A screen for modifiers of epigenetic reprogramming

Epigenetic modifiers are the proteins involved in establishing and maintaining the epigenome of an organism. They are particularly important for development. Changes in epigenetic modifiers have been shown be lethal, or cause diseases. Our laboratory has developed an ENU mutagenesis screen to produce mouse mutants displaying altered epigenetic gene silencing. The screen relies on a GFP transgene that is expressed in red blood cells in a variegated manner. In the orginal transgenic FVB mice expression occurs in approximately 55% of red blood cells. During the course of my Masters, I characterised four different Mommes (Modifiers of murine metastable epiallele), MommeD32, MommeD33, MommeD35 and MommeD36. For each Momme, I identified the underlying mutation, and observed the corresponding phenotype. In MommeD32 the causative mutation is in Dnmt1, (DNA methyltransferase 1). This gene was previously identified in the screen, as MommeD2, and the new allele, MommeD32 has a change in the BAH domain of the protein. MommeD33 is the result of a change at the transgene itself. MommeD35 carries a mutation in Suv39h1 (suppressor of variegation 3-9 homolog 1). This gene has not previously been identified in the screen, but it is a known epigenetic modifier. MommeD36 had the same ENU treated sire as MommeD32, and I found that it has the same mutation as MommeD32. These mutant strains provide valuable tools that can be used to further our knowledge of epigenetic reprogramming. An example being the cancer study done with MommeD9 which has a mutation in Trim28. By crossing MommeD9+/- mutant mice with Trp53+/- mice, it can be seen if Trim28 has an effect on the rate of tumour genesis. However no clear effect of Trim28 haploinsufficiency can be observed in Trp53+/- mice.

Identifying customer behaviour and dwell time using soft biometrics

In a commercial environment, it is advantageous to know how long it takes customers to move between different regions, how long they spend in each region, and where they are likely to go as they move from one location to another. Presently, these measures can only be determined manually, or through the use of hardware tags (i.e. RFID). Soft biometrics are characteristics that can be used to describe, but not uniquely identify an individual. They include traits such as height, weight, gender, hair, skin and clothing colour. Unlike traditional biometrics, soft biometrics can be acquired by surveillance cameras at range without any user cooperation. While these traits cannot provide robust authentication, they can be used to provide identification at long range, and aid in object tracking and detection in disjoint camera networks. In this chapter we propose using colour, height and luggage soft biometrics to determine operational statistics relating to how people move through a space. A novel average soft biometric is used to locate people who look distinct, and these people are then detected at various locations within a disjoint camera network to gradually obtain operational statistics

Influence of plate bending stiffness on mechanical properties of healing fracture, mouse femora

Mechanically well-defined stabilization systems have only recently become available, providing standardized conditions for studying the role of the mechanical environment on mouse bone fracture healing. The aim of this study was to characterize the time course of strength recovery and callus development of mouse femoral osteotomies stabilized with either low or high flexibility (in bending and torsion) internal fixation plates. Animals were euthanized and femora excised at 14, 21, and 28 days post-osteotomy for microCT analysis and torsional strength testing. While a larger mineralized callus was observed in osteotomies under more flexible conditions at all time points, the earlier bridging of the mineralized callus under less flexible conditions by 1 week resulted in an earlier recovery of torsional strength in mice stabilized with low flexibility fixation. Ultimate torque values for these bones were significantly higher at 14 and 21 days post-osteotomy compared to bones with the more flexible stabilization. Our study confirms the high reproducibility of the results that are achieved with this new implant system, therefore making it ideal for studying the influence of the mechanical environment on murine fracture healing under highly standardized conditions.

Zoledronic acid preserves bone structure and increases survival but does not limit tumour incidence in a prostate cancer bone metastasis model

Background The bisphosphonate, zoledronic acid (ZOL), can inhibit osteoclasts leading to decreased osteoclastogenesis and osteoclast activity in bone. Here, we used a mixed osteolytic/osteoblastic murine model of bone-metastatic prostate cancer, RM1(BM), to determine how inhibiting osteolysis with ZOL affects the ability of these cells to establish metastases in bone, the integrity of the tumour-bearing bones and the survival of the tumour-bearing mice. Methods The model involves intracardiac injection for arterial dissemination of the RM1(BM) cells in C57BL/6 mice. ZOL treatment was given via subcutaneous injections on days 0, 4, 8 and 12, at 20 and 100 µg/kg doses. Bone integrity was assessed by micro-computed tomography and histology with comparison to untreated mice. The osteoclast and osteoblast activity was determined by measuring serum tartrate-resistant acid phosphatase 5b (TRAP 5b) and osteocalcin, respectively. Mice were euthanased according to predetermined criteria and survival was assessed using Kaplan Meier plots. Findings Micro-CT and histological analysis showed that treatment of mice with ZOL from the day of intracardiac injection of RM1(BM) cells inhibited tumour-induced bone lysis, maintained bone volume and reduced the calcification of tumour-induced endochondral osteoid material. ZOL treatment also led to a decreased serum osteocalcin and TRAP 5b levels. Additionally, treated mice showed increased survival compared to vehicle treated controls. However, ZOL treatment did not inhibit the cells ability to metastasise to bone as the number of bone-metastases was similar in both treated and untreated mice. Conclusions ZOL treatment provided significant benefits for maintaining the integrity of tumour-bearing bones and increased the survival of tumour bearing mice, though it did not prevent establishment of bone-metastases in this model. From the mechanistic view, these observations confirm that tumour-induced bone lysis is not a requirement for establishment of these bone tumours.

Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production

Background Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system. Results Transgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs. Conclusion Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.

Chitosan adhesive for laser tissue repair: In vitro characterization

Background and Objectives Laser tissue repair usually relies on hemoderivate protein solders, based on serum albumin. These solders have intrinsic limitations that impair their widespread use, such as limited tensile strength of repaired tissue, poor solder solubility, and brittleness prior to laser denaturation. Furthermore, the required activation temperature of albumin solders (between 65 and 70°C) can induce significant thermal damage to tissue. In this study, we report on the design of a new polysaccharide adhesive for tissue repair that overcomes some of the shortcomings of traditional solders. Study Design/Materials and Methods Flexible and insoluble strips of chitosan adhesive (elastic modulus ~6.8 Mpa, surface area ~34 mm2, thickness ~20 µm) were bonded onto rectangular sections of sheep intestine using a diode laser (continuous mode, 120 ± 10 mW, = λ 808 nm) through a multimode optical fiber with an irradiance of ~15 W/cm2. The adhesive was based on chitosan and also included indocyanin green dye (IG). The temperature between tissue and adhesive was measured using a small thermocouple (diameter ~0.25 mm) during laser irradiation. The repaired tissue was tested for tensile strength by a calibrated tensiometer. Murine fibroblasts were cultured in extracted media from chitosan adhesive to assess cytotoxicity via cell growth inhibition in a 48 hours period. Results Chitosan adhesive successfully repaired intestine tissue, achieving a tensile strength of 14.7 ± 4.7 kPa (mean ± SD, n = 30) at a temperature of 60-65°C. Media extracted from chitosan adhesive showed negligible toxicity to fibroblast cells under the culture conditions examined here. Conclusion A novel chitosan-based adhesive has been developed, which is insoluble, flexible, and adheres firmly to tissue upon infrared laser activation.

Micromarrows—Three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates—which we term “micromarrows.” Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.