Naoyuki Nishimura

Safe clinical use of carbon nanotubes as innovative biomaterials.

Carbon nanotubes (CNTs) are structurally described as sheets of six-membered carbon atom rings (i.e., graphene) rolled up into cylinders. CNTs with only one layer are known as single-walled CNTs (SWCNTs), and those with two or more layers are known as multiwalled CNTs (MWCNTs). Cup-stacked carbon nanotubes and carbon nanohorns are also sometimes called CNTs.1−3 Currently, these very attractive carbon materials and nanomaterials are a subject of vigorous product development in a broad range of fields.4−11 The reasons are that CNTs have useful electrical, thermal, and mechanical characteristics, and their base material performance can be improved by combination with other materials.12−23 A recent industrial application of CNTs as an electrode additive to lithium-ion batteries is based on their excellent electrical characteristics. Addition of CNTs prevents battery deterioration and substantially lengthens time to recharging. It is doubtless that the demand for high-performance batteries will grow increasingly with multifunctionalization of personal computers and mobile phones, development of new mobile terminals, spread of electric vehicles, and other factors.24−30 Composite materials with the excellent mechanical characteristics of CNTs have already been used in sporting goods such as golf clubs, tennis rackets, and bicycles. CNTs are also expected to have applications that reduce the weight of aircraft and automobiles.10,14,31−35 A wide variety of advantages are gained from the use CNTs in precision parts as well. CNTs are also used in transistors and memory devices, and enhance their efficiency. The use of CNTs in various displays and TV screens continues to increase in rate. CNTs are also widely used in products designed to prevent static electricity, to shield electromagnetic waves, to store electricity, and for other purposes.36−45 Furthermore, Japan is now facing nuclear energy issues stemming from the accident at Tokyo Electric Power Company’s Fukushima No. 1 nuclear power plant. As a result, CNTs are expected to play a major role in developing new energy sources such as solar photovoltaic power generation and wind power generation.46−52 In the medical field, extensive research activities are underway to develop new CNTs biomaterials for use in the treatment and diagnosis of disease. For example, application of CNTs to cancer treatment and diagnosis, such as in drug delivery systems (DDSs) for treatment of cancer, hyperthermia, and in vivo imaging, has been investigated.53−57 In a study that aimed at applying CNTs to regenerative medicine, CNTs were found to work excellently as scaffold materials for nerve and bone tissue regeneration.58−63 Furthermore, R&D activities are underway to improve the mechanical strength and durability of implants by combining CNTs with existing biomaterials.64−67 Besides, numerous ideas have been put forth about how CNTs can be used in the treatment of a variety of diseases. Figure ​Figure11 shows the trend in the number of articles found in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) (accessed 20 March 2014) by searches using “carbon nanotubes” and “biomaterials” as keywords. The number has been soaring since 2005, suggesting that CNTs research has become a highly competitive field worldwide over the past few years. Of course, numerous articles on the biological applications of CNTs do exist that cannot be captured with these two simple keywords, and the graphic representation of this trend is no more than an indicator of the increase in this research over time. Figure 1 Time trends for the number of articles found in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) (accessed 20 March 2014) by search using “carbon nanotubes” and “biomaterials” as keywords. Recent years have seen ...

Natural disturbance and tree species coexistence in an old-growth beech - dwarf bamboo forest, southwestern Japan

