Heather A. Lindfors

Evidence for de Novo Evolution of Testis-Expressed Genes in the Drosophila yakuba/Drosophila erecta Clade

The mutational origin and subsequent evolution of de novo genes, which are hypothesized to be genes of recent origin that are not obviously related to ancestral coding sequence, are poorly understood. However, accumulating evidence suggests that such genes may often function in male reproduction. Here we use testis-derived expressed sequence tags (ESTs) from Drosophila yakuba to identify genes that have likely arisen either in D. yakuba or in the D. yakuba/D. erecta ancestor. We found several such genes, which show testis-biased expression and are often X-linked. Comparative data indicate that three of these genes have very short open reading frames, which suggests the possibility that a significant number of testis-biased de novo genes in the D. yakuba/D. erecta clade may be noncoding RNA genes. These data, along with previously published data from D. melanogaster, support the idea that many de novo Drosophila genes function in male reproduction and that a small region of the X chromosome in the melanogaster subgroup may be a hotspot for the evolution of novel testis-biased genes.

Recently Evolved Genes Identified From Drosophila yakuba and D. erecta Accessory Gland Expressed Sequence Tags

The fraction of the genome associated with male reproduction in Drosophila may be unusually dynamic. For example, male reproduction-related genes show higher-than-average rates of protein divergence and gene expression evolution compared to most Drosophila genes. Drosophila male reproduction may also be enriched for novel genetic functions. Our earlier work, based on accessory gland protein genes (Acps) in D. simulans and D. melanogaster, suggested that the melanogaster subgroup Acps may be lost and/or gained on a relatively rapid timescale. Here we investigate this possibility more thoroughly through description of the accessory gland transcriptome in two melanogaster subgroup species, D. yakuba and D. erecta. A genomic analysis of previously unknown genes isolated from cDNA libraries of these species revealed several cases of genes present in one or both species, yet absent from ingroup and outgroup species. We found no evidence that these novel genes are attributable primarily to duplication and divergence, which suggests the possibility that Acps or other genes coding for small proteins may originate from ancestrally noncoding DNA.

A Radio-Frequency Coupling Network for Heating of Citrate-Coated Gold Nanoparticles for Cancer Therapy: Design and Analysis

Gold nanoparticles (GNPs) are nontoxic, can be functionalized with ligands, and preferentially accumulate in tumors. We have developed a 13.56-MHz RF-electromagnetic field (RFEM) delivery system capable of generating high E-fleld strengths required for noninvasive, noncontact heating of GNPs. The bulk heating and specific heating rates were measured as a function of NP size and concentration. It was found that heating is both size and concentration dependent, with 5 nm particles producing a 50.6 ± 0.2°C temperature rise in 30 s for 25 μg/mL gold (125 W input). The specific heating rate was also size and concentration dependent, with 5 nm particles producing a specific heating rate of 356 ± 78 kW/g gold at 16 μg/mL (125 W input). Furthermore, we demonstrate that cancer cells incubated with GNPs are killed when exposed to 13.56 MHz RF-EM fields. Compared to cells that were not incubated with GNPs, three out of four RF-treated groups showed a significant enhancement of cell death with GNPs (p <; 0.05). GNP-enhanced cell killing appears to require temperatures above 50°C for the experimental parameters used in this study. Transmission electron micrographs show extensive vacuolization with the combination of GNPs and RF treatment.

Copper-doxorubicin as a nanoparticle cargo retains efficacy with minimal toxicity.

Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.

Dynamic imaging of arginine-rich heart-targeted vehicles in a mouse model

Efficacious delivery of drugs and genes to the heart is an important goal. Here, a radiolabeled peptide-targeted liposome was engineered to bind to the heart, and the biodistribution and pharmacokinetics were determined by dynamic positron emission tomography in the FVB mouse. Efficient targeting occurred only with an exposed ligand and a dense concentration of peptide (6000 peptides/particles). Liposomes targeted with CRPPR or other arginine-rich peptides with an exposed guanidine moiety bound within 100 s after intravenous injection and remained stably bound. With CRPPR-targeted particles, the radioisotope density in the heart averaged 44 +/- 9% injected dose/gram of tissue, more than 30-fold higher than in skeletal muscle. The rapid and efficient targeting of these particles can be exploited in drug and gene delivery systems and with dynamic positron emission tomography provides a model system to optimize targeting of engineered particles.

Novel Ultrasound and DCE-MRI Analyses after Antiangiogenic Treatment with a Selective VEGF Receptor Inhibitor

We report a comparison between tumor perfusion estimates acquired using contrast-enhanced MRI and motion-corrected contrast-enhanced ultrasound before and after treatment with AG-028262, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor. Antiangiogenic activity was determined by assessing weekly ultrasound and MRI images of rats with bilateral hind flank mammary adenocarcinomas before and after treatment with AG-028262. Images were acquired with a spoiled gradient, 1.5 T magnetic resonance sequence and a destruction-replenishment ultrasound protocol. For ultrasound, a time to 80% contrast replenishment was calculated for each tumor voxel; for MR imaging, a measure of local flow rate was estimated from a linear fit of minimum to maximum intensities. AG-028262 significantly decreased tumor growth and increased the time required to replenish tumor voxels with an ultrasound contrast agent from 2.66 to 4.54 s and to fill with an MR contrast agent from 29.5 to 50.8 s. Measures of flow rate derived from MRI and ultrasound demonstrated a positive linear correlation of r2 = 0.86.

