Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation^1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)⁴. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces^5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs^4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating⁹. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed.After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.     

Comparison of F(ST) outlier tests for SNP loci under selection

Genome scans with many genetic markers provide the opportunity to investigate local adaptation in natural populations and identify candidate genes under selection. In particular, SNPs are dense throughout the genome of most organisms and are commonly observed in functional genes making them ideal markers to study adaptive molecular variation. This approach has become commonly employed in ecological and population genetics studies to detect outlier loci that are putatively under selection. However, there are several challenges to address with outlier approaches including genotyping errors, underlying population structure and false positives, variation in mutation rate and limited sensitivity (false negatives). In this study, we evaluated multiple outlier tests and their type I (false positive) and type II (false negative) error rates in a series of simulated data sets. Comparisons included simulation procedures (FDIST2, ARLEQUIN v.3.5 and BAYESCAN) as well as more conventional tools such as global F(ST) histograms. Of the three simulation methods, FDIST2 and BAYESCAN typically had the lowest type II error, BAYESCAN had the least type I error and Arlequin had highest type I and II error. High error rates in Arlequin with a hierarchical approach were partially because of confounding scenarios where patterns of adaptive variation were contrary to neutral structure; however, Arlequin consistently had highest type I and type II error in all four simulation scenarios tested in this study. Given the results provided here, it is important that outlier loci are interpreted cautiously and error rates of various methods are taken into consideration in studies of adaptive molecular variation, especially when hierarchical structure is included.     

Assay of the multiple energy-producing pathways of mammalian cells

Background

To elucidate metabolic changes that occur in diabetes, obesity, and cancer, it is important to understand cellular energy metabolism pathways and their alterations in various cells.

Methodology and Principal Findings

Here we describe a technology for simultaneous assessment of cellular energy metabolism pathways. The technology employs a redox dye chemistry specifically coupled to catabolic energy-producing pathways. Using this colorimetric assay, we show that human cancer cell lines from different organ tissues produce distinct profiles of metabolic activity. Further, we show that murine white and brown adipocyte cell lines produce profiles that are distinct from each other as well as from precursor cells undergoing differentiation.

Conclusions

This technology can be employed as a fundamental tool in genotype-phenotype studies to determine changes in cells from shared lineages due to differentiation or mutation.     

Chromosomal instability by mutations in the novel minor spliceosome component CENATAC

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12‐dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT‐AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT‐AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.     

Retinoids for treatment of Alzheimer's disease

Retinoids are Vitamin A derivatives involved in cellular regulatory processes including cell differentiation, neurite outgrowth and defense against oxidative stress. Retinoids may also influence Amyloid beta processing upregulation of alpha secretase via ADAM10. Vitamin A and other retinoids also directly inhibit formation of Amyloid fibrils in vivo. These properties of retinoids are relevant to theories of Alzheimer's disease pathogenesis. Retinoids are already used in treatment of acne vulgaris, psoriasis, neuroblastoma and acute promyelocytic leukemia. Clinical studies involving in cognitively impaired older adults with Alzheimer's disease are beginning with a variety of retinoids. These studies need to address safety issues of retinoids in older populations, and hold hope for demonstrating efficacy in translating these basic mechanisms to treatment of a widespread dementing illness.     

Potent and orally efficacious benzothiazole amides as TRPV1 antagonists

Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.     

Supportive breeding boosts natural population abundance with minimal negative impacts on fitness of a wild population of Chinook salmon

While supportive breeding programmes strive to minimize negative genetic impacts to populations, case studies have found evidence for reduced fitness of artificially produced individuals when they reproduce in the wild. Pedigrees of two complete generations were tracked with molecular markers to investigate differences in reproductive success (RS) of wild and hatchery‐reared Chinook salmon spawning in the natural environment to address questions regarding the demographic and genetic impacts of supplementation to a natural population. Results show a demographic boost to the population from supplementation. On average, fish taken into the hatchery produced 4.7 times more adult offspring, and 1.3 times more adult grand‐offspring than naturally reproducing fish. Of the wild and hatchery fish that successfully reproduced, we found no significant differences in RS between any comparisons, but hatchery‐reared males typically had lower RS values than wild males. Mean relative reproductive success (RRS) for hatchery F₁ females and males was 1.11 (P = 0.84) and 0.89 (P = 0.56), respectively. RRS of hatchery‐reared fish (H) that mated in the wild with either hatchery or wild‐origin (W) fish was generally equivalent to W × W matings. Mean RRS of H × W and H × H matings was 1.07 (P = 0.92) and 0.94 (P = 0.95), respectively. We conclude that fish chosen for hatchery rearing did not have a detectable negative impact on the fitness of wild fish by mating with them for a single generation. Results suggest that supplementation following similar management practices (e.g. 100% local, wild‐origin brood stock) can successfully boost population size with minimal impacts on the fitness of salmon in the wild.     

Nuclear-Cytoplasmic Trafficking of NTF2, the Nuclear Import Receptor for the RanGTPase, Is Subjected to Regulation

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).