DNA Marker Transmission and Linkage Analysis in Populations Derived from a Sugarcane (Saccharum spp.) x Erianthus arundinaceus Hybrid.

Introgression of Erianthus arundinaceus has been the focus of several sugarcane breeding programs in the world, because the species has desirable traits such as high biomass production, vigour, ratooning ability and good resistance to environmental stresses and disease. In this study four genetic maps were constructed for two intergeneric populations. The first population (BC1) was generated from a cross between an Erianthus/Saccharum hybrid YC96-40 and a commercial sugarcane variety CP84-1198. The second population (BC2) was generated from a cross between YCE01-116, a progeny of the BC1 cross and NJ57-416, a commercial sugarcane cultivar. Markers across both populations were generated using 35 AFLP and 23 SSR primer pairs. A total of 756 and 728 polymorphic markers were scored in the BC1 and BC2 populations, respectively. In the BC1 population, a higher proportion of markers was derived from the Erianthus ancestor than those from the Saccharum ancestor Badila. In the BC2 population, both the number and proportion of markers derived from Erianthus were approximately half of those in the BC1 population. Linkage analysis led to the construction of 38, 57, 36 and 47 linkage groups (LGs) for YC96-40, CP84-1198, YCE01-116, and NJ57-416, encompassing 116, 174, 97 and 159 markers (including single dose, double dose and bi-parental markers), respectively. These LGs could be further placed into four, five, five and six homology groups (HGs), respectively, based on information from multi-allelic SSR markers and repulsion phase linkages detected between LGs. Analysis of repulsion phase linkage indicated that Erianthus behaved like a true autopolyploid.

The influence on cell cycle and cell division by various cadmium-containing quantum dots.

Quantum dots (QDs) have attracted great attention because of their favorable optical properties and have been widely applied in biomedical fields. However, in recent years, there have been an increasing number of reports about the cytotoxicity of QDs, especially cadmium-containing QDs, which may release cadmium ions to induce cytotoxicity. Importantly, the chemical composition and surface modifications of cadmium-based QDs determine the amount of Cd(2+) released inside the cell. Thus, there is an urgent need for more systematic work to study the relationship between cytotoxicity and the surface properties of QDs. In this article, the cytotoxicity of seven cadmium-containing QDs with different constituent elements and surface chemistries are compared. The results show that the cytotoxicity of QDs is closely related to their constituent elements and surface properties: First, CdTe@ZnS core-shell QDs show much lower cytotoxicity than naked ones when they have similar surface modifications; second, the positively charged QDs are more toxic than the negatively charged ones. Moreover, both positively and negatively charged QDs without ZnS coatings lead to multipolar spindles, misaligned chromosomes, and G2/M checkpoint failures. Interestingly, although CdSe QDs with a PEG coating cause no apparent cytotoxicity in any of the cell lines studied, they can localize near the contractile ring during cytokinesis and then block contractile ring disassembly. The cellular effect of CdTe QDs comes not only from the release of cadmium ions but also the intracellular distribution of QD nanoparticles in cells and the associated nanoscale effects. It is also found that QD-caused cytokinesis failure is closely related to the decreased expression of Cyclin A and Cyclin B. Taken together, the above findings provide new insight into the dynamic fate of QDs during cell mitosis, and are important for understanding the intracellular effects of QDs on the mitotic spindle and chromosomes during cell division. Furthermore, this kind of cytotoxicity evaluation method should be applicable to studies of the biological effects and health impacts of other nanomaterials.

Intracellular dynamics of cationic and anionic polystyrene nanoparticles without direct interaction with mitotic spindle and chromosomes.

