Christopher Osgood

IN VIVO IMAGING OF TRANSPORT AND BIOCOMPATIBILITY OF SINGLE SILVER NANOPARTICLES IN EARLY DEVELOPMENT OF ZEBRAFISH EMBRYOS

Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5-46 nm) are transported into and out of embryos through chorion pore canals (CPCs) and exhibit Brownian diffusion (not active transport), with the diffusion coefficient inside the chorionic space (3 x 10(-9) cm(2)/s) approximately 26 times lower than that in egg water (7.7 x 10(-8) cm(2)/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities.

A new pulsed electric field therapy for melanoma disrupts the tumor's blood supply and causes complete remission without recurrence

We have discovered a new, ultrafast therapy for treating skin cancer that is extremely effective with a total electric field exposure time of only 180 μsec. The application of 300 high‐voltage (40 kV/cm), ultrashort (300 nsec) electrical pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. None of these melanomas recurred during a 4‐month period after the initial melanoma had disappeared. These pulses generate small, long‐lasting, rectifying nanopores in the plasma membrane of exposed cells, resulting in increased membrane permeability to small molecules and ions, as well as an increase in intracellular Ca2+, DNA fragmentation, disruption of the tumors blood supply and the initiation of apoptosis. Apoptosis was indicated by a 3‐fold increase in Bad labeling and a 72% decrease in Bcl‐2 labeling. In addition, microvessel density within the treated tumors fell by 93%. This new therapy utilizing nanosecond pulsed electric fields has the advantages of highly localized targeting of tumor cells and a total exposure time of only 180 μsec. These pulses penetrate into the interior of every tumor cell and initiate DNA fragmentation and apoptosis while at the same time reducing blood flow to the tumor. This new physical tumor therapy is drug free, highly localized, uses low energy, has no significant side effects and results in very little scarring.

Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection

OBJECTIVE To determine aneuploidy frequencies in pellet and swim-up semen fractions from 10 infertile men with severe oligoasthenoteratozoospermia (OAT) who were donating sperm for intracytoplasmic sperm injection and to determine whether the swim-up isolation method would successfully separate aneuploid from haploid sperm. DESIGN Prospective study. SETTING Infertility clinic and molecular genetics laboratory. PATIENT(S) Ten patients with severe OAT. INTERVENTION(S) Cytogenetic analyses by fluorescence in situ hybridization to determine aneuploidy frequencies for chromosomes 1, 13, 18, 21, X, and Y in sperm from swim-up and pellet fractions. MAIN OUTCOME MEASURE(S) Gametic aneuploidy was scored in sperm fractions separated by the swim-up technique and clinical results after intracytoplasmic sperm injection were tabulated. RESULT(S) In all cases, chromosome aneuploidy levels in patients were significantly greater than in controls. The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm, which remained in the pellet fraction. After ET, 2 (20%) of 10 couples achieved successful pregnancies. CONCLUSION(S) The data show significantly higher rates of diploidy, autosomal disomy and nullisomy, sex chromosome disomy and nullisomy, and total aneuploidy in sperm from all separated fractions obtained from all patients with OAT versus controls. This patient population with OAT may be at increased risk of producing aneuploid offspring.

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Biochemical Characterization of Repair-Deficient Mutants of Drosophila

DNA metabolism is being analyzed in cell cultures derived from the available mutagen-sensitive stocks. Thus far, mutants occurring at eleven different genetic loci in Drosophila melanogaster have been shown to be defective in DNA synthesis or repair. Mutants associated with the following genetic loci exhibit defects in the corresponding metabolic functions: Excision Repair — mei-9, mus (2)201, mus (2)205, mus(3)308 Postreplication Repair — mei-41, mus(1)101, mus(1)104, mus (2)205, mus (3)302, mus (3)310, mus (3)311 DNA Synthesis — mus(1)101, mus(1)104, mus (2)205, mus (3)307, mus (3)308

Current status of aneuploidy testing in Drosophila.

Based on the literature on file at EMIC, 181 papers contained material on aneuploidy testing. Initial screening rejected papers providing no data, no negative control and/or poorly designed genetic schemes; 67 papers representing tests of 76 compounds were reported on. Statistical classifications were established as follows: (+)=a statistically significant difference at the 5% level between the treated and control frequencies; (-)=no significant difference at the 5% level when the number of offspring tested was sufficient to have identified an increase of 0.2% over the control with a power of 75%; I=inconclusive= (a) no significant difference at the 5% level but the number of offspring tested was below that necessary to detect an increase of 0.2% with a power of 75%; (b) the nature of apparent complete loss is undetermined; or (c) the nature of the germ cells sampled is not determined. Of the 76 compounds analyzed, calls were made on 34 compounds. 17/34 compounds were positive for chromosome gain (11/34 for chromosome gain and chromosome loss, 6/34 for chromosome gain only). 17/34 compounds were negative for chromosome gain (11/34 for chromosome gain and chromosome loss and 6 for chromosome gain only). Are any fo the compounds found to induce aneuploidy specific for aneuploid induction? 7 or the compounds positive for chromosome gain were positive in one or more tests assaying for other genetic endpoints, and no reliable data exists regarding results in other tests for the remaining 10 compounds; accordingly, the answer to the question awaits further work.

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development.

Cocaine increases intracellular calcium and reactive oxygen species, depolarizes mitochondria, and activates genes associated with heart failure and remodeling

To determine the cardiovascular molecular events associated with acute exposure to cocaine, the present study utilized in vivo analysis of left-ventricular heart function in adult rabbits fluorescence confocal microscopy of fluo-2, rhod-2, (5-(and-6) carboxy 2′, 7′ dichlorodihydrofluores-cein diacetate (carboxy-H2DCFDA), and JC-1 in H9C2 cells and gene expression microarray technology for analysis of gene activation in both rabbit ventricular tissue and H9C2 cells. In the rabbit, acute cocaine exposure (2 mg/kg) caused left-ventricular dysfunction and 0.1–10 mM cocaine increased cytosolic and mitochondrial calcium activity and mitochondrial membrane depolarization in H9C2 cells. A 3-min pretreatment of H9C2 cells by 10 μM verapamil, nifedipine, or nadolol inhibited calcium increases, but only 1 mM N-acetylcysteine (NAC) or 1 mM glutathione blocked mitochondrial membrane depolarization. Cocaine induced activation of genes in the rabbit heart and H9C2 cells including angiotensinogen, ADRB1, and c-reactive protein (CRP). In H9C2 cells NAC pretreatment blocked cocaine-mediated increases in CRP, FAS, FAS ligand, and cytokine receptor-like factor 1 (CRLF1) expression. Collectively, these data suggest that acute cocaine administration initiates cellular and genetic changes that, if chronically manifested, could cause cardiac deficits similar to those seen in heart failure and ischemia, such as ventricular dysfunction, cardiac arrhythmias, and cardiac remodeling.

3,4-Methylenedioxymethamphetamine activates nuclear factor-κB, increases intracellular calcium, and modulates gene transcription in rat heart cells

Abstract3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug that has gained immense popularity among teenagers and young adults. The cardiovascular toxicological consequences of abusing this compound have not been fully characterized. The present study utilized a transient transfection/dual luciferase genetic reporter assay, fluorescence confocal microscopy, and gene expression macroarray technology to determine nuclear factor-κB (NF-κB) activity, intracellular calcium balance, mitochondrial depolarization, and gene transcription profiles, respectively, in cultured rat striated cardiac myocytes (H9c2) exposed to MDMA. At concentrations of 1×10−3M and 1×10−2M, MDMA significantly enhanced NF-κB reporter activity compared with 0 M (medium only) control. This response was mitigated by cotransfection with IκB for 1×10−3M but not 1×10−2M MDMA. MDMA significantly increased intracellular calcium at concentrations of 1×10−3M and 1×10−2M and caused mitochondrial depolarization at 1×10−2M. MDMA increased the transcription of genes that are considered to be biomarkers in cardiovascular disease and genes that respond to toxic indults. Selected gene activation was verified via temperature-gradient RT-PCR conducted with annealing temperatures ranging from 50°C to 65°C. Collectively, these results suggest that MDMA may be toxic to the heart through its ability to activate the myocardial NF-κB response, disrupt cytosolic calcium and mitochondrial homeostasis, and alter gene transcription.