Steric Interaction of Polyglycerol-Functionalized Detonation Nanodiamonds.

Detonation nanodiamonds have found numerous potential applications in a diverse array of fields such as biomedical imaging and drug delivery. Here, we systematically characterized non-functionalized and polyglycerol-functionalized detonation nanodiamond particles (DNPs) dispersed in aqueous suspensions at different ionic strengths (∼1.0 × 10-7 to 1.0 × 10-2 M) via dynamic light scattering and cryogenic transmission electron microscopy. For these colloidal suspensions, the total potential energies of interactions between a pair of DNPs were theoretically calculated using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory plus the fitting of the Boltzmann distribution to the interparticle spacing distribution of the colloidal DNPs. These investigations revealed that the non-functionalized DNPs are dispersed in aqueous media through the long-range (>10 nm) and weak (<7 kBT) electrical double-layer repulsive interaction, while the driving force on dispersion of polyglycerol-functionalized DNPs is mostly derived from the short-range (<2 nm) and strong (∼55 kBT) steric repulsive potential barrier generated by the polyglycerol. Moreover, our results show that the truly monodispersed and individually dispersed DNP colloids, forming no aggregates in aqueous suspensions, are available by both functionalizing DNPs by polyglycerol and increasing ionic strength of suspending media to ≳1.0 × 10-2 M.

Diamond Colloidal Probe Force Spectroscopy.

Diamond is a highly attractive coating material as it is characterized by a wide optical transparency window, a high thermal conductivity, and an extraordinary robustness due to its mechanical properties and its chemical inertness. In particular, the latter has aroused a great deal of interest for scanning probe microscopy applications in recent years. In this study, we present a novel method for the fabrication of atomic force microscopy (AFM) probes for force spectroscopy using robust diamond-coated spheres, i.e., colloidal particles. The so-called colloidal probe technique is commonly used to study interactions of single colloidal particles, e.g., on biological samples like living cells, or to measure mechanical properties like the Young's modulus. Under physiological measurement conditions, contamination of the particle often strongly limits the measurement time and often impedes reusability of the probe. Diamond as a chemically inert material allows treatment with harsh chemicals without degradation to refurbish the probe. Apart from that, the large surface area of spherical probes makes sensitive studies on surface interactions possible. This provides detailed insight into the interface of diamond with other materials and/or solvents. To fabricate such probes, silica microspheres were coated with a nanocrystalline diamond film and attached to tipless cantilevers. Measurements on soft polydimethylsiloxane (PDMS) show that the manufactured diamond spheres, even though possessing a rough surface, can be used to determine the Young's modulus from a Derjaguin-Muller-Toporov (DMT) fit. By means of force spectroscopy, they can readily probe force interactions of diamond with different substrate materials under varying conditions. The influence of the surface termination of the diamond was investigated concerning the interaction with flat diamond substrates in air. Additionally, measurements in solution, using varying salt concentrations, were carried out, which provide information on double-layer and van-der-Waals forces at the interface. The developed technique offers detailed insight into surface chemistry and physics of diamond with other materials concerning long and short-range force interactions and may provide a valuable probe for investigations under harsh conditions but also on biological samples, e.g., living cells, due to the robustness, chemical inertness, and biocompatibility of diamond.

Expression of Two Classes of Pax6 Transcripts in Reprogramming Retinal Pigment Epithelium Cells of the Adult Newt.

The adult newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown. Here, to approach this issue we focused on Pax6, a transcription factor known to be expressed in RPESCs. We first identified four classes (v1, v2, v3 and v4) of Pax6 variants in the eye of adult newt, Cynops pyrrhogaster. These variants were expressed in most tissues of the intact eye in different combinations but not in the RPE, choroid or sclera. On the basis of this information, we investigated the expression of Pax6 in RPE cells after the NR was removed from the eye by surgery (retinectomy), and found that two classes (v1 and v2) of Pax6 variants were newly expressed in RPE cells 10 days after retinectomy, both in vivo and in vitro (RLEC system). In the RLEC system, we found that Pax6 expression is mediated through a pathway separate from the MEK-ERK pathway, which is required for cell cycle re-entry of RPE cells. These results predict the existence of a pathway that may be of fundamental importance to a better understanding of the reprogramming of RPE cells in vivo.

Appropriate salt concentration of nanodiamond colloids for electrostatic self-assembly seeding of monosized individual diamond nanoparticles on silicon dioxide surfaces.

Monosized (∼4 nm) diamond nanoparticles arranged on substrate surfaces are exciting candidates for single-photon sources and nucleation sites for ultrathin nanocrystalline diamond film growth. The most commonly used technique to obtain substrate-supported diamond nanoparticles is electrostatic self-assembly seeding using nanodiamond colloidal suspensions. Currently, monodisperse nanodiamond colloids, which have a narrow distribution of particle sizes centering on the core particle size (∼4 nm), are available for the seeding technique on different substrate materials such as Si, SiO2, Cu, and AlN. However, the self-assembled nanoparticles tend to form small (typically a few tens of nanometers or even larger) aggregates on all of those substrate materials. In this study, this major weakness of self-assembled diamond nanoparticles was solved by modifying the salt concentration of nanodiamond colloidal suspensions. Several salt concentrations of colloidal suspensions were prepared using potassium chloride as an inserted electrolyte and were examined with respect to seeding on SiO2 surfaces. The colloidal suspensions and the seeded surfaces were characterized by dynamic light scattering and atomic force microscopy, respectively. Also, the interaction energies between diamond nanoparticles in each of the examined colloidal suspensions were compared on the basis of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. From these investigations, it became clear that the appropriate salt concentration suppresses the formation of small aggregates during the seeding process owing to the modified electrostatic repulsive interaction between nanoparticles. Finally, monosized (<10 nm) individual diamond nanoparticles arranged on SiO2 surfaces have been successfully obtained.