The fate of nanomaterials with different sizes and charges in mitotic cells is of great importance but seldom explored. Herein we investigate the intracellular fate of negatively charged carboxylated polystyrene (COOH-PS) and positively charged amino-modified polystyrene (NH(2)-PS) nanoparticles of three different diameters (50, 100 and 500 nm) on cancer HeLa cells and normal NIH 3T3 cells during the cell cycles. The results showed that all the fluorescent PS nanoparticles differing in size and/or charge did not interact with chromosome reorganization and cytoskeleton assembly during the mitotic process in live cells. They neither disturbed chromosome reorganization nor affected the cytoskeleton reassembly in both normal and cancer cells. However, NH(2)-PS at the size of 50 nm caused G1 phase delay and a decrease of cyclin (D, E) expression, respectively. Moreover, NH(2)-PS displayed higher cellular toxicity and NH(2)-PS of 50 nm disturbed the integrity of cell membranes. Both cationic and anionic PS nanoparticles had a more pronounced effect on normal NIH 3T3 cells than cancer HeLa cell. Our research provides insight into the dynamic fate, intracellular behavior, and the effects of nanoparticles on spindle and chromosomes during cell division, which will enable the optimization of design and selection of much safer nanoparticles for lower risk to human health and widely medical applications.

Time-series investigation of fused vesicles in microvessel endothelial cells with atomic force microscopy.

Vesicles or caveolae within endothelial cells, fusing together to form vacuolar organelles, are implicated in macromolecular transport and cellular element transmigration across the blood-brain barrier (BBB) during inflammation and ischemia. Vacuolar organelles have been described by transmission electron microscopy and immunofluorescence, but the details of their dynamics have not been well addressed yet. Herein, by using tapping mode atomic force microscopy (AFM), we observed the time-series changes of fused vesicles within the serum-free cultured rat cerebral microvessel endothelial cells. The fused vesicles were certainly proved by fluorescent staining of Fm4-64 combining simultaneous AFM imaging, as well as the field emission scanning electron microscopy technique. And energy dispersive spectrum results additionally implied that there may be specific structure and compositions around the vesicle region. These results indicate that increased vesicles in BBB may contribute to the formation of fused vesicles and a higher probability to construct the trans-endothelial channel across endothelium layer. Furthermore, the AFM application may open up a new approach to investigate the details of transcellular process by fused vesicles.

Fullerene nanoparticles selectively enter oxidation-damaged cerebral microvessel endothelial cells and inhibit JNK-related apoptosis.

There is a dearth in fundamental cellular-level understanding of how nanoparticles interact with the cells of the blood brain barrier (BBB), particularly under the oxidative environment. The apoptosis of cerebral microvessel endothelial cells (CMECs) induced by oxidative stress injury plays a key role in the dysfunction of BBB. By use of CMECs as an in vitro BBB model, we show for the first time that C(60)(C(COOH)(2))(2) nanoparticles can selectively enter oxidized CMECs rather than normal cells, and maintain CMECs integrity by attenuating H(2)O(2)-induced F-actin depolymerization via the observation of several state-of-the art microscopic techniques. Additionally, we have found that C(60)(C(COOH)(2))(2) nanoparticles greatly inhibit the apoptosis of CMECs induced by H(2)O(2), which is related to their modulation of the JNK pathway. C(60)(C(COOH)(2))(2) nanoparticles can regulate several downstream signaling events related to the JNK pathway, including reduction of JNK phosphorylation, activation of activator protein 1 (AP-1) and caspase-3, and inhibition of polyADP-ribose polymerase (PARP) cleavage and mitochondrial cytochrome c release. Our results indicate that C(60)(C(COOH)(2))(2) nanoparticles possess a novel ability of selectively entering oxidation-damaged cerebral endothelial cells rather than normal endothelial cells and then protecting them from apoptosis.

Immunostimulatory properties and enhanced TNF- alpha mediated cellular immunity for tumor therapy by C60(OH)20 nanoparticles.