. The structure and composition of a cool-temperate old-growth beech (Fagus crenata) - dwarf bamboo (Sasa spp.) forest, partially affected by landslide disturbance, in the Daisen Forest Reserve of southwestern Japan, were investigated in relation to forest floor and canopy conditions. All stems ≥ 4 cm DBH were mapped on a 4-ha plot and analyses were made of population structure, spatial distribution and spatial association of major tree species. The dominant species, F. crenata, which had the maximum DBH among the species present, had the highest stem density. However, for other species, larger-sized species had lower stem density with few smaller stems or saplings, while smaller-sized species had higher stem density with many smaller stems or saplings. Canopy trees of F. crenata were distributed randomly in the plot, while its stems in the other layers and all other species were distributed patchily. Small patches represent gap-phase regeneration. Larger patches correlate with landslide disturbance, difference in soil age, or the presence/absence of Sasa. Cluster analysis for spatial associations among species and stems in the different layers revealed that the forest community consists of several groups. One main group was formed on sites not covered with Sasa. This group contained a successional subgroup (from Betula grossa to Acer mono and/or F. crenata) initiated by landslide disturbance and a subgroup of tree species that avoid Sasa. Another group was formed on sites with mature soils covered largely with Sasa. This contained associations of canopy trees of F. crenata and smaller-sized tree species such as Acanthopanax sciadophylloides and Acer japonicum. It is found that the community of this old-growth beech forest is largely organized by natural disturbance and heterogeneous conditions of the forest floor (difference in soil age and presence/absence of Sasa). The existence of these different factors and the different responses of species to them largely contribute to the maintenance of tree species diversity in this forest.; Keywords: Cluster analysis; Fagus crenata; Forest dynamics; Gap; Landslide; Spatial pattern.

Tree competition and species coexistence in a cool-temperate old-growth forest in southwestern Japan

The growth dynamics and mode of competition between adult trees > 4 cm in DBH (stem diameter at breast height 1.3 m) of eight abundant species occupying ca. 90 % of the total basal area were investigated in a 4-ha study plot (200 m x 200 m) of a cool-temperate, old-growth forest on Mount Daisen, southwestern Japan. In the study plot, 30 tree species with individuals > 4.0 cm DBH co-occurred. A bimo- dal DBH distribution showing upper and lower-canopy layers was found for the most dominant and largest species, Fagus crenata (ca. 78 % of the total basal area), whilst other tree species showed unimodal DBH distributions corresponding mostly to the lower-canopy layer. We developed a model for individual growth incorporating both intra and interspecific competition and the degree of competitive asymmetry. One- sided interspecific competition was detected only from Fagus crenata (upper-canopy species) to Acer japonicum and Acan- thopanax sciadophylloides (lower-canopy species) on the scale of the 4-ha study plot. Only Acanthopanax sciadophylloides showed symmetric intraspecific competition. However, a posi- tive (non-competitive) interspecific relationship between adult trees prevailed over a competitive relationship; for example, individual DBH growth rate of Fagus crenata (especially lower-canopy trees) was correlated with the abundance of Acer mono. The positive relationship represented a group of species with similar habitat preference (soil type (mature or immature) caused by landslide disturbance and the presence/ absence of Sasa dwarf bamboos in the understorey), where tree densities were not so high as to bring about competition. Competitive interactions between adult trees > 4 cm in DBH occurred only locally between a few specific species and were suggested to be almost irrelevant to the variation in species coexistence on the 4-ha scale of cool-temperate forest. Rather, the coexistence of 30 tree species (species diversity) on this large scale was suggested to be governed by the regeneration pattern of each component species (habitat preference, seed- ling establishment, sapling competition) with respect to land- slide disturbance.

EFFECTS OF KIN-STRUCTURED SEED DISPERSAL ON THE GENETIC STRUCTURE OF THE CLONAL DIOECIOUS SHRUB ILEX LEUCOCLADA

Abstract Nonrandom patterns of gene dispersal have been identified as possible causes of genetic structuring within populations. Attempts to model these patterns have generally focused solely on the effects of isolation by distance, but the processes involved are more complex than such modeling suggests. Here, we extend considerations of gene dispersal processes beyond simple isolation by distance effects by directly evaluating the effects of kin-structured gene dispersal mediated by the group dispersal of related seeds within fruits (i.e., kin-structured seed dispersal) by birds on genetic structure in Ilex leucoclada, a clonal dioecious shrub. To examine the genetic structure patterns, we established two 30×30 m plots (one with immature soils in old-growth forest and one in secondary forest, designated IM and SC, respectively) with different I. leucoclada stem densities. In these two plots 145 and 510 stems were found, representing 78 and 85 genets, respectively, identified by analyzing their genotypes at eight microsatellite loci. The clonal structure was stronger in the SC plot than in the IM plot. Correlograms of coancestry for genets in both plots exhibited significant, positive, high values in the shortest distance class, indicating the presence of strong genetic structure. However, Sp statistics revealed that the pattern of the genetic structure differed between the plots. In addition, to estimate the family structure within fruits, we sampled forty fruits, in total, from 15 randomly selected plants in the area around the IM and SC plots, and found that 80% of the fruits were multiseeded and 42–100% of the multiseeded fruits contained at least one pair of full sibs. Simulations based on these estimates demonstrated that the group dispersal of related seeds produced through correlated mating both within and across fruits, but not unstructured half-sib dispersal, could generate the observed magnitude and trends of genetic structure found in the IM plot. Furthermore, in addition to kin-structured seed dispersal, isolation by distance processes is also likely to promote genetic substructuring in the SC plot. After discussing possible ecological factors that may have contributed to the observed genetic structure, we contrast our results with those predicted by general isolation by distance models, and propose that kin-structured seed dispersal should promote some evolutionary phenomena, and thus should be incorporated, where appropriate, in models of gene dispersal in natural plant populations.

