ALBERT

Description: We prove a spectral flow formula for one-parameter families of Hamiltonian systems under homoclinic boundary conditions, which relates the spectral flow to the relative Maslov index of a pair of curves of Lagrangians induced by the stable and unstable subspaces, respectively. Finally, we deduce sufficient conditions for bifurcation of homoclinic trajectories of one-parameter families of non-autonomous Hamiltonian vector fields.

Description: We consider the Schur–Horn problem for normal operators in von Neumann algebras, which is the problem of characterizing the possible diagonal values of a given normal operator based on its spectral data. For normal matrices, this problem is well known to be extremely difficult, and in fact, it remains open for matrices of size greater than $3$ . We show that the infinite-dimensional version of this problem is more tractable, and establish approximate solutions for normal operators in von Neumann factors of type I $_\infty$ , II, and III. A key result is an approximation theorem that can be seen as an approximate multivariate analogue of Kadison's Carpenter Theorem.

Description: We study the rate of convergence to zero of the tail entropy of $C^\infty$ maps. We give an upper bound of this rate in terms of the growth in $k$ of the derivative of order $k$ and give examples showing the optimality of the established rate of convergence. We also consider the case of multimodal maps of the interval. Finally, we prove that homoclinic tangencies give rise to $C^r$ $(r\geqslant 2)$ robustly non- $h$ -expansive dynamical systems.

Description: Let ${{\mathscr {C}}}^0_{{{\mathfrak {g}}}}$ be the category of finite-dimensional integrable modules over the quantum affine algebra $U_{q}'({{\mathfrak {g}}})$ and let $R^{A_\infty }{\mbox {-}\mathrm {gmod}}$ denote the category of finite-dimensional graded modules over the quiver Hecke algebra of type $A_{\infty }$ . In this paper, we investigate the relationship between the categories ${{\mathscr {C}}}^0_{A_{N-1}^{(1)}}$ and ${{\mathscr {C}}}^0_{A_{N-1}^{(2)}}$ by constructing the generalized quantum affine Schur–Weyl duality functors ${\mathcal {F}}^{(t)}$ from $R^{A_\infty }{\mbox {-}\mathrm {gmod}}$ to ${{\mathscr {C}}}^0_{A_{N-1}^{(t)}}\ (t=1,2)$ .

Description: We present new constructions of complex and $p$ -adic Darmon points on elliptic curves over base fields of arbitrary signature. We conjecture that these points are global and present numerical evidence to support our conjecture.

Description: It has been hypothesised that positive associations between age and levels of oxidative stress-generated damage to DNA may be related to an age-dependent decline in DNA repair activity. The objective of this study was to investigate the association between age and repair activity of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18–83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16–1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio ( P 〈 0.05) and plasma concentrations of glycosylated hemoglobin ( P = 0.07). However, multivariate regression analysis only showed an inverse association between age and DNA repair activity ( P 〈 0.05), indicating that the decline in repair activity was not mediated by metabolic risk factors. In summary, the results show an inverse association between age and DNA repair activity of oxidatively damaged DNA.

Description: Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55–83 years and body mass index above 25kg/m 2 were exposed for 5h to either particle-filtered or sham-filtered air from a busy street with number of concentrations and PM 2.5 levels of 1800/cm 3 versus 23 000/cm 3 and 3 µg/m 3 versus 24 µg/m 3 , respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo exposure to nanosized carbon black as well as expression of genes related to inflammation ( chemokine (C-C motif) ligand 2 , interleukin-8 and tumour necrosis factor ), oxidative stress response ( heme oxygenase (decycling)-1 ) and DNA repair ( oxoguanine DNA glycosylase ). DNA strand breaks and oxidised purines were assayed by the alkaline comet assay. No statistically significant differences were found for any biomarker immediately after exposure to PM from urban street air although strand breaks and oxidised purines combined were significantly associated with the particle number concentration during exposure. In conclusion, 5h of controlled exposure to PM from urban traffic did not change the gene expression related to inflammation, oxidative stress or DNA repair, ROS production or oxidatively damaged DNA in PBMCs from elderly overweight human subjects.

Description: Ionising radiation causes free radical–mediated damage in cellular DNA. This damage is manifested as chromosomal aberrations and micronuclei (MN) in proliferating cells. Sesamol, present in sesame seeds, has the potential to scavenge free radicals; therefore, it can reduce radiation-induced cytogenetic damage in cells. The aim of this study was to investigate the radioprotective potential of sesamol in bone marrow cells of mice and related haematopoietic system against radiation-induced genotoxicity. A comparative study with melatonin was designed for assessing the radioprotective potential of sesamol. C57BL/6 mice were administered intraperitoneally with either sesamol or melatonin (10 and 20mg/kg body weight) 30min prior to 2-Gy whole-body irradiation (WBI) and sacrificed after 24h. Total chromosomal aberrations (TCA), MN and cell cycle analyses were performed using bone marrow cells. The comet assay was performed on bone marrow cells, splenocytes and lymphocytes. Blood was drawn to study haematological parameters. Prophylactic doses of sesamol (10 and 20mg/kg) in irradiated mice reduced TCA and micronucleated polychromatic erythrocyte frequency in bone marrow cells by 57% and 50%, respectively, in comparison with radiation-only groups. Sesamol-reduced radiation-induced apoptosis and facilitated cell proliferation. In the comet assay, sesamol (20mg/kg) treatment reduced radiation-induced comets (% DNA in tail) compared with radiation only ( P 〈 0.05). Sesamol also increased granulocyte populations in peripheral blood similar to melatonin. Overall, the radioprotective efficacy of sesamol was found to be similar to that of melatonin. Sesamol treatment also showed recovery of relative spleen weight at 24h of WBI. The results strongly suggest the radioprotective efficacy of sesamol in the haematopoietic system of mice.

Description: We introduce a new framework for the analysis of the stability of solitons for the one-dimensional Gross–Pitaevskii equation. In particular, we establish the asymptotic stability of the black soliton with zero speed.

Description: Let $k$ and $n$ be positive even integers. For a cuspidal Hecke eigenform $h$ in the Kohnen plus space of weight $k-n/2+1/2$ for $\varGamma _0(4),$ let $I_n(h)$ be the Duke–Imamo $\bar {{\text {g}}}$ lu–Ikeda lift of $h$ in the space of cusp forms of weight $k$ for ${\rm Sp}_n({{\bf{Z}}}),$ and $f$ be the primitive form of weight $2k-n$ for ${\rm SL}_2({{\bf{Z}}})$ corresponding to $h$ under the Shimura correspondence. We then express the ratio $\displaystyle {\langle I_n(h), I_n(h) \rangle / \langle h, h \rangle }$ of the period of $I_n(h)$ to that of $h$ in terms of special values of certain $L$ -functions of $f$ . This proves the conjecture proposed by Ikeda concerning the period of the Duke–Imamo $\bar {{\text {g}}}$ lu–Ikeda lift.

Description: The Mediterranean region is a hot spot of climate change vulnerable to increased droughts and heat waves. Scaling carbon fluxes from leaf to landscape levels is particularly challenging under drought conditions. We aimed to improve the mechanistic understanding of the seasonal acclimation of photosynthesis and morphology in sunlit and shaded leaves of four Mediterranean trees ( Quercus ilex L., Pinus halepensis Mill., Arbutus unedo L. and Quercus pubescens Willd.) under natural conditions. V c,max and J max were not constant, and mesophyll conductance was not infinite, as assumed in most terrestrial biosphere models, but varied significantly between seasons, tree species and leaf position. Favourable conditions in winter led to photosynthetic recovery and growth in the evergreens. Under moderate drought, adjustments in the photo/biochemistry and stomatal/mesophyllic diffusion behaviour effectively protected the photosynthetic machineries. Severe drought, however, induced early leaf senescence mostly in A. unedo and Q. pubescens , and significantly increased leaf mass per area in Q. ilex and P. halepensis . Shaded leaves had lower photosynthetic potentials but cushioned negative effects during stress periods. Species-specificity, seasonal variations and leaf position are key factors to explain vegetation responses to abiotic stress and hold great potential to reduce uncertainties in terrestrial biosphere models especially under drought conditions.

Description: Plants experiencing drought stress are frequently more susceptible to pathogens, likely via alterations in physiology that create favorable conditions for pathogens. Common plant responses to drought include the production of reactive oxygen species (ROS) and the accumulation of free amino acids (AAs), particularly proline. These same phenomena also frequently occur during pathogenic attack. Therefore, drought-induced perturbations in AA and ROS metabolism could potentially contribute to the observed enhanced susceptibility. Furthermore, nitrogen (N) availability can influence AA accumulation and affect plant resistance, but its contributions to drought-induced susceptibility are largely unexplored. Here we show that drought induces accumulation of hydrogen peroxide (H 2 O 2 ) in Austrian pine ( Pinus nigra Arnold) shoots, but that shoot infection by the blight and canker pathogen Diplodia sapinea (Fr.) Fuckel leads to large reductions in H 2 O 2 levels in droughted plants. In in vitro assays, H 2 O 2 was toxic to D. sapinea , and the fungus responded to this oxidative stress by increasing catalase and peroxidase activities, resulting in substantial H 2 O 2 degradation. Proline increased in response to drought and infection when examined independently, but unlike all other AAs, proline further increased in infected shoots of droughted trees. In the same tissues, the proline precursor, glutamate, decreased significantly. Proline was found to protect D. sapinea from H 2 O 2 damage, while also serving as a preferred N source in vitro. Fertilization increased constitutive and drought-induced levels of some AAs, but did not affect plant resistance. A new model integrating interactions of proline and H 2 O 2 metabolism with drought and fungal infection of plants is proposed.

Description: Small differences in the sensitivity of stomatal conductance to light intensity on leaf surfaces may lead to large differences in total canopy transpiration ( E C ) with increasing canopy leaf area ( L ). Typically, the increase of L would more than compensate for the decrease of transpiration per unit of leaf area ( E L ), resulting in concurrent increase of E C . However, highly shade-intolerant species, such as Larix principis-rupprechtii Mayr., may be so sensitive to increased shading that such compensation is not complete. We hypothesized that in such a stand, windfall-induced spatial variation at a decameter scale would result in greatly reduced E L in patches of high L leading to lower E C than low competition patches of sparse canopy. We further hypothesized that quicker extraction of soil moisture in patches of lower competition will result in earlier onset of drought symptoms in these patches. Thus, patches of low L will transition from light to soil moisture as the factor dominating E L . This process should progressively homogenize E C in the stand even as the variation of soil moisture is increasing. We tested the hypotheses utilizing sap flux of nine trees, and associated environmental and stand variables. The results were consistent with only some of the expectations. Under non-limiting soil moisture, E L was very sensitive to the spatial variation of L , decreasing sharply with increasing L and associated decrease of mean light intensity on leaf surfaces. Thus, under the conditions of ample soil moisture maximum E C decreased with increasing patch-scale L . Annual E C and biomass production also decreased with L , albeit more weakly. Furthermore, variation of E C among patches decreased as average stand soil moisture declined between rain events. However, contrary to expectation, high L plots which transpired less showed a greater E L sensitivity to decreasing stand-scale soil moisture, suggesting a different mechanism than simple control by decreasing soil moisture. We offer potential explanations to the observed phenomenon. Our results demonstrate that spatial variation of L at decameter scale, even within relatively homogeneous, single-species, even-aged stands, can produce large variation of transpiration, soil moisture and biomass production and should be considered in 1-D soil–plant–atmosphere models.

Description: The main goal of this study was to develop a method for the extraction and indirect estimation of the quantity of calcium oxalate (CaOx) in the foliage of trees. Foliar tissue was collected from a single tree of each species (five conifers and five hardwoods) for comparison of extractions in different solvents using 10 replicates per species from the same pool of tissue. For each species, calcium (Ca) and oxalate were extracted sequentially in double deionized water and 2N acetic acid, and finally, five replicate samples were extracted in 5% (0.83N) perchloric acid (PCA) and the other five in 2N hydrochloric acid (HCl); three cycles of freezing and thawing were used for each solvent. Total ions were extracted by microwave digestion. Calcium was quantified with an inductively coupled plasma emission spectrophotometer method and oxalate was eluted and quantified using a high performance liquid chromatography method. This experiment was repeated again with two conifer and two hardwood species using four trees per species, and two analytical replicates for each tree. We report here that, regardless of age of individual trees within a species, time of collection or species type, the third extraction in PCA or HCl resulted in near equimolar quantities of Ca and oxalate ( r 2  ≥ 0.99). This method provides an easy estimate of the quantity of CaOx crystals using a small sample of foliar tissue. An additional benefit of PCA is that it precipitates the nucleic acids and proteins, allowing the quantification of several free/soluble metabolites such as amino acids, polyamines, organic acids and inorganic elements all from a single sample extract.

Description: The white-rot fungus Heterobasidion parviporum Niemelä & Korhonen establishes a necrotrophic interaction with Norway spruce ( Picea abies (L.) H.Karst.) causing root and butt rot and growth losses in living trees. The interaction occurs first with the bark and the outer sapwood, as the pathogen enters the tree via wounds or root-to-root contacts. Later, when the fungus reaches the heartwood, it spreads therein creating a decay column, and the interaction mainly occurs in the inner sapwood where the tree creates a reaction zone. While bark and outer sapwood interactions are well studied, little is known about the nature of the transcriptional responses leading to the creation of a reaction zone. In this study, we sampled bark and sapwood both proximal and distal to the reaction zone in artificially inoculated and naturally infected trees. We quantified gene expression levels of candidate genes in secondary metabolite, hormone biosynthesis and signalling pathways using quantitative polymerase chain reaction. An up-regulation of mainly the phenylpropanoid pathway and jasmonic acid biosynthesis was found at the inoculation site, when inoculations were compared with wounding. We found that transcriptional responses in inner sapwood were similar to those reported upon infection through the bark. Our data suggest that the defence mechanism is induced due to direct fungal contact irrespective of the tissue type. Understanding the nature of these interactions is important when considering tree breeding-based resistance strategies to reduce the spread of the pathogen between and within trees.

Description: Global warming and associated decreases in summer rainfall may threaten tree vitality and forest productivity in many regions of the temperate zone in the future. One option for forestry to reduce the risk of failure is to plant genotypes which combine high productivity with drought tolerance. Growth experiments with provenances from different climates indicate that drought exposure can trigger adaptive drought responses in temperate trees, but it is not well known whether and to what extent regional precipitation reduction can increase the drought resistance of a species. We conducted a common garden growth experiment with five European beech ( Fagus sylvatica L.) populations from a limited region with pronounced precipitation heterogeneity (816–544 mm year –1 ), where phylogenetically related provenances grew under small to large water deficits. We grew saplings of the five provenances at four soil moisture levels (dry to moist) and measured ~30 morphological (leaf and root properties, root : shoot ratio), physiological (leaf water status parameters, leaf conductance) and growth-related traits (above- and belowground productivity) with the aim to examine provenance differences in the drought response of morphological and physiological traits and to relate the responsiveness to precipitation at origin. Physiological traits were more strongly influenced by provenance (one-third of the studied traits), while structural traits were primarily affected by water availability in the experiment (two-thirds of the traits). The modulus of leaf tissue elasticity reached much higher values late in summer in plants from moist origins resulting in more rapid turgor loss and a higher risk of hydraulic failure upon drought. While experimental water shortage affected the majority of morphological and productivity-related traits in the five provenances, most parameters related to leaf water status were insensitive to water shortage. Thus, plant morphology, and root growth in particular, did respond to reduced water availability with higher phenotypic plasticity than did physiology. We conclude that beech provenances exposed to different precipitation regimes have developed some genotypic differences with respect to leaf water status regulation, but these adaptations are associated with only minor adaptation in plant morphology and they do not affect the growth rate of the saplings.

Description: Many studies have demonstrated linkages between the occurrence of fog and ecophysiological functioning in cloud forests, but few have investigated hydraulic functioning as a determining factor that explains sharp changes in vegetation. The objective of this study was to compare the plant water status during cloud-immersed and non-immersed conditions and hydraulic vulnerability in branches and roots of species across a temperate, mountain fog ecotone. Because cloud forests are often dark, cool and very moist, we expected cloud forest species to have less drought-tolerant characteristics (i.e., lower P e and P 50 —the pressures required to induce a 12 and 50% loss in hydraulic conductivity, respectively) relative to non-cloud forest species in adjacent (lower elevation) forests. Additionally, due to the ability of cloud forest species to absorb cloud-fog water, we predicted greater improvements in hydraulic functioning during fog in cloud forest species relative to non-cloud forest species. Across the cloud forest ecotone, most species measured were very resistant to losses in conductivity with branch P 50 values from –4.5 to –6.0 MPa, hydraulic safety margins ( min – P 50 ) 〉1.5 MPa and low calculated hydraulic conductivity losses. Roots had greater vulnerabilities, with P 50 values ranging from –1.4 to –2.5 MPa, leading to greater predicted losses in conductivity (~20%). Calculated values suggested strong losses of midday leaf hydraulic conductance in three of the four species, supporting the hydraulic segmentation hypothesis. In both cloud forest and hardwood species, s were greater on foggy days than sunny days, demonstrating the importance of fog periods to plant water balance across fog regimes. Thus, frequent fog did not result in systemic changes in hydraulic functioning or vulnerability to embolism across our temperate cloud forest ecotone. Finally, roots functioned with lower hydraulic conductivity than branches, suggesting that they may serve as more sensitive indicators of hydraulic functioning in these mesic, foggy ecosystems.

Description: Climate warming is having an impact on distribution, acclimation and defence capability of plants. We compared the emission rate and composition of volatile organic compounds (VOCs) from silver birch ( Betula pendula (Roth)) provenances along a latitudinal gradient in a common garden experiment over the years 2012 and 2013. Micropropagated silver birch saplings from three provenances were acquired along a gradient of 7° latitude and planted at central (Joensuu 62°N) and northern (Kolari 67°N) sites. We collected VOCs emitted by shoots and assessed levels of herbivore damage of three genotypes of each provenance on three occasions at the central site and four occasions at the northern site. In 2012, trees of all provenances growing at the central site had higher total VOC emission rates than the same provenances growing at the northern site; in 2013 the reverse was true, thus indicating a variable effect of latitude. Trees of the southern provenance had lower VOC emission rates than trees of the central and northern provenances during both sampling years. However, northward or southward translocation itself had no significant effect on the total VOC emission rates, and no clear effect on insect herbivore damage. When VOC blend composition was studied, trees of all provenances usually emitted more green leaf volatiles at the northern site and more sesquiterpenes at the central site. The monoterpene composition of emissions from trees of the central provenance was distinct from that of the other provenances. In summary, provenance translocation did not have a clear effect in the short-term on VOC emissions and herbivory was not usually intense at the lower latitude. Our data did not support the hypothesis that trees growing at lower latitudes would experience more intense herbivory, and therefore allocate resources to chemical defence in the form of inducible VOC emissions.

Description: Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P.   x   canescens . Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium -infiltrated into leaves of the two poplar species. Agrobacterium -mediated delivery of the shorter insert, TRV2 -PePDS 266 , into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS 558 . The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV–VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65–73 and 83–94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P.   x   canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P.   x   canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P.   x   canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P.   x   canescens using three different statistical approaches, geNorm, NormFinder and BestKeeper. Quantitative real-time PCR showed significant reductions in PDS transcripts (55–64%) in the photobleached leaves of both VIGS-treated poplar species. Our results demonstrate that the TRV-based VIGS provides a practical tool for gene functional analysis in Populus sp., especially in those poplar species which are otherwise recalcitrant to transformation.

Description: Latex, the cytoplasm of laticiferous cells localized in the inner bark of rubber trees ( Hevea brasiliensis Müll. Arg.), is collected by tapping the bark. Following tapping, latex flows out of the trunk and is regenerated, whereas in untapped trees, there is no natural exudation. It is still unknown whether the carbohydrates used for latex regeneration in tapped trees is coming from recent photosynthates or from stored carbohydrates, and in the former case, it is expected that latex carbon isotope composition of tapped trees will vary seasonally, whereas latex isotope composition of untapped trees will be more stable. Temporal variations of carbon isotope composition of trunk latex ( 13 C-L), leaf soluble compounds ( 13 C-S) and bulk leaf material ( 13 C-B) collected from tapped and untapped 20-year-old trees were compared. A marked difference in 13 C-L was observed between tapped and untapped trees whatever the season. Trunk latex from tapped trees was more depleted (1.6 on average) with more variable 13 C values than those of untapped trees. 13 C-L was higher and more stable across seasons than 13 C-S and 13 C-B, with a maximum seasonal difference of 0.7 for tapped trees and 0.3 for untapped trees. 13 C-B was lower in tapped than in untapped trees, increasing from August (middle of the rainy season) to April (end of the dry season). Differences in 13 C-L and 13 C-B between tapped and untapped trees indicated that tapping affects the metabolism of both laticiferous cells and leaves. The lack of correlation between 13 C-L and 13 C-S suggests that recent photosynthates are mixed in the large pool of stored carbohydrates that are involved in latex regeneration after tapping.

Description: Trees contain non-structural carbon (NSC), but it is unclear for how long these reserves are stored and to what degree they are used to support plant activity. We used radiocarbon ( 14 C) to show that the carbon (C) in stemwood NSC can achieve ages of several decades in California oaks. We separated NSC into two fractions: soluble (~50% sugars) and insoluble (mostly starch) NSC. Soluble NSC contained more C than insoluble NSC, but we found no consistent trend in the amount of either pool with depth in the stem. There was no systematic difference in C age between the two fractions, although ages increased with stem depth. The C in both NSC fractions was consistently younger than the structural C from which they were extracted. Together, these results indicate considerable inward mixing of NSC within the stem and rapid exchange between soluble and insoluble pools, compared with the timescale of inward mixing. We observed similar patterns in sympatric evergreen and deciduous oaks and the largest differences among tree stems with different growth rates. The 14 C signature of carbon dioxide (CO 2 ) emitted from tree stems was higher than expected from very recent photoassimilates, indicating that the mean age of C in respiration substrates included a contribution from C fixed years previously. A simple model that tracks NSC produced each year, followed by loss (through conversion to CO 2 ) in subsequent years, matches our observations of inward mixing of NSC in the stem and higher 14 C signature of stem CO 2 efflux. Together, these data support the idea of continuous accumulation of NSC in stemwood and that ‘vigor’ (growth rate) and leaf habit (deciduous vs evergreen) control NSC pool size and allocation.

Description: Gibberellins (GAs) are important regulators of plant shoot biomass growth, and GA 20-oxidase (GA20ox) is one of the major regulatory enzymes in the GA biosynthetic pathway. Previously, we showed that the expression levels of a putative GA20ox1 (i.e., PdGA20ox1 ) in stem tissue of 3-month-old seedlings of 12 families of Pinus densiflora were positively correlated with stem diameter growth across those same families growing in an even-aged 32-year-old pine forest (Park EJ, Lee WY, Kurepin LV, Zhang R, Janzen L, Pharis RP (2015) Plant hormone-assisted early family selection in Pinus densiflora via a retrospective approach. Tree Physiol 35:86–94). To further investigate the molecular function of this gene in the stem wood growth of forest trees, we produced transgenic poplar lines expressing PdGA20ox1 under the control of the 35S promoter (designated as 35S::PdGA20ox1). By age 3 months, most of the 35S::PdGA20ox1 poplar trees were showing an exceptional enhancement of stem wood growth, i.e., up to fourfold increases in stem dry weight, compared with the nontransformed control poplar plants. Significant increases in endogenous GA 1 , its immediate precursor (GA 20 ) and its catabolite (GA 8 ) in elongating internode tissue accompanied the increased stem growth in the transgenic lines. Additionally, the development of gelatinous fibers occurred in vertically grown stems of the 35S::PdGA20ox1 poplars. An analysis of the cell wall monosaccharide composition of the 35S::PdGA20ox1 poplars showed significant increases in xylose and glucose contents, indicating a qualitative increase in secondary wall depositions. Microarray analyses led us to find a total of 276 probe sets that were upregulated (using threefold as a threshold) in the stem tissues of 35S::PdGA20ox1 poplars relative to the controls. ‘Cell organization or biogenesis’- and ‘cell wall’-related genes were overrepresented, including many of genes that are involved in cell wall modification. Several transcriptional regulators, which positively regulate cell elongation through GA signaling, were also upregulated. In contrast, genes involved in defense signaling were appreciably downregulated in the 35S::PdGA20ox1 stem tissues, suggesting a growth versus defense trade-off. Taken together, our results suggest that PdGA20ox1 functions to promote stem growth and wood formation in poplar, probably by activating GA signaling while coincidentally depressing defense signaling.

Description: Bark beetles (Coleoptera: Curculionidae, Scolytinae) cause widespread tree mortality in coniferous forests worldwide. Constitutive and induced host defenses are important factors in an individual tree’s ability to survive an attack and in bottom-up regulation of bark beetle population dynamics, yet quantifying defense levels is often difficult. For example, in Pinus spp., resin flow is important for resistance to bark beetles but is extremely variable among individuals and within a season. While resin is produced and stored in resin ducts, the specific resin duct metrics that best correlate with resin flow remain unclear. The ability and timing of some pine species to produce induced resin is also not well understood. We investigated (i) the relationships between ponderosa pine ( Pinus ponderosa Lawson & C. Lawson) resin flow and axial resin duct characteristics, tree growth and physiological variables, and (ii) if mechanical wounding induces ponderosa pine resin flow and resin ducts in the absence of bark beetles. Resin flow increased later in the growing season under moderate water stress and was highest in faster growing trees. The best predictors of resin flow were nonstandardized measures of resin ducts, resin duct size and total resin duct area, both of which increased with tree growth. However, while faster growing trees tended to produce more resin, models of resin flow using only tree growth were not statistically significant. Further, the standardized measures of resin ducts, density and duct area relative to xylem area, decreased with tree growth rate, indicating that slower growing trees invested more in resin duct defenses per unit area of radial growth, despite a tendency to produce less resin overall. We also found that mechanical wounding induced ponderosa pine defenses, but this response was slow. Resin flow increased after 28 days, and resin duct production did not increase until the following year. These slow induced responses may allow unsuccessfully attacked or wounded trees to resist future bark beetle attacks. Forest management that encourages healthy, vigorously growing trees will also favor larger resin ducts, thereby conferring increased constitutive resistance to bark beetle attacks.

Description: Temperature responses and sensitivity of photosynthesis ( A n _ T ) and respiration for leaves at different ages are crucial to modeling ecosystem carbon (C) cycles and productivity of evergreen forests. Understanding the mechanisms and processes of temperature sensitivity may further shed lights on temperature acclimation of photosynthesis and respiration with leaf aging. The current study examined temperature responses of photosynthesis and respiration of young leaves (YLs) (fully expanded in current growth season) and old leaves (OLs) (fully expanded in last growth season) of Quercus aquifolioides Rehder and E.H. Wilson in an alpine oak forest, southwestern China. Temperature responses of dark respiration ( R dark ), net assimilation ( A n ), maximal velocity of carboxylation ( V cmax ) and maximum rate of electron transport ( J max ) were significantly different between the two leaf ages. Those differences implied different temperature response parameters should be used for leaves of different ages in modeling vegetation productivity and ecosystem C cycles in Q. aquifolioides forests and other evergreen forests. We found that RuBP carboxylation determined the downward shift of A n _ T in OLs, while RuBP regeneration and the balance between Rubisco carboxylation and RuBP regeneration made little contribution. Sensitivity of stomatal conductance to vapor pressure deficit changed in OLs and compensated part of the downward shift. We also found that OLs of Q. aquifolioides had lower A n due to lower stomatal conductance, higher stomatal conductance limitation and deactivation of the biochemical processes. In addition, the balance between R dark and A n changed between OLs and YLs, which was represented by a higher R dark / A n ratio for OLs.

Description: Plants allocate carbon (C) to sink tissues depending on phenological, physiological or environmental factors. We still have little knowledge on C partitioning into various cellular compounds and metabolic pathways at various ecophysiological stages. We used compound-specific stable isotope analysis to investigate C partitioning of freshly assimilated C into tree compartments (needles, branches and stem) as well as into needle water-soluble organic C (WSOC), non-hydrolysable structural organic C (stOC) and individual chemical compound classes (amino acids, hemicellulose sugars, fatty acids and alkanes) of Norway spruce ( Picea abies ) following in situ 13 C pulse labelling 15 days after bud break. The 13 C allocation within the above-ground tree biomass demonstrated needles as a major C sink, accounting for 86% of the freshly assimilated C 6 h after labelling. In needles, the highest allocation occurred not only into the WSOC pool (44.1% of recovered needle 13 C) but also into stOC (33.9%). Needle growth, however, also caused high 13 C allocation into pathways not involved in the formation of structural compounds: (i) pathways in secondary metabolism, (ii) C-1 metabolism and (iii) amino acid synthesis from photorespiration. These pathways could be identified by a high 13 C enrichment of their key amino acids. In addition, 13 C was strongly allocated into the n -alkyl lipid fraction (0.3% of recovered 13 C), whereby 13 C allocation into cellular and cuticular exceeded that of epicuticular fatty acids. 13 C allocation decreased along the lipid transformation and translocation pathways: the allocation was highest for precursor fatty acids, lower for elongated fatty acids and lowest for the decarbonylated n -alkanes. The combination of 13 C pulse labelling with compound-specific 13 C analysis of key metabolites enabled tracing relevant C allocation pathways under field conditions. Besides the primary metabolism synthesizing structural cell compounds, a complex network of pathways consumed the assimilated 13 C and kept most of the assimilated C in the growing needles.

Description: Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g –1 for soluble sugars, 6–533 (mean = 94) mg g –1 for starch and 53–649 (mean = 153) mg g –1 for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R 2 = 0.05–0.12 for soluble sugars, 0.10–0.33 for starch and 0.01–0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g –1 for total NSC, compared with the range of laboratory estimates of 596 mg g –1 . Laboratories were reasonably consistent in their ranks of estimates among tissues for starch ( r = 0.41–0.91), but less so for total NSC ( r = 0.45–0.84) and soluble sugars ( r = 0.11–0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Description: We employed the warm temperate conifer Cunninghamia lanceolata (Lamb.) Hook. as a model of plantation forest species to investigate ecophysiological responses to root treatments (control (0%), and ~25, 50 or 75% of the initial root mass) under well-watered and water-limited conditions. Our results indicated that total root dry mass accumulation was negatively associated with the severity of root pruning, but there was evidence of multiple compensatory responses. The plants exhibited higher instantaneous and long-term (assessed by carbon isotope composition, 13 C) water-use efficiency in pruning treatments, especially under low water availability. Root pruning also increased the fine root/total root mass ratio, specific root length and fine root vitality in both water availability treatments. As a result of the compensatory responses, under well-watered conditions, height, stem dry mass accumulation, leaf/fine root biomass ratio (L/FR), transpiration rate, photosynthetic capacity and photosynthetic nitrogen-use efficiency ( E N ) were the highest under 25% pruning. Yet, all these traits except L/FR and foliage nitrogen content were severely reduced under 75% pruning. Drought negatively affected growth and leaf gas exchange rates, and there was a greater negative effect on growth, water potential, gas exchange and E N when 〉25% of total root biomass was removed. The stem/aboveground mass ratio was the highest under 25% pruning in both watering conditions. These results indicate that the responses to root severance are related to the excision intensity and soil moisture content. A moderate root pruning proved to be an effective means to improve stem dry mass accumulation.