Publications concerning the mechanism of biological activity, especially the immunological mechanism of C(60)(OH)(20) nanoparticles, are relatively limited. However, the structure and characteristics of this carbon allotrope have been widely investigated. In this paper, we have demonstrated that water-soluble C(60)(OH)(20) nanoparticles have an efficient anti-tumor activity in vivo, and show specific immunomodulatory effects to the immune cells, such as T cells and macrophages, both in vivo and in vitro. For example, C(60)(OH)(20) nanoparticles can increase the production of T-helper cell type 1 (Th1) cytokines (IL-2, IFN- gamma and TNF-alpha), and decrease the production of Th2 cytokines (IL-4, IL-5 and IL-6) in serum samples. On the other hand, C(60)(OH)(20) nanoparticles show almost no adverse effect to the viability of immune cells in vitro but stimulate the immune cells to release more cytokines, in particular TNF- alpha, which plays a key role in the cellular immune process to help eliminate abnormal cells. TNF- alpha production increased almost three-fold in treated T lymphocytes and macrophages. Accordingly, we conclude that C(60)(OH)(20) nanoparticles have an efficient anti-tumor activity and this effect is associated with an increased CD(4)(+)/CD(8)(+) lymphocyte ratio and the enhancement of TNF- alpha production. The data suggest that C(60)(OH)(20) nanoparticles can improve the immune response to help to scavenge and kill tumor cells.

The effect of Gd@C82(OH)22 nanoparticles on the release of Th1/Th2 cytokines and induction of TNF-alpha mediated cellular immunity.

It is known that down-regulation of the immune response may be associated with the progenesis, development and prognosis of cancer or infectious diseases. Up-regulating the immune response in vivo is therefore a desirable strategy for clinical treatment. Here we report that poly-hydroxylated metallofullerenol (Gd@C(82)(OH)(22)) has biomedical functions useful in anticancer therapy arising from immunomodulatory effects observed both in vivo and in vitro. We found that metallofullerenol can inhibit the growth of tumors, and shows specific immunomodulatory effects on T cells and macrophages. These effects include polarizing the cytokine balance towards Th1 (T-helper cell type 1) cytokines, decreasing the production of Th2 cytokines (IL-4, IL-5 and IL-6), and increasing the production of Th1 cytokines (IL-2, IFN-gamma and TNF-alpha) in the serum samples. Immune-system regulation by this nanomaterial showed dose-dependent behavior: at a low concentration, Gd@C(82)(OH)(22) nanoparticles slightly affected the activity of immune cells in vitro, while at a high concentration, they markedly enhanced immune responses and stimulated immune cells to release more cytokines, helping eliminate abnormal cells. Gd@C(82)(OH)(22) nanoparticles stimulated T cells and macrophages to release significantly greater quantities of TNF-alpha, which plays a key role in cellular immune processes. Gd@C(82)(OH)(22) nanoparticles are more effective in inhibiting tumor growth in mice than some clinical anticancer drugs but have negligible side effects. The underlying mechanism for high anticancer activity may be attributed to the fact that this water-soluble nanomaterial effectively triggers the host immune system to scavenge tumor cells.

Fullerene derivatives protect endothelial cells against NO-induced damage.

Functional fullerene derivatives have been demonstrated with potent antioxidation properties. Nitric oxide (NO) is a free radical that plays a part in leading to brain damage when it is accumulated to a high concentration. The possible scavenging activity of NO by the hydroxylated fullerene derivative C60(OH)22 and malonic acid derivative C60(C(COOH)2)2 was investigated using primary rat brain cerebral microvessel endothelial cells (CMECs). Results demonstrate that sodium nitroprusside (SNP), used as an NO donor, caused a marked decrease in cell viability and an increase in apoptosis. However, fullerene derivatives can remarkably protect against the apoptosis induced by NO assault. In addition, fullerene derivatives can also prevent NO-induced depolymerization of cytoskeleton and damage of the nucleus and accelerate endothelial cell repair. Further investigation shows that the sudden increase of the intercellular reactive oxygen species (ROS) induced by NO was significantly attenuated by post-treatment with fullerene derivatives. Our results suggest that functional fullerene derivatives are potential applications for NO-related disorders.

The scavenging of reactive oxygen species and the potential for cell protection by functionalized fullerene materials.