Tree competition and species coexistence in a warm-temperate old-growth evergreen broad-leaved forest in Japan

The growth dynamics and mode of competition between adult trees ≥ 5.0cm in diameter at breast height (DBH) of nine abundant treespeciesoccupying ca. 85% of the total basal area were investigated in a 4ha study plot (200 m × 200 m) of awarm-temperate old-growth evergreen broad-leaved forest in the Tatera ForestReserve of Tsushima Island, southwestern Japan. In the plot, adult trees ≥5.0 cm DBH co-occurred with 35 woody plant species (except forwoody vine species). The most dominant and largest species,Castanopsis cuspidata var. sieboldiiexhibited a bimodal DBH distribution; it was found in both the upper and lowervertical layers. Other tree species had unimodal DBH distributionscorrespondingmostly to the lower vertical layer. We developed a model for individual growthincorporating both intra- and interspecific competition and degree ofcompetitive asymmetry. One-sided interspecific competition was detected in 17cases out of the 66 possible combinations on the scale of the 4 hastudy plot. The direction of interspecific competition was generally one-sidedfrom layer-I species to layer-II and III ones. The effects of two-sidedcompetition were detected only in layer-II and III species. OnlyDistylium racemosum exhibited one-sided intraspecificcompetition. We also found 11 cases of positive interspecific relationships.Generally, competitive relationships prevailed over positive relationshipsbetween adult trees in this warm-temperate evergreen broad-leaved forest.Competition between adult trees ≥ 5.0 cm in DBH did not occurinthe same vertical layer, but occurred only between trees in different verticallayers. This suggests that competition between adult trees ≥ 5.0cm in DBH plays a key role in the variation in species coexistencebetween different vertical layers on the 4 ha scale of thewarm-temperate evergreen broad-leaved forests. Moreover, it was found bycomparing with three different forest types that interspecific competition ismore intense in warm-temperate forests than in cool-temperate or sub-borealforests. We conclude that, compared to cool-temperate or sub-boreal forests(which have little interspecific competition), warm-temperate forests supportmore complex interspecific relationships and species-specific habitatpreferences that result in higher species diversity.

Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice

The application of carbon nanotubes (CNTs) as biomaterials is of wide interest, and studies examining their application in medicine have had considerable significance. Biological safety is the most important factor when considering the clinical application of CNTs as biomaterials, and various toxicity evaluations are required. Among these evaluations, carcinogenicity should be examined with the highest priority; however, no report using transgenic mice to evaluate the carcinogenicity of CNTs has been published to date. Here, we performed a carcinogenicity test by implanting multi-walled CNTs (MWCNTs) into the subcutaneous tissue of rasH2 mice, using the carbon black present in black tattoo ink as a reference material for safety. The rasH2 mice did not develop neoplasms after being injected with MWCNTs; instead, MWCNTs showed lower carcinogenicity than carbon black. Such evaluations should facilitate the clinical application and development of CNTs for use in important medical fields.

