of individual chromosomes is likely to provide valuable information for the systematic sequencing, assembly and scaffolding of large genomes

The Lamprey Genome: Illuminating Genomic Change across Eons and Embryogenesis

The lamprey genome provides unique insights into both the deep evolutionary history of vertebrate genomes and the maintenance of genome structure/integrity over development. The lamprey lineage diverged from all other vertebrates approximately 500 million years ago. As such, comparisons between lamprey and other vertebrates permit reconstruction of ancient duplication and rearrangement events that defined the fundamental architecture and gene content of all extant vertebrate genomes. Lamprey also undergoes programmatic changes genome structure that result in the physical elimination of ~20% of its genomic DNA (~0.5Gb from a ~2 Gb genome) from all somatic cell lineages during early embryonic development. Here, we outline recent progress in assembly and analysis of the lamprey germline genome, and progress in the development of methods for characterizing the cellular events that mediate DNA elimination. We have integrated information from several sampling approaches and sequencing technologies to achieve a highly contiguous assembly of lamprey genome (including: Illumina fragments/mate pairs, 20X coverage in Pacific Biosciences reads, dense meiotic maps and optical mapping data). This genome assembly has dramatically improved our ability to dissect the molecular basis and genetic outcomes of programmed genome rearrangements (PGRs), and has improved our understanding of the tempo and mode of large-­scale duplications and translocations within the ancestral vertebrate lineage. Analysis of the germline genome identifies several genes that are expressed in germline but physically eliminated from all somatic tissues. These eliminated genes correspond to several known oncogenes and appear to identify several other novel oncogene candidates. Complementing this assembly, the development of approaches to in situ analysis of 3D preserved cells has revealed that PGR unfolds through a series of dramatic cellular events that involve the programmatic alteration of several fundamental mechanisms of genome maintenance, including: alignment of chromosomes at metaphase, chromatid cohesion, separation and segregation, and nuclear envelope formation