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster.

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory. In these animals, survival drops drastically after metamorphosis as their foraging behaviour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined metamorphosis inhibition (Ml) protocols to bypass the period (four months to two years after hatching) in which the animal feeds exclusively on moving foods. We found that approximately 30% of animals survived after 2-year Ml, and that the survivors continuously grew, only with static food while maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamorphosis with TU allows newts to be reared with the same static food under aqueous conditions, providing an alternative rearing protocol that offers the advantage of bypassing the critical period and obtaining animals that have grown sufficiently for use in regeneration studies.

Musashi-1, an RNA-binding protein, is indispensable for survival of photoreceptors.

Musashi-1 (Msi1), an RNA-binding protein (RBP), has been postulated to play important roles in the maintenance of the stem-cell state, differentiation, and tumorigenesis. However, the expression and function of Msi1 in differentiated cells remain obscure. Here we show that Msi1 is expressed in mature photoreceptors and retinal pigment epithelium (RPE) cells, and is indispensable for the survival of photoreceptors. We found in the adult newt eye that Msi1 is expressed in all photoreceptors and RPE cells as well as in the retinal stem/progenitor cells in the ciliary marginal zone (CMZ). We found in the analyses of the newt normal and regenerating retinas that the expression profiles of the Msi1 transcripts and protein isoforms in the photoreceptors are different from those in the retinal stem/progenitor cells. Furthermore, we found that all photoreceptors and RPE cells of the adult mice also express Msi1, and that Msi1 knockout (Msi1-KO) results in degeneration of photoreceptors and a lack of a visual cycle protein RPE65 in the microvilli of RPE cells. Taken together, our current results demonstrate that the expression of Msi1 in mature photoreceptors and RPE cells is evolutionarily conserved, and that Msi1 bears essential functions for vision. Considering such an Msi1-KO phenotype in the retina, it is now reasonable to address whether defects of the Msi1 functions are responsible for inherited retinal diseases. Studying the regulation of Msi1 and the target RNAs of Msi1 in photoreceptors and RPE cells might contribute to fundamental and clinical studies of retinal degeneration.

Electrostatic Self-Assembly of Diamond Nanoparticles onto Al- and N-Polar Sputtered Aluminum Nitride Surfaces.

Electrostatic self-assembly of diamond nanoparticles (DNPs) onto substrate surfaces (so-called nanodiamond seeding) is a notable technique, enabling chemical vapor deposition (CVD) of nanocrystalline diamond thin films on non-diamond substrates. In this study, we examine this technique onto differently polarized (either Al- or N-polar) c-axis oriented sputtered aluminum nitride (AlN) film surfaces. This investigation shows that Al-polar films, as compared to N-polar ones, obtain DNPs with higher density and more homogeneously on their surfaces. The origin of these differences in density and homogeneity is discussed based on the hydrolysis behavior of AlN surfaces in aqueous suspensions.

MEK-ERK signaling in adult newt retinal pigment epithelium cells is strengthened immediately after surgical induction of retinal regeneration.

Adult newt retinal pigment epithelium (RPE) cells are mitotically quiescent in the physiological condition, but upon a traumatic injury of the neural retina (NR) they re-enter the cell-cycle and eventually regenerate the missing NR. Here, to understand the mechanism underlying the cell-cycle re-entry of RPE cells following NR injury, we first investigated changes in MEK-ERK signaling activity in RPE cells upon removal of the NR (retinectomy) from the eye of living animals, and found that ERK-mediated signaling activity is elevated quickly (in 30 min) upon retinectomy. In addition, we found, in in vitro analyses, that immediate early activation of MEK-ERK signaling may occur in RPE cells upon NR injury, intensifying the MEK-ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway, and that 1-h blockade of such early MEK-ERK signaling interferes with the cell-cycle re-entry, which occurs 5-10 days later. Together, these results provide us with insight that elevation of MEK-ERK signaling activity upon NR injury may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle, leading to retinal regeneration.

MEK-ERK and heparin-susceptible signaling pathways are involved in cell-cycle entry of the wound edge retinal pigment epithelium cells in the adult newt.

The onset mechanism of proliferation in mitotically quiescent retinal pigment epithelium (RPE) cells is still obscure in humans and newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. To address this issue, we investigated factors or signaling pathways involved in the first cell-cycle entry of RPE cells upon retinal injury using a newt retina-less eye-cup culture system in which the cells around the wound edge of the RPE exclusively enter the cell cycle. We found that MEK-ERK signaling is necessary for their cell-cycle entry, and signaling pathways whose activities can be modulated by heparin, such as Wnt-, Shh-, and thrombin-mediated pathways, are capable of regulating the cell-cycle entry. Furthermore, we found that the cells inside the RPE have low proliferation competence even in the presence of serum, suggesting inversely that a loss of cell-to-cell contact would allow the cells to enter the cell cycle.