An 8-(phenyl)-2′-deoxyguanosine derivative self-assembles in aqueous media into discrete hexadecamers that further self-assemble above 32 °C into microglobules that encapsulate the drug doxorubicin.Advances in supramolecular chemistry have led to the design of a variety of biomimetic materials that are suitable for the development of stimuli responsive nanocarrier systems. [1][2][3] Light, pH, magnetic fields and temperature are among the most frequently used stimuli. 4 Nano-and microscopic globular assemblies made from thermoresponsive polymers are likewise promising systems for responsive drug carriers. 5,6 Besides the Elastin-Like Polypeptides, 7 most of the work in this area has relied on the poly(N-isopropylacrylamide) 8 scaffold, and its copolymers, as environmentally responsive materials due to their sharp coilto-globule transition at biocompatible temperatures of around 32 °C. 9 Supramolecular self-assembly offers a complement ary strategy to the use of polymers for the development of functional nanostructures. 10 To circumvent some of the limitations shown by polymers (e.g., polydispersity, lack of self-correcting synthesis) and to obtain new thermoresponsives scaffolds, recently, our lab developed an 8-(meta-acetylphenyl)-2′-deoxyguanosine (mAG) derivative that self-assembles in aqueous media into discrete supramolecular hexadecamers that exhibit the Lower Critical Solution Temperature (LCST) phenomenon. 11 Such LCST phenomenon occurs with a transition temperature (T t ) of 58 °C, above which the supramolecular hexadecamers engage in a temperature induced assembly to form solid nanoscopic globules of low polydispersity. 11 We hypothesized that these globules could provide a versatile scaffold for host-guest recognition in aqueous media. Furthermore, if the T t were reduced to a value closer to and below body temperature, these systems may become suitable to prepare thermoresponsive nano-or microcarriers for bioactive materials such as drugs. Herein, we report our initial attempts towards achieving these goals.Controlling the T t via intrinsic parameters (i.e., structural information in the building blocks of supramolecules) enables the reliable construction of nanostructures of well-defined size Correspondence to: José M. Rivera, jmrivortz@mac.com. NIH Public Access Author ManuscriptChem Commun (Camb). Author manuscript; available in PMC 2011 December 7. and composition. In recent years we have developed a family of 8-aryl-2′-deoxyguanosine (8ArG) derivatives as versatile recognition motifs for the construction of supramolecular nanostructures in both organic 12,13 and aqueous media 14 (Scheme 1). Our studies suggest that properly placed functional groups can increase the stability and specificity of the resulting G-quadruplex supramolecules by enhancing non-covalent interactions such as hydrogen bonds and pi-stacking. 15 To this end, we synthesized the 8-(metaethoxycarbonylphenyl)-2′-deoxyguanosine (mECGD2OH, 1) derivative, which shows a lower T t than our previous mAG-based system. This new 8...

Here we review the literature of a male poecillid's sexually dimorphic body plan, behavior, and nervous system, including work dating from the mid 1800s to the mid 1990s as well as work in press or in preparation for publication. Rosa-Molinar described the remodeling of the sexually dimorphic anal fin appendicular support, confirmed earlier claims about the development of the male and female secondary sex characteristics in the Western Mosquitofish, Gambusia affinis and provided for the first time direct embryonic evidence suggesting that remodeling of the sexually dimorphic anal fin appendicular support is biphasic. The first process begins in embryos and proceeds similarly in immature males and females; the second process occurs only in males and results in the anterior transposition of the anal fin and its appendicular support to the level of vertebra 11 [Rosa-Molinar E, Hendricks SE, Rodriguez-Sierra JF, Fritzsch B. 1994. Development of the anal fin appendicular support in the western mosquitofish, Gambusia affinis (Baird and Girard, 1854): a reinvestigation and reinterpretation. Acta Anat 151:20-35.] and the formation of a gonopodium used for internal fertilization. Studies using high-speed video cameras confirmed and extended Peden's and others' observations of copulatory behavior. The cameras showed that circumduction is a complex movement combining in a very fast sequence abduction, extension and pronation, S-start-type fast-start (defined as torque-thrust), and adduction movements. Recent work on the nervous system demonstrated dye-coupling between motor neurons and interneurons via gap junctions, suggesting an attractive substrate for the rapid motions involved in poecillid copulatory reflexes.

