Michelle Pine

Insulin-Like Growth Factor-I Activates KiSS-1 Gene Expression in the Brain of the Prepubertal Female Rat

KiSS-1 gene expression has been shown to increase as puberty approaches, and its peptide products, kisspeptins, are involved in LHRH secretion at puberty. Factors contributing to increased KiSS-1 expression, however, have not been identified; thus, the purpose of this study was to assess whether IGF-I could induce transcription of this gene in prepubertal female rats. IGF-I or saline was centrally administered to immature rats that were killed 2, 4, and 6 h later. Real-time PCR revealed that IGF-I induced (P < 0.01) KiSS-1 gene expression at 6 h in a tissue fragment that contained both the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. Subsequently, the AVPV and ARC nuclei were separated to assess whether region-specific effects could be identified. IGF-I stimulated (P < 0.01) KiSS-1 gene expression in the AVPV nucleus at 6 h after injection, with no change observed in the ARC nucleus. Serum estradiol (E2) levels were not altered at any time point after IGF-I, demonstrating that the increased KiSS-1 expression observed was not caused by an elevation in E2. Additionally, the IGF-I action to induce KiSS-1 gene expression in the AVPV nucleus was further demonstrated when the IGF-I was administered systemically. E2 appears to play an important permissive role because 1-d ovariectomized rats responded to IGF-I with increased (P < 0.01) KiSS-1 expression, whereas, 20 d after ovariectomy, when the E2 levels had fallen below assay sensitivity, the IGF-I was unable to induce KiSS-1 expression. The IGF-I effect was further demonstrated by showing that the IGF-I receptor antagonist, JB-1, blocked the IGF-I-induced increase in KiSS-1 expression. Collectively, these data indicate that IGF-I is an activator of the KiSS-1 gene in the prepubertal female rat.

The pyrethroid metabolites 3-phenoxybenzoic acid and 3-phenoxybenzyl alcohol do not exhibit estrogenic activity in the MCF-7 human breast carcinoma cell line or Sprague–Dawley rats

Synthetic pyrethroids are one of the most frequently and widely used class of insecticides, primarily because they have a higher insect to mammalian toxicity ratio than organochlorines or organophosphates. The basic structure of pyrethroids can be characterized as an acid joined to an alcohol by an ester bond. Pyrethroid degradation occurs through either oxidation at one or more sites located in the alcohol or acid moieties or hydrolysis at the central ester bond, the latter reaction being important for mammalian metabolism of most pyrethroids. The primary alcohol liberated from the ester cleavage is hydroxylated to 3-phenoxybenzyl alcohol, which for most pyrethroids is then oxidized to 3-phenoxybenzoic acid. These products may then be conjugated with amino acids, sulfates, sugars, or sugar acids. In vitro studies have suggested that some of the pyrethroids may have estrogenic activity. Interestingly, the chemical structure of specific pyrethroid metabolites indicates that they may be more likely to interact with the estrogen receptor than the parent compounds. Two of the pyrethroid metabolites, 3-phenoxybenzoic acid (3PBA) and 3-phenoxybenzyl alcohol (3PBalc) have been reported to have endocrine activity using a yeast based assay. 3PBAlc exhibited estrogenic activity with reported EC(50)s of 6.67 x 10(-6) and 2 x 10(-5) while 3PBAcid exhibited anti-estrogenic activity with a calculated IC(50) of 6.5 x 10(-5). To determine if the metabolites were able to cause the same effects in a mammalian system, the estrogen-dependent cell line, MCF-7, was utilized. Cells were treated with 1.0, 10.0 or 100.0 microM concentrations of each metabolite and cytotoxicity was assessed. The two lowest concentrations of both metabolites did not induce cell death and even appeared to increase proliferation over that of the control cells. However, when cellular proliferation was measured using a Coulter counter neither metabolite stimulated proliferation (1.0 nM, 10.0 nM, or 10.0 microM) or induced an estrogen receptor alpha/ERE-controlled luciferase reporter in the MCF-7 cells. Following the in vitro screenings, the metabolites were then evaluated for estrogenic activity in vivo using the uterotrophic assay in Sprague-Dawley rats. Animals were orally gavaged (10.0, 5.0, and 1.0 mg/kg) once daily for 3 days. Neither metabolite had any effect on uterine wet weight, body weight, or organ weight. Lastly, in order to determine if either metabolite was able to alter the onset of puberty, immature female rats were orally gavaged (10.0, 5.0, and 1.0 mg/kg) once a day with the metabolites beginning 1 day post-weaning until the onset of puberty as evidenced by vaginal opening (VO). Again, neither metabolite had any effect on the onset of VO.

The Pyrethroid Pesticide Esfenvalerate Suppresses the Afternoon Rise of Luteinizing Hormone and Delays Puberty in Female Rats

Background One of the most widely used classes of insecticides is the synthetic pyrethroids. Although pyrethroids are less acutely toxic to humans than to insects, in vitro studies have suggested that pyrethroids may be estrogenic. Objectives We assessed pubertal effects by orally administering 0.5, 1.0, and 5.0 mg/kg/day of the type II pyrethroid esfenvalerate (ESF) to female rats beginning on postnatal day (PND) 22 until vaginal opening. ESF administration suppresses serum estradiol and delays pubertal onset. Materials and methods To assess possible hypothalamic and/or pituitary effects, animals received 0.5 or 1.0 mg/kg ESF or corn oil on PNDs 22–29. On PND30, we drew three blood samples (200 μL) from each rat at 15-min intervals beginning at 1000 hours, and again at 1500 hours. To test hypothalamic responsiveness, after the third afternoon sample, all animals received an intravenous injection of N-methyl-d,l-aspartic acid (NMA; 40 mg/kg), and then we drew two more samples. We performed a second experiment as above except that animals received luteinizing hormone–releasing hormone (LHRH; 25 ng/rat) to test pituitary responsiveness. Results Basal levels of luteinizing hormone (LH) in the afternoon hours were higher in control animals than in animals treated with 1.0 mg/kg ESF (p < 0.05). Furthermore, NMA- and LHRH-stimulated LH release was similar in control and ESF-treated animals, indicating that both hypothalamic and pituitary responsiveness, respectively, were unaffected. Conclusions Although the hypothalamus is able to respond to exogenous stimuli, absence of a normal afternoon rise in LH would indicate a hypothalamic deficit in ESF-treated animals.

Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone‐releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30‐day‐old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N‐monomethyl‐l‐arginine (NMMA); 300 or 500 μm) or a soluble guanylyl cyclase (sGC) inhibitor (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ); 100 or 250 μm) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 μm) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 μm MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300–500 μm NMMA to the medium did not block Mn2+‐stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 μm MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+‐stimulated secretion of LHRH in a dose‐dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 μm) inhibited Mn2+‐induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Surface functionalization of silver nanoparticles: Novel applications for insect vector control:

Every day, people and animals contract debilitating and life threatening diseases due to bites from infected flies, ticks, and mosquitoes. The current methods utilized to fight against these diseases are only partially effective or safe for humans and animals. When it comes to insect vector control, a conceptual paradigm shift is urgently needed. This work proposes a novel synthetic scheme to produce a nanoparticle-pesticide core-shell conjugate to be used as an active agent against arthropod vectors, such as mosquitoes. As a proof of concept, we conjugated nanosilver to the pyrethroid pesticide deltamethrin. First, electron microscopy and Fourier transform infrared spectroscopy verified the presence of a 15 nm nanosilver core surrounded by deltamethrin. Second, when the conjugate was exposed to mosquitoes for a 24 h bioassay, mortality was observed at 9 × 10(-4) M. Silver was detected in the hemolymph of mosquitoes exposed to the conjugate. We concluded that the newly developed nanoconjugate did not inactivate the primary function of the pesticide and was effective in killing mosquitoes at low concentrations. These results demonstrate the potential to use nanoparticle surfaces to kill insects, specifically vectors of human pathogens.

Anatomy builder VR: Applying a constructive learning method in the virtual reality canine skeletal system

We present Anatomy Builder VR that examines how a virtual reality (VR) system can support embodied learning in anatomy education. The backbone of the project is to pursue an alternative constructivist pedagogical model for learning canine anatomy. The main focus of the study was to identify and assemble bones in the live-animal orientation, using real thoracic limb bones in a bone box and digital pelvic limb bones in the Anatomy Builder VR. Eleven college students participated in the study. The pilot study showed that participants most enjoyed interacting with anatomical contents within the VR program. Participants spent less time assembling bones in the VR, and instead spent a longer time tuning the orientation of each VR bone in the 3D space. This study showed how a constructivist method could support anatomy education while using virtual reality technology in an active and experiential way.

ARnatomy: tangible AR app for learning gross anatomy

classroom use is granted without fee provided that copies are not made or distributed for commercial advantage and that copies bear this notice and the full citation on the first page. Copyrights for third-party components of this work must be honored. For all other uses, contact the Owner/Author. SIGGRAPH 2014, August 10 – 14, 2014, Vancouver, British Columbia, Canada. 2014 Copyright held by the Owner/Author. ACM 978-1-4503-2958-3/14/08 ARnatomy: Tangible AR App for learning Gross Anatomy Jinsil Hwaryoung Seo, James Storey, John Chavez, Diana Reyna Texas A&M University College Station, TX, USA hwaryoung@tamu.edu Jinkyo Suh Simon Fraser University Burnaby, BC, Canada jks28@sfu.ca Michelle Pine Texas A&M University College Station, TX, USA MPine@cvm.tamu.edu

Development of an in vitro model of excess intracellular reactive oxygen species

These investigations characterize an in vitro model for generating excess intracellular reactive oxygen species (ROS). This novel model may be useful in the identification and delineation of cellular mechanisms associated with aging due to the link between age and excess oxidative events. The human cell line, MCF7, was stably transfected using the pSV3.neo plasmid housing a gene encoding the Aequorea victoria green fluorescent protein (GFP). Transfected cells were analyzed for maintenance of GFP over time, showing stability of the GFP gene. These studies demonstrate that the presence of fluorescing GFP significantly increases intracellular ROS, creating oxidative stress in these cells. Antioxidant supplementation was evaluated to determine the effectiveness of intracellular H2O2 reduction. The results demonstrate that supplementation with a potent antioxidant, such as reduced glutathione, protects cells from oxidative damage by decreasing intracellular concentrations of H2O2. This model for intracellular generation of excess ROS establishes a clear method by which the utility of antioxidant supplementation to protect against intracellularly generated reactive oxygen species may be evaluated.

Kinetic Pelvic Limb Model to Support Students' Understanding of Spatial Visualization in Gross Anatomy

Visual aids currently being used for anatomy education are often only designed to help identify structures and, in some cases, to facilitate understanding of the spatial relationships amongst structures. However, a knowledge of the basic functions of structures is crucial to understanding anatomy and is vastly neglected in the design of instructional tools. One major concept which current tools lack the ability to demonstrate is basic biomechanics. A possible solution to address this oversight is the creation of a kinetic, interactive model that demonstrates movement. Creation of this type of model should incorporate aspects of many different disciplines and should facilitate learning by providing engaging and intuitive interaction. To test this hypothesis, a physically based interactive kinetic simulation model of the canine pelvic limb was constructed. From the user studies, positive student feedback as well as improved quiz scores show that the interactive simulation model had a positive effect on student comprehension in anatomy education.

Anatomy builder VR: comparative anatomy lab promoting spatial visualization through constructionist learning

Anatomy Builder VR is a comparative anatomy learning application that examines how a virtual reality (VR) system can support spatial visualization through embodied learning methods. The backbone of the project is to pursue an alternative constructionist pedagogical model for learning canine anatomy. The main focus of the project is to identify, sort, and assemble bones in the canine and human orientations using 3D scanned human and canine bones.