We demonstrated that three different types of water-soluble fullerenes materials can intercept all of the major physiologically relevant ROS. C(60)(C(COOH)(2))(2), C(60)(OH)(22), and Gd@C(82)(OH)(22) can protect cells against H(2)O(2)-induced oxidative damage, stabilize the mitochondrial membrane potential and reduce intracellular ROS production with the following relative potencies: Gd@C(82)(OH)(22)> or =C(60)(OH)(22)>C(60)(C(COOH)(2))(2). Consistent with their cytoprotective abilities, these derivatives can scavenge the stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH), and the reactive oxygen species (ROS) superoxide radical anion (O(2)(*-)), singlet oxygen, and hydroxyl radical (HO(*)), and can also efficiently inhibit lipid peroxidation in vitro. The observed differences in free radical-scavenging capabilities support the hypothesis that both chemical properties, such as surface chemistry induced differences in electron affinity, and physical properties, such as degree of aggregation, influence the biological and biomedical activities of functionalized fullerenes. This represents the first report that different types of fullerene derivatives can scavenge all physiologically relevant ROS. The role of oxidative stress and damage in the etiology and progression of many diseases suggests that these fullerene derivatives may be valuable in vivo cytoprotective and therapeutic agents.

Potential neurological lesion after nasal instillation of TiO(2) nanoparticles in the anatase and rutile crystal phases.

Nanoscale titanium dioxide (TiO(2)) is massively produced and widely used in living environment, which hence make the potential risk to human health. Central nervous system (CNS) is the potential susceptible target of inhaled nanoparticles, but the studies on this aspect are limited so far. We report the accumulation and toxicity results in vivo of two crystalline phases of TiO(2) nanoparticles (80nm, rutile and 155nm, anatase; purity >99%). The female mice were intranasally instilled with 500microg of TiO(2) nanoparticles suspension every other day for 30 days. Synchrotron radiation X-ray fluorescence analysis (SRXRF) and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine the contents of titanium in murine brain. Then, the pathological examination of brain tissue, oxidative stress-mediated responses, and levels of neurochemicals in the brain of exposed mice were also analyzed. The obvious morphological changes of hippocampal neurons and increased GFAP-positive astrocytes in the CA4 region were observed, which were in good agreements with higher Ti contents in the hippocampus region. Oxidative stress occurred obviously in whole brain of exposed mice such as lipid peroxidation, protein oxidation and increased activities of catalase, as well as the excessive release of glutamic acid and nitric oxide. These findings indicate anatase TiO(2) nanoparticles exhibited higher concern on some tested biological effects. To summarize, results provided the preliminary evidence that nasal instilled TiO(2) nanoparticles could be translocated into the central nervous system and cause potential lesion of brain, and the hippocampus would be the main target within brain.

Quantitative analysis of metal impurities in carbon nanotubes: efficacy of different pretreatment protocols for ICPMS spectroscopy.

Metal impurities in carbon nanotubes (CNTs) are undesirable for their uses in diverse applications, for instance, they may potentially have a negative health impact when using in biomedical fields. However, so far there is a lack of analysis methods able to quantify metallic impurities in CNTs. In this paper, using the neutron activation analysis (NAA) technique as a nondestructive standard quantification method and inductively coupled plasma mass spectrometry (ICPMS) as a practical approach, we established an analytical method for quantitative determination of metallic impurities in CNTs. ICPMS, one of the most sensitive analytical techniques used for coincident multielement measurements, has become a common tool in many laboratory, and thus it is easily available and a good selection for determining the metal impurities in CNTs. However, because of their extremely stable structure and the encapsulated metals in the defect structure, CNTs must undergo special pretreatments before ICPMS. We investigated different sample pretreatment procedures for ICPMS analysis, including dry ashing coupled with acid extraction, wet digestion, and a combination of dry ashing with acid digestion. With the reference data from the nondestructive analytical method of NAA, we found that the quantitative determination of metal impurities in CNTs is highly dependent on the sample pretreatment in which the conditions are largely different from those used for conventional biological samples or environmental materials. This paper not only provides the practical method and analysis conditions for quantifying the metal impurities of CNTs but also the first protocol for pretreatment processes of CNT samples.

Time-dependent translocation and potential impairment on central nervous system by intranasally instilled TiO(2) nanoparticles.

Nanoparticles can be administered via nasal, oral, intraocular, intratracheal (pulmonary toxicity), tail vein and other routes. Here, we focus on the time-dependent translocation and potential damage of TiO(2) nanoparticles on central nervous system (CNS) through intranasal instillation. Size and structural properties are important to assess biological effects of TiO(2) nanoparticles. In present study, female mice were intranasally instilled with two types of well-characterized TiO(2) nanoparticles (i.e. 80 nm, rutile and 155 nm, anatase; purity>99%) every other day. Pure water instilled mice were served as controls. The brain tissues were collected and evaluated for accumulation and distribution of TiO(2), histopathology, oxidative stress, and inflammatory markers at post-instillation time points of 2, 10, 20 and 30 days. The titanium contents in the sub-brain regions including olfactory bulb, cerebral cortex, hippocampus, and cerebellum were determined by inductively coupled plasma mass spectrometry (ICP-MS). Results indicated that the instilled TiO(2) directly entered the brain through olfactory bulb in the whole exposure period, especially deposited in the hippocampus region. After exposure for 30 days, the pathological changes were observed in the hippocampus and olfactory bulb using Nissl staining and transmission electron microscope. The oxidative damage expressed as lipid peroxidation increased significantly, in particular in the exposed group of anatase TiO(2) particles at 30 days postexposure. Exposure to anatase TiO(2) particles also produced higher inflammation responses, in association with the significantly increased tumor necrosis factor alpha (TNF-alpha) and interleukin (IL-1 beta) levels. We conclude that subtle differences in responses to anatase TiO(2) particles versus the rutile ones could be related to crystal structure. Thus, based on these results, rutile ultrafine-TiO(2) particles are expected to have a little lower risk potential for producing adverse effects on central nervous system. Although understanding the mechanisms requires further investigation, the present results suggest that we should pay attention to potential risk of occupational exposure for large-scaled production of TiO(2) nanoparticles.

Inhibition of tumor growth by endohedral metallofullerenol nanoparticles optimized as reactive oxygen species scavenger.

Intraperitoneal injection of [Gd@C82(OH)22]n nanoparticles decreased activities of enzymes associated with the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice. Several physiologically relevant ROS were directly scavenged by nanoparticles, and lipid peroxidation was inhibited in this study. [Gd@C82(OH)22]n nanoparticles significantly reduced the electron spin resonance (ESR) signal of the stable 2,2-diphenyl-1-picryhydrazyl radical measured by ESR spectroscopy. Like-wise, studies using ESR with spin-trapping demonstrated efficient scavenging of superoxide radical anion, hydroxyl radical, and singlet oxygen (1O2) by [Gd@C82(OH)22]n nanoparticles. In vitro studies using liposomes prepared from bovine liver phosphatidylcholine revealed that nanoparticles also had a strong inhibitory effect on lipid peroxidation. Consistent with their ability to scavenge ROS and inhibit lipid peroxidation, we determined that [Gd@C82(OH)22]n nanoparticles also protected cells subjected in vitro to oxidative stress. Studies using human lung adenocarcinoma cells or rat brain capillary endothelial cells demonstrated that [Gd@C82(OH)22]n nanoparticles reduced H2O2-induced ROS formation and mitochondrial damage. [Gd@C82(OH)22]n nanoparticles efficiently inhibited the growth of malignant tumors in vivo. In summary, the results obtained in this study reveal antitumor activities of [Gd@C82(OH)22]n nanoparticles in vitro and in vivo. Because ROS are known to be implicated in the etiology of a wide range of human diseases, including cancer, the present findings demonstrate that the potent inhibition of [Gd@C82(OH)22]n nanoparticles on tumor growth likely relates with typical capacity of scavenging reactive oxygen species.

The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis.

Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C(60)(C(COOH)(2))(2)](n) nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C(60)(C(COOH)(2))(2)](n) nanoparticles entering cells are mainly via endocytosis with time-, temperature- and energy-dependent manners. The cellular uptake of [C(60)(C(COOH)(2))(2)](n) nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside cells.