Promotion of species co-existence in old-growth coniferous forest through interplay of life-history strategy and tree competition

Abstract Question: Does the interplay of life-history strategy and tree competition promote tree species co-existence? Using a growth dynamics model, we investigated mechanisms of co-existence among major tree species in a sub-alpine old-growth coniferous forest. Location: Sub-alpine old-growth coniferous forest at 1850–1920 m a.s.l. in the Ontake Forest Reserve of central Japan. Methods: We investigated the growth and mortality rates of trees ≥ 5.0 cm stem DBH and recruitment processes in a 2-ha study plot, and developed a model for individual growth that incorporated both intra and interspecific competition and analysed the direction and degree of competitive effect. Results: Four species, Picea jezoensis var. hondoensis, Tsuga diversifolia, Abies mariesii and Abies veitchii co-occurred as dominant species in the canopy layer. P. jezoensis var. hondoensis and T. diversifolia had low stem densities and bell shaped DBH distributions. In contrast, A. mariesii and A. veitchii had high stem densities and inverse J-shaped DBH distributions. The growth of the species with inverse J-shaped DBH distributions (Abies spp.) was governed by the abundances of the species with bell shaped DBH distributions (Picea and Tsuga). However, Picea and Tsuga were inferior to Abies spp. in terms of recruitment rate (the number of juveniles that grow up to 5.0 cm DBH per year). Therefore, it was suggested that there was a trade-off between recruitment pattern and interspecific competition for species co-existence. Conclusions: Picea and Tsuga, with inferior recruitment, coexisted with Abies spp., with superior recruitment, by suppressing the growth of potential successors of Abies spp. The interplay of life-history strategies (recruitment and longevity) and interspecific competition therefore plays an important role in promoting species co-existence in this sub-alpine old-growth coniferous forest. Abbreviations: GLM = General linear model. Nomenclature: Ohwi & Kitagawa (1992).

Evaluation of CNT toxicity by comparison to tattoo ink

The absence of an optimal nano-sized reference material has been the biggest obstacle in evaluating the safety of carbon nanotubes as biomaterials. In this study, black tattoo inks, which have a long history of use by humans, are shown to be suitable reference materials composed of nano-sized carbon black particles. We have also demonstrated that multi-walled carbon nanotubes have comparable basic safety properties to those of tattoo inks when used as biomaterials.

Long-term canopy dynamics analyzed by aerial photographs and digital elevation data in a subalpine old-growth coniferous forest

ABSTRACT We analyzed the long-term canopy dynamics of a 2-ha permanent plot in subalpine old-growth coniferous forest over 43 y using digital surface models (DSMs) and a digital elevation model (DEM). The models contoured canopy and ground surface elevation, respectively. Abies mariesii, A. veitchii, Betula ermanii, Picea jezoensis var. hondoensis, and Tsuga diversifolia were the main species in the forest canopy. Canopy surface DSMs (2.5- × 2.5-m grids) were constructed of the area including the 2-ha plot using aerial photographs from 1959, 1969, 1979, 1989, and 2002, and a DEM was constructed from ground survey data collected in 1991. Canopy height profiles were obtained by calculating the difference between the canopy and the ground surface, and the status of the forest in each grid cell for each year was classified as gap or closed canopy, depending on whether the canopy height was ≤ 25 m or > 25 m, respectively. Tree census data were collected in 2000. The threshold value was decided by comparing the gap from digital elevation data with the result of the field survey. The total gap area in 1959 was greater than 1 ha, indicating that some disturbances had occurred in this plot, probably related to the Isewan Typhoon. A large change occurred during 1969–1989, when mean canopy closure rates were significantly higher than mean gap formation rates. Abies mariesii and B. ermanii tended to occur in the canopy layer in grid cells that contained gaps in 1959 and closed canopy in 2002. The presence of A. veitchii in the canopy layer was also associated with the change from gap to closed canopy, although not significantly so. These results suggest that Abies spp. regenerate more effectively than the other species by establishing seedling or sapling banks before gap formation. Large-scale disturbances, such as the Isewan Typhoon, do not favour the regeneration of spruce over subalpine fir, and species other than spruce are responsible for recovery following such disturbances, according to our analyses of long-term canopy dynamics.

CORRIGENDUM: Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice

CORRIGENDUM: Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice