Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

Understanding the Genetic Basis of C4 Kranz Anatomy with a View to Engineering C3 Crops.

One of the most remarkable examples of convergent evolution is the transition from C3 to C4 photosynthesis, an event that occurred on over 60 independent occasions. The evolution of C4 is particularly noteworthy because of the complexity of the developmental and metabolic changes that took place. In most cases, compartmentalized metabolic reactions were facilitated by the development of a distinct leaf anatomy known as Kranz. C4 Kranz anatomy differs from ancestral C3 anatomy with respect to vein spacing patterns across the leaf, cell-type specification around veins, and cell-specific organelle function. Here we review our current understanding of how Kranz anatomy evolved and how it develops, with a focus on studies that are dissecting the underlying genetic mechanisms. This research field has gained prominence in recent years because understanding the genetic regulation of Kranz may enable the C3-to-C4 transition to be engineered, an endeavor that would significantly enhance crop productivity.

Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW during leaf patterning in C4 grasses.

Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf tissues depending on whether the leaf carries out C3 or C4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C3 species veins are separated by a greater number of intervening mesophyll cells than in derived C4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C3 species rice. Here we show that SCR is required for inner leaf patterning in the C4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C4 but not C3 grasses. Together our results provide insight into the evolution of cell patterning in grass leaves and demonstrate a novel patterning role for IDD genes in C4 leaves.

Redundant SCARECROW genes pattern distinct cell layers in roots and leaves of maize.

The highly efficient C4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature.

SHORTROOT-Mediated Increase in Stomatal Density Has No Impact on Photosynthetic Efficiency.

The coordinated positioning of veins, mesophyll cells, and stomata across a leaf is crucial for efficient gas exchange and transpiration and, therefore, for overall function. In monocot leaves, stomatal cell files are positioned at the flanks of underlying longitudinal leaf veins, rather than directly above or below. This pattern suggests either that stomatal formation is inhibited in epidermal cells directly in contact with the vein or that specification is induced in cell files beyond the vein. The SHORTROOT pathway specifies distinct cell types around the vasculature in subepidermal layers of both root and shoots, with cell type identity determined by distance from the vein. To test whether the pathway has the potential to similarly pattern epidermal cell types, we expanded the expression domain of the rice (Oryza sativa ssp japonica) OsSHR2 gene, which we show is restricted to developing leaf veins, to include bundle sheath cells encircling the vein. In transgenic lines, which were generated using the orthologous ZmSHR1 gene to avoid potential silencing of OsSHR2, stomatal cell files were observed both in the normal position and in more distant positions from the vein. Contrary to theoretical predictions, and to phenotypes observed in eudicot leaves, the increase in stomatal density did not enhance photosynthetic capacity or increase mesophyll cell density. Collectively, these results suggest that the SHORTROOT pathway may coordinate the positioning of veins and stomata in monocot leaves and that distinct mechanisms may operate in monocot and eudicot leaves to coordinate stomatal patterning with the development of underlying mesophyll cells.

The WIP6 transcription factor TOO MANY LATERALS specifies vein type in C4 and C3 grass leaves.

Grass leaves are invariantly strap shaped with an elongated distal blade and a proximal sheath that wraps around the stem. Underpinning this shape is a scaffold of leaf veins, most of which extend in parallel along the proximo-distal leaf axis. Differences between species are apparent both in the vein types that develop and in the distance between veins across the medio-lateral leaf axis. A prominent engineering goal is to increase vein density in leaves of C3 photosynthesizing species to facilitate the introduction of the more efficient C4 pathway. Here, we discover that the WIP6 transcription factor TOO MANY LATERALS (TML) specifies vein rank in both maize (C4) and rice (C3). Loss-of-function tml mutations cause large lateral veins to develop in positions normally occupied by smaller intermediate veins, and TML transcript localization in wild-type leaves is consistent with a role in suppressing lateral vein development in procambial cells that form intermediate veins. Attempts to manipulate TML function in rice were unsuccessful because transgene expression was silenced, suggesting that precise TML expression is essential for shoot viability. This finding may reflect the need to prevent the inappropriate activation of downstream targets or, given that transcriptome analysis revealed altered cytokinin and auxin signaling profiles in maize tml mutants, the need to prevent local or general hormonal imbalances. Importantly, rice tml mutants display an increased occupancy of veins in the leaf, providing a step toward an anatomical chassis for C4 engineering. Collectively, a conserved mechanism of vein rank specification in grass leaves has been revealed.

Tumors induce complex DNA damage in distant proliferative tissues in vivo.

That tumors cause changes in surrounding tissues is well documented, but whether they also affect distant tissues is uncertain. Such knowledge may be important in understanding the relationship between cancer and overall patient health. To address this question, we examined tissues distant to sites of implanted tumors for genomic damage using cohorts of C57BL/6 and BALB/c mice with early-stage subcutaneous syngeneic grafts, specifically, B16 melanoma, MO5076 sarcoma, and COLON26 carcinoma. Here we report that levels of two serious types of DNA damage, double-strand breaks (DSBs) measured by γ-H2AX focus formation and oxidatively induced non-DSB clustered DNA lesions (OCDLs), were elevated in tissues distant from the tumor site in tumor-bearing mice compared with their age- and sex-matched controls. Most affected were crypts in the gastrointestinal tract organs and skin, both highly proliferative tissues. Further investigation revealed that, compared with controls, tumor-bearing mice contained elevated amounts of activated macrophages in the distant gastrointestinal tissues, as well as elevated serum levels of several cytokines. One of these cytokines, CCL2/MCP-1, has been linked to several inflammation-related conditions and macrophage recruitment, and strikingly, CCL2-deficient mice lacked increased levels of DSBs and OCDLs in tissues distant from implanted tumors. Thus, this study is unique in being a direct demonstration that the presence of a tumor may induce a chronic inflammatory response in vivo, leading to increased systemic levels of DNA damage. Importantly, these findings suggest that tumors may have more profound effects on their hosts than heretofore expected.

Effect of Titanium and Molybdenum Cover on the Surface Restructuration of Diamond Single Crystal during Annealing.

Diamond is an important material for electrical and electronic devices. Because the diamond is in contact with the metal in these applications, it becomes necessary to study the metal-diamond interaction and the structure of the interface, in particular, at elevated temperatures. In this work, we study the interaction of the (100) and (111) surfaces of a synthetic diamond single crystal with spattered titanium and molybdenum films. Atomic force microscopy reveals a uniform coating of titanium and the formation of flattened molybdenum nanoparticles. A thin titanium film is completely oxidized upon contact with air and passes from the oxidized state to the carbide state upon annealing in an ultrahigh vacuum at 800 °C. Molybdenum interacts with the (111) diamond surface already at 500 °C, which leads to the carbidization of its nanoparticles and catalytic graphitization of the diamond surface. This process is much slower on the (100) diamond surface; sp2-hybridized carbon is formed on the diamond and the top of molybdenum carbide nanoparticles, only when the annealing temperature is raised to 800 °C. The conductivity of the resulting sample is improved when compared to the Ti-coated diamond substrates and the Mo-coated (111) substrate annealed at 800 °C. The presented results could be useful for the development of graphene-on-diamond electronics.

Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks.

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

Effect of Toluene Addition in an Electric Arc on Morphology, Surface Modification, and Oxidation Behavior of Carbon Nanohorns and Their Sedimentation in Water.

Carbon nanohorns (CNHs) are attractive for various applications, where a high specific surface area and long dispersion stability in water are important. In the present work, we study these parameters of CNHs prepared by arc evaporation of graphite depending on the conditions of the synthesis and subsequent oxidation in air. It is shown that the addition of toluene in the reactor during the arcing allows obtaining CNHs functionalized with -CHx groups. Heating of CNHs in air at 400 °C leads to substitution of -CHx groups for oxygen-containing groups. Moreover, the CNH endcaps are opened at 500 °C, and as a result, the specific surface area of CNHs increases 4 times. Aqueous suspensions with a concentration of oxidized CNHs of 100 µg/mL are stable for 8 months.

Laser Patterning of Aligned Carbon Nanotubes Arrays: Morphology, Surface Structure, and Interaction with Terahertz Radiation.

The patterning of arrays of aligned multi-walled carbon nanotubes (MWCNTs) allows creating metastructures for terahertz (THz) applications. Here, the strips and columns from MWCNTs vertically grown on silicon substrates are prepared using CO2 laser treatment. The tops of the patterned arrays are flat when the laser power is between 15 and 22 W, and craters appear there with increasing power. Laser treatment does not destroy the alignment of MWCNTs while removing their poorly ordered external layers. The products of oxidative destruction of these layers deposit on the surfaces of newly produced arrays. The oxygen groups resulting from the CO2 laser treatment improve the wettability of nanotube arrays with an epoxy resin. We show that the patterned MWCNT arrays absorb the THz radiation more strongly than the as-synthesized arrays. Moreover, the pattern influences the frequency behavior of the absorbance.

microRNAome changes in bystander three-dimensional human tissue models suggest priming of apoptotic pathways.

The radiation-induced bystander effect (RIBE) is a phenomenon whereby unexposed cells exhibit molecular symptoms of stress exposure when adjacent or nearby cells are traversed by ionizing radiation (IR). Recent data suggest that RIBE may be epigenetically mediated by microRNAs (miRNAs), which are small regulatory molecules that target messenger RNA transcripts for translational inhibition. Here, we analyzed microRNAome changes in bystander tissues after α-particle microbeam irradiation of three-dimensional artificial human tissues using miRNA microarrays. Our results indicate that IR leads to a deregulation of miRNA expression in bystander tissues. We report that major bystander end points, including apoptosis, cell cycle deregulation and DNA hypomethylation, may be mediated by altered expression of miRNAs. Specifically, c-MYC-mediated upregulation of the miR-17 family was associated with decreased levels of E2F1 and RB1, suggesting a switch to a proliferative state in bystander tissues, while priming these cells for impending death signals. Upregulation of the miR-29 family resulted in decreased levels of its targets DNMT3a and MCL1, consequently affecting DNA methylation and apoptosis. Altered expression of miR-16 led to changes in expression of BCL2, suggesting modulation of apoptosis. Thus, our data clearly show that miRNAs play a profound role in the manifestation of late RIBE end points. In summary, this study creates a roadmap for understanding the role of microRNAome in RIBE and for developing novel RIBE biomarkers.

H2AX: functional roles and potential applications.

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed gamma-H2AX, is easily identified with antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of gamma-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that gamma-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.

Role of oxidatively induced DNA lesions in human pathogenesis.

Genome stability is essential for maintaining cellular and organismal homeostasis, but it is subject to many threats. One ubiquitous threat is from a class of compounds known as reactive oxygen species (ROS), which can indiscriminately react with many cellular biomolecules including proteins, lipids, and DNA to produce a variety of oxidative lesions. These DNA oxidation products are a direct risk to genome stability, and of particular importance are oxidative clustered DNA lesions (OCDLs), defined as two or more oxidative lesions present within 10 bp of each other. ROS can be produced by exposure of cells to exogenous environmental agents including ionizing radiation, light, chemicals, and metals. In addition, they are produced by cellular metabolism including mitochondrial ATP generation. However, ROS also serve a variety of critical cellular functions and optimal ROS levels are maintained by multiple cellular antioxidant defenses. Oxidative DNA lesions can be efficiently repaired by base excision repair or nucleotide excision repair. If ROS levels increase beyond the capacity of its antioxidant defenses, the cell's DNA repair capacity can become overwhelmed, leading to the accumulation of oxidative DNA damage products including OCDLs, which are more difficult to repair than individual isolated DNA damage products. Here we focus on the induction and repair of OCDLs and other oxidatively induced DNA lesions. If unrepaired, these lesions can lead to the formation of mutations, DNA DSBs, and chromosome abnormalities. We discuss the roles of these lesions in human pathologies including aging and cancer, and in bystander effects.

Light-Induced Sulfur Transport inside Single-Walled Carbon Nanotubes.

Filling of single-walled carbon nanotubes (SWCNTs) and extraction of the encapsulated species from their cavities are perspective treatments for tuning the functional properties of SWCNT-based materials. Here, we have investigated sulfur-modified SWCNTs synthesized by the ampoule method. The morphology and chemical states of carbon and sulfur were analyzed by transmission electron microscopy, Raman scattering, thermogravimetric analysis, X-ray photoelectron and near-edge X-ray absorption fine structure spectroscopies. Successful encapsulation of sulfur inside SWCNTs cavities was demonstrated. The peculiarities of interactions of SWCNTs with encapsulated and external sulfur species were analyzed in details. In particular, the donor-acceptor interaction between encapsulated sulfur and host SWCNT is experimentally demonstrated. The sulfur-filled SWCNTs were continuously irradiated in situ with polychromatic photon beam of high intensity. Comparison of X-ray spectra of the samples before and after the treatment revealed sulfur transport from the interior to the surface of SWCNTs bundles, in particular extraction of sulfur from the SWCNT cavity. These results show that the moderate heating of filled nanotubes could be used to de-encapsulate the guest species tuning the local composition, and hence, the functional properties of SWCNT-based materials.

Intercellular communication of cellular stress monitored by gamma-H2AX induction.

When cells are exposed to ionizing radiation (IR), unexposed cells that share media with damaged cells exhibit similar effects to irradiated cells including increased levels of DNA double-strand breaks (DSBs). Hypothesizing that this effect, known as the radiation-induced bystander effect, may be a specific instance of communication between damaged and undamaged cells regardless of damage source, we demonstrated that exposure of target cells to non-IR induces bystander damage in non-targeted cells as measured by gamma-H2AX and 53BP1 focal formation. Initially, bystander damage was found primarily in S-phase cells, but at later times, non-S-phase cells were also affected. In addition, media from undamaged malignant and senescent cells also was found to induce DSBs in primary cultures. Media conditioned on cells targeted with either ionizing or non-IR as well as on undamaged malignant and senescent cells contained elevated levels of several cytokines. One of these, transforming growth factor beta (TGF-beta), and nitric oxide (NO) were found to elevate numbers of gamma-H2AX/53BP1 foci in normal cell cultures similar to levels found in bystander cells, and this elevation was abrogated by NO synthase inhibitors, TGF-beta blocking antibody and antioxidants. These findings support the hypothesis that damage in bystander cells results from their exposure to cytokines or reactive compounds released from stressed cells, regardless of damage source. These results have implications for oncogenesis in that they indicate that damaged normal cells or undamaged tumor cells may induce genomic instability, leading to an increased risk of oncogenic transformation in other cells with which they share media or contact directly.

Accumulation of oxidatively induced clustered DNA lesions in human tumor tissues.

Increased levels of oxidatively induced DNA damage have been reported in various cases of human pathogenesis like age-related and chronic diseases. Advances in experimental carcinogenesis associate high oxidative stress with genome instability and oncogenic transformation. Cancer biomarkers are helpful for early tumor diagnostics, prediction of tumor development, and analysis of individual tumors' response to therapy as well as recurrence. The repair resistant oxidatively induced clustered DNA lesions (OCDLs) could serve as a common indicator of oxidative stress in human malignant cells or tissues. To test this hypothesis, we assessed the levels of endogenous OCDLs in several human tumor and adjacent normal tissues from patients with liver, ovary, kidney, breast and colon cancer. These tumor tissues have already been shown to accumulate higher endogenous levels of gamma-H2AX foci. For the detection of clustered DNA lesions we used the human repair enzymes APE1, OGG1 and NTH1 as well as the Escherichia coli homologue Endonuclease III. In the majority of cases we detected higher levels of OCDLs in tumor vs. normal tissues but not always with a statistically significant difference and not with uniform tissue dependence. These data suggest for the first time the importance of endogenous non-DSB clusters in human cancer and their potential use as cancer biomarkers.

γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin.

Ionizing radiation (IR) exposure is inevitable in our modern society and can lead to a variety of deleterious effects including cancer and birth defects. A reliable, reproducible and sensitive assessment of exposure to IR and the individual response to that exposure would provide much needed information for the optimal treatment of each donor examined. We have developed a diagnostic test for IR exposure based on detection of the phosphorylated form of variant histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). The cell responds to a nascent DSB through the phosphorylation of thousands of H2AX molecules flanking the damaged site. This highly amplified response can be visualized as a γ-H2AX focus in the chromatin that can be detected in situ with the appropriate antibody. Here we assess the usability of γ-H2AX focus formation as a possible biodosimeter for human exposure to IR using peripheral blood lymphocytes irradiated ex vivo and three-dimensional artificial models of human skin biopsies. In both systems, the tissues were exposed to 0.2-5 Gy, doses of IR that might be realistically encountered in various scenarios such as cancer radiotherapies or accidental exposure to radiation. Since the γ-H2AX response is maximal 30 minutes after exposure and declines over a period of hours as the cells repair the damage, we examined the time limitations of the useful detectibility of γ-H2AX foci. We report that a linear response proportional to the initial radiation dose was obtained 48 hours and 24 hours after exposure in blood samples and skin cells respectively. Thus, detection of γ-H2AX formation to monitor DNA damage in minimally invasive blood and skin tests could be useful tools to determine radiation dose exposure and analyze its effects on humans.

DNA double-strand breaks form in bystander cells after microbeam irradiation of three-dimensional human tissue models.

The "radiation-induced bystander effect," in which irradiated cells can induce genomic instability in unirradiated neighboring cells, has important implications for cancer radiotherapy and diagnostic radiology as well as for human health in general. Although the mechanisms of this effect remain to be elucidated, we reported previously that DNA double-strand breaks (DSBs), directly measured by gamma-H2AX focus formation assay, are induced in bystander cultured cells. To overcome the deficiencies of cultured cell studies, we examined alpha-particle microbeam irradiation-induced bystander effects in human tissue models, which preserve the three-dimensional geometric arrangement and communication of cells present in tissues in vivo. In marked contrast to DNA DSB dynamics in irradiated cells, in which maximal DSB formation is seen 30 min after irradiation, the incidence of DSBs in bystander cells reached a maximum by 12 to 48 h after irradiation, gradually decreasing over the 7-day time course. At the maxima, 40% to 60% of bystander cells were affected, a 4- to 6-fold increase over controls. These increases in bystander DSB formation were followed by increased levels of apoptosis and micronucleus formation, by loss of nuclear DNA methylation, and by an increased fraction of senescent cells. These findings show the involvement of DNA DSBs in tissue bystander responses and support the notion that bystander DNA DSBs are precursors to widespread downstream effects in human tissues. Bystander cells exhibiting postirradiation signs of genomic instability may be more prone than unaffected cells to become cancerous. Thus, this study points to the importance of considering the indirect biological effects of radiation in cancer risk assessment.

Bromine polycondensation in pristine and fluorinated graphitic carbons.

Despite decades of study the precise behavior of bromine in graphitic carbons remains unclear. In this report, using Raman spectroscopy, we reveal two types of bromine structure in graphitic carbon materials. Between fluorinated graphene layers with a composition close to C2F, Br2 molecules are intercalated in a form similar to liquid bromine. Bromination of pristine and low-fluorinated graphitic carbons behaves very differently with distinct Br-related Raman spectra. With the guidance of density functional theory (DFT) calculations, all Raman features are assigned to normal vibration modes of specific bromine species over graphene and fluorinated graphene. When intercalated between extended non-fluorinated sp2-hybridized carbon regions, physisorbed Br2 molecules move freely across the non-functionalized region toward the CF border. Multiple Br2 molecules then combine spontaneously into Br3-based chains, whose coupling activates otherwise Raman inactive modes. Significant charge transfer to bromine species occurs in this case. DFT calculated frequencies match precisely the experimental Br-related Raman bands observed in the intercalation carbon compounds. The fluorine-catalyzed bromine chain-formation process shown here is general and should also operate with edges and other defect species.