Description: The timing of wood formation is crucial to determine how environmental factors affect tree growth. The long-lived bristlecone pine ( Pinus longaeva D. K. Bailey) is a foundation treeline species in the Great Basin of North America reaching stem ages of about 5000 years. We investigated stem cambial phenology and radial size variability to quantify the relative influence of environmental variables on bristlecone pine growth. Repeated cellular measurements and half-hourly dendrometer records were obtained during 2013 and 2014 for two high-elevation stands included in the Nevada Climate-ecohydrological Assessment Network. Daily time series of stem radial variations showed rehydration and expansion starting in late April–early May, prior to the onset of wood formation at breast height. Formation of new xylem started in June and lasted until mid-September. There were no differences in phenological timing between the two stands, or in the air and soil temperature thresholds for the onset of xylogenesis. A multiple logistic regression model highlighted a separate effect of air and soil temperature on xylogenesis, the relevance of which was modulated by the interaction with vapor pressure and soil water content. While air temperature plays a key role in cambial resumption after winter dormancy, soil thermal conditions coupled with snowpack dynamics also influence the onset of wood formation by regulating plant–soil water exchanges. Our results help build a physiological understanding of climate–growth relationships in P. longaeva , the importance of which for dendroclimatic reconstructions can hardly be overstated. In addition, environmental drivers of xylogenesis at the treeline ecotone, by controlling the growth of dominant species, ultimately determine ecosystem responses to climatic change.

Description: Seasonal analyses of cambial cell production and day-by-day stem radial increment can help to elucidate how climate modulates wood formation in conifers. Intra-annual dynamics of wood formation were determined with microcores and dendrometers and related to climatic signals in Norway spruce ( Picea abies (L.) Karst.). The seasonal dynamics of these processes were observed at two sites of different altitude, Savignano (650 m a.s.l.) and Lavazè (1800 m a.s.l.) in the Italian Alps. Seasonal dynamics of cambial activity were found to be site specific, indicating that the phenology of cambial cell production is highly variable and plastic with altitude. There was a site-specific trend in the number of cells in the wall thickening phase, with the maximum cell production in early July (DOY 186) at Savignano and in mid-July (DOY 200) at Lavazè. The formation of mature cells showed similar trends at the two sites, although different numbers of cells and timing of cell differentiation were visible in the model shapes; at the end of ring formation in 2010, the number of cells was four times higher at Savignano (106.5 cells) than at Lavazè (26.5 cells). At low altitudes, microcores and dendrometers described the radial growth patterns comparably, though the dendrometer function underlined the higher upper asymptote of maximum growth in comparison with the cell production function. In contrast, at high altitude, these functions exhibited different trends. The best model was obtained by fitting functions of the Gompertz model to the experimental data. By combining radial growth and cambial activity indices we defined a model system able to synchronize these processes. Processes of adaptation of the pattern of xylogenesis occurred, enabling P. abies to occupy sites with contrasting climatic conditions. The use of daily climatic variables in combination with plant functional traits obtained by sensors and/or destructive sampling could provide a suitable tool to better investigate the effect of disturbances on response strategies in trees and, consequently, contribute to improving our prediction of tree growth and species resilience based on climate scenarios.

Description: In deciduous trees growing in temperate forests, bud break and growth in spring must rely on intrinsic carbon (C) reserves. Yet it is unclear whether growth and C storage occur simultaneously, and whether starch C in branches is sufficient for refoliation. To test in situ the relationships between growth, phenology and C utilization, we monitored stem growth, leaf phenology and stem and branch nonstructural carbohydrate (NSC) dynamics in three deciduous species: Carpinus betulus L., Fagus sylvatica L. and Quercus petraea (Matt.) Liebl. To quantify the role of NSC in C investment into growth, a C balance approach was applied. Across the three species, 〉95% of branchlet starch was consumed during bud break, confirming the importance of C reserves for refoliation in spring. The C balance calculation showed that 90% of the C investment in foliage (7.0–10.5 kg tree –1 and 5–17 times the C needed for annual stem growth) was explained by simultaneous branchlet starch degradation. Carbon reserves were recovered sooner than expected, after leaf expansion, in parallel with stem growth. Carpinus had earlier leaf phenology (by ~25 days) but delayed cambial growth (by ~15 days) than Fagus and Quercus , the result of a competitive strategy to flush early, while having lower NSC levels.