“Dye-coupling”, whole-mount immunohistochemistry for gap junction channel protein connexin 35 (Cx35), and freeze-fracture replica immunogold labeling (FRIL) reveal an abundance of electrical synapses/gap junctions at glutamatergic mixed synapses in the 14th spinal segment that innervates the adult male gonopodium of Western Mosquitofish, Gambusia affinis (Mosquitofish). To study gap junctions’ role in fast motor behavior, we used a minimally-invasive neural-tract-tracing technique to introduce gap junction-permeant or -impermeant dyes into deep muscles controlling the gonopodium of the adult male Mosquitofish, a teleost fish that rapidly transfers (complete in <20 mS) spermatozeugmata into the female reproductive tract. Dye-coupling in the 14th spinal segment controlling the gonopodium reveals coupling between motor neurons and a commissural primary ascending interneuron (CoPA IN) and shows that the 14th segment has an extensive and elaborate dendritic arbor and more gap junctions than do other segments. Whole-mount immunohistochemistry for Cx35 results confirm dye-coupling and show it occurs via gap junctions. Finally, FRIL shows that gap junctions are at mixed synapses and reveals that >50 of the 62 gap junctions at mixed synapses are in the 14th spinal segment. Our results support and extend studies showing gap junctions at mixed synapses in spinal cord segments involved in control of genital reflexes in rodents, and they suggest a link between mixed synapses and fast motor behavior. The findings provide a basis for studies of specific roles of spinal neurons in the generation/regulation of sex-specific behavior and for studies of gap junctions’ role in regulating fast motor behavior. Finally, the CoPA IN provides a novel candidate neuron for future studies of gap junctions and neural control of fast motor behaviors.

Conjugations of colloidal gold (18.0 nm) to the cholera toxin B-subunit (CTB) of green fluorescent Alexa Fluor® 488 (ex 495 nm/em 519 nm) and to the CTB of the red-fluorescent Alexa Fluor® 594 (ex 590 nm/em 617 nm) show promise for correlative light (i.e. differential interference contrast [DIC]), fluorescence, and electron microscopy. Commercially available (Molecular Probes, Inc., Eugene, OR) Alexa Fluor 488 conjugated to CTB and Alexa Fluor 594 conjugated to CTB were separately dialyzed in distilled water for 1hr and separately added to 18.0 nm colloidal gold (pH 8.18) to a concentration of 200 mgm per ml of gold, spun down, and re-suspended in 0.1M phosphate buffer at pH 7.4. The final concentrations of Alexa Fluor 488 and Alexa Fluor 594 conjugates of colloidal gold CTB were approximately 5 x 10 twelfth particles/ml. Filter paper saturated with Alexa Fluor 488 or Alexa Fluor 594 colloidal gold conjugates of CTB was immediately applied to both ends of peripheral nerve stumps of the anal fin appendicular support of newborn, immature and adult Western Mosquitofish, Gambusia affinis affinis. The cut peripheral nerves were exposed for 1 minute, the time found to be sufficient to retrogradely label even the most finely axonal fibers and dendritic branches (Figs. 2-3). G. a. affinis were revived, allowed to survive for 6-24 hours, then euthanized, perfusion fixed with 4% paraformaldehyde, washed, bleached, cleared, and visualized using DIC and fluorescence microscopy.Alexa Fluor 488 and Alexa Fluor 594 colloidal gold conjugates of CTB labeled Mauthner cells and their axons within the hindbrain (Figs. 1-2) and secondary spinal motor neuron cell bodies and their extensive dendritic arbors (Figs. 2-3). The tyramide signal amplification procedure for Alexa Fluor 594 (Molecular Probes, Inc., Eugene OR) combined with a previously described whole-mount clear and stain procedure [1] labeled peripheral nerve fibers of the ano-urogenital plexus at all stages and made them easy to visualize (Fig. 4).Our results demonstrate that the conjugations of colloidal gold to the CTB of Alexa Fluor 488 and to the CTB of Alexa Fluor 594 have properties that permit their identification in light and fluorescence microscopy and show promise for electron microscopy and correlative studies.

Background: Microwave technology has revolutionized histological processing by reducing processing time and improving tissue integrity. We describe the first microwave-accelerated tissue processing procedure for healthy and diseased tissue fragments of two species of corals: the gorgonian Gorgonia ventalina and the scleractinian Acropora cervicornis. Methods: Fourteen tissue samples from sea fans (eleven healthy and three diseased), and one tissue sample from a healthy A. cervicornis, were decalcified and processed using microwaves. Histological slices were stained using hematoxylin and eosin and immunostained using an antibody against Aspergillus. Results: By using microwave technology, the decalcification time, as well as the steps and time for tissue processing were significantly reduced while maintaining the integrity of the tissue. Conclusions: This technique accelerates the chemical processing of coral tissue, while providing high quality and optimal resolution of histological sections.