Abstract 5538: Enhanced tumor cytotoxicity of a combinational therapy using liposomal copper-doxorubicin, rapamycin, and ultrasound

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We introduce an effective local therapeutic strategy using a combined treatment with: a) increased and stable loading of doxorubicin (Dox) in liposomes using a complex of Dox and copper (II), b) suppression of tumor proliferation using rapamycin, c) insonation of the entire tumor to increase microvascular permeability, extravasation and accumulation of liposomes. Liposomes composed of HSPC: Cholesterol: DSPE-PEG2k, 56: 39: 5 were prepared in the presence of 100 mM copper(II) gluconate and 270 mM triethanolamine (TEA), pH 7.4 and extruded with 100 nm membrane filters. Cu-liposomes were loaded with Dox up to a maximum concentration of 0.6 mg-drug/mg-lipid with 100% loading. We studied the efficacy of Cu-Dox liposomes and optimized the treatment strategy using the highly invasive and metastatic Met-1 tumor, a syngeneic model of human breast carcinoma which was transplanted bilaterally into the mouse mammary fat pad. Over the 28 days of the study, Cu-Dox liposomes were administrated intravenously (iv) two times per week at a therapeutic level of 6 mg-drug/kg-body weight and rapamycin was injected intraperitoneally (ip) three times per week at 0.9 mg-drug/kg-body weight. Ultrasound was applied using a peak negative pressure of 1.23 MPa (MI=0.99), cycle length of 100 and pulse repetition rate maintained between 0.1 and 5 kHz, resulting in a temperature of 42°C for a two-minute duration. A dotted and diffuse structure of the Cu- Dox complex within the liposomes was verified by cryo-electron microscopy. For the Met-1 tumor line, the IC50 for liposomal doxorubicin with an identical lipid formulation decreased from 2.2±0.91 to 0.412±0.16 µM in the presence of copper gluconate during loading. The toxicity of copper-loaded liposomes (without doxorubicin) was not increased over the control (no copper, no doxorubicin) liposome formulation. Insonifying one tumor per mouse after systemic injection of Cu-Dox liposomes resulted in an increase in local accumulation of liposomes from 7.7±8 to 14±9 %ID/g-tumor as detected using ICP-MS. Although single therapy using Cu-Dox liposomes suppressed tumor growth, significant tumor regression was accomplished only when Cu-Dox liposomes were combined with rapamycin and tumor insonation. At 15 days following the onset of therapy, the percent change in tumor diameter was −20±42, 114±250, 234±213, 833±247, 5533±4200 for treatments of Cu-Dox liposomes/rapamycin/ultrasound, Cu-Dox liposomes/rapamycin, Cu-Dox liposomes, rapamycin, rapamycin diluent, respectively. Diluent-treated animals survived 18 days after the onset of treatment; all animals receiving the full combined therapy survived throughout the 28-day course of treatment, demonstrating a lower fraction of angiogenic vessels and decreased tumor proliferation upon immunohistochemistry. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5538.

Recently evolved genes identified from Drosophila yakuba and D. erecta accessory gland ESTs

The fraction of the genome associated with male reproduction in Drosophila may be unusually dynamic. For example, male reproduction-related genes show higher-than-average rates of protein divergence and gene expression evolution compared to most Drosophila genes. Drosophila male reproduction may also be enriched for novel genetic functions. Our earlier work, based on accessory gland protein genes (Acps) in D. simulans and D. melanogaster, suggested that the melanogaster subgroup Acps may be lost and/or gained on a relatively rapid timescale. Here we investigate this possibility more thoroughly through description of the accessory gland transcriptome in two melanogaster subgroup species, D. yakuba and D. erecta. A genomic analysis of previously unknown genes isolated from cDNA libraries of these species revealed several cases of genes present in one or both species, yet absent from ingroup and outgroup species. We found no evidence that these novel genes are attributable primarily to duplication and divergence, which suggests the possibility that Acps or other genes coding for small proteins may originate from ancestrally noncoding DNA.

Novel melanogaster subgroup genes identified from Drosophila yakuba and D. erecta accessory gland ESTs

The fraction of the genome associated with male reproduction in Drosophila may be unusually dynamic. For example, male reproduction-related genes show higher-than-average rates of protein divergence and gene expression evolution compared to most Drosophila genes. Drosophila male reproduction may also be enriched for novel genetic functions. Our earlier work, based on accessory gland protein genes (Acps) in D. simulans and D. melanogaster, suggested that the melanogaster subgroup Acps may be lost and/or gained on a relatively rapid timescale. Here we investigate this possibility more thoroughly through description of the accessory gland transcriptome in two melanogaster subgroup species, D. yakuba and D. erecta. A genomic analysis of previously unknown genes isolated from cDNA libraries of these species revealed several cases of genes present in one or both species, yet absent from ingroup and outgroup species. We found no evidence that these novel genes are attributable primarily to duplication and divergence, which suggests the possibility that Acps or other genes coding for small proteins may originate from ancestrally noncoding DNA.