Description: Fungal infections result in decreases in photosynthesis, induction of stress and signaling volatile emissions and reductions in constitutive volatile emissions, but the way different physiological processes scale with the severity of infection is poorly known. We studied the effects of infection by the obligate biotrophic fungal pathogen Melampsora larici-populina Kleb., the causal agent of poplar leaf rust disease, on photosynthetic characteristics, and constitutive isoprene and induced volatile emissions in leaves of Populus balsamifera var. suaveolens (Fisch.) Loudon. exhibiting different degrees of damage. The degree of fungal damage, quantified by the total area of chlorotic and necrotic leaf areas, varied between 0 (noninfected control) and ~60%. The rates of all physiological processes scaled quantitatively with the degree of visual damage, but the scaling with damage severity was weaker for photosynthetic characteristics than for constitutive and induced volatile release. Over the whole range of damage severity, the net assimilation rate per area ( A A ) decreased 1.5-fold, dry mass per unit area 2.4-fold and constitutive isoprene emissions 5-fold, while stomatal conductance increased 1.9-fold and dark respiration rate 1.6-fold. The emissions of key stress and signaling volatiles (methanol, green leaf volatiles, monoterpenes, sesquiterpenes and methyl salicylate) were in most cases nondetectable in noninfested leaves, and increased strongly with increasing the spread of infection. The moderate reduction in A A resulted from the loss of photosynthetically active biomass, but the reduction in constitutive isoprene emissions and the increase in induced volatile emissions primarily reflected changes in the activities of corresponding biochemical pathways. Although all physiological alterations in fungal-infected leaves occurred in a stress severity-dependent manner, modifications in primary and secondary metabolic pathways scaled differently due to contrasting operational mechanisms.

Description: Current knowledge of the genetic mechanisms underlying the inheritance of photosynthetic activity in forest trees is generally limited, yet it is essential both for various practical forestry purposes and for better understanding of broader evolutionary mechanisms. In this study, we investigated genetic variation underlying selected chlorophyll a fluorescence (ChlF) parameters in structured populations of Scots pine ( Pinus sylvestris L.) grown on two sites under non-stress conditions. These parameters were derived from the OJIP part of the ChlF kinetics curve and characterize individual parts of primary photosynthetic processes associated, for example, with the exciton trapping by light-harvesting antennae, energy utilization in photosystem II (PSII) reaction centers (RCs) and its transfer further down the photosynthetic electron-transport chain. An additive relationship matrix was estimated based on pedigree reconstruction, utilizing a set of highly polymorphic single sequence repeat markers. Variance decomposition was conducted using the animal genetic evaluation mixed-linear model. The majority of ChlF parameters in the analyzed pine populations showed significant additive genetic variation. Statistically significant heritability estimates were obtained for most ChlF indices, with the exception of DI 0 /RC, D0 and P0 ( F v / F m ) parameters. Estimated heritabilities varied around the value of 0.15 with the maximal value of 0.23 in the ET 0 /RC parameter, which indicates electron-transport flux from Q A to Q B per PSII RC. No significant correlation was found between these indices and selected growth traits. Moreover, no genotype  x  environment interaction (G  x  E) was detected, i.e., no differences in genotypes’ performance between sites. The absence of significant G  x  E in our study is interesting, given the relatively low heritability found for the majority of parameters analyzed. Therefore, we infer that polygenic variability of these indices is selectively neutral.

Description: The ethylene response factor (ERF) family is one of the largest plant-specific transcription factor families, playing an important role in plant development and response to stresses. The ERF76 gene is a member of the poplar ERF transcription factor gene family. First, we validated that the ERF76 gene expressed in leaf and root tissues is responsive to salinity stress. We then successfully cloned the ERF76 cDNA fragment containing an open reading frame from di-haploid Populus simonii   x   Populus nigra and proved that ERF76 protein is targeted to the nucleus. Finally, we transferred the gene into the same poplar clone by the Agrobacterium -mediated leaf disc method. Using both RNA-Seq and reverse transcription-quantitative polymerase chain reaction, we validated that expression level of ERF76 is significantly higher in transgenic plants than that in the nontransgenic control. Using RNA-Seq data, we have identified 375 genes that are differentially expressed between the transgenic plants and the control under salt treatment. Among the differentially expressed genes, 16 are transcription factor genes and 45 are stress-related genes, both of which are upregulated significantly in transgenic plants, compared with the control. Under salt stress, the transgenic plants showed significant increases in plant height, root length, fresh weight, and abscisic acid (ABA) and gibberellin (GA) concentration compared with the control, suggesting that overexpression of ERF76 in transgenic poplar upregulated the expression of stress-related genes and increased the ability of ABA and GA biosynthesis, which resulted in stronger tolerance to salt stress.

Description: Summer droughts are likely to increase in frequency and intensity across Europe, yet long-lived trees may have a limited ability to tolerate drought. It is therefore critical that we improve our understanding of phenotypic plasticity to drought in natural populations for ecologically and economically important trees such as Populus nigra L. A common garden experiment was conducted using ~500 wild P. nigra trees, collected from 11 river populations across Europe. Phenotypic variation was found across the collection, with southern genotypes from Spain and France characterized by small leaves and limited biomass production. To examine the relationship between phenotypic variation and drought tolerance, six genotypes with contrasting leaf morphologies were subjected to a water deficit experiment. ‘North eastern’ genotypes were collected at wet sites and responded to water deficit with reduced biomass growth, slow stomatal closure and reduced water use efficiency (WUE) assessed by 13 C. In contrast, ‘southern’ genotypes originating from arid sites showed rapid stomatal closure, improved WUE and limited leaf loss. Transcriptome analyses of a genotype from Spain (Sp2, originating from an arid site) and another from northern Italy (Ita, originating from a wet site) revealed dramatic differences in gene expression response to water deficit. Transcripts controlling leaf development and stomatal patterning, including SPCH , ANT , ER , AS1 , AS2 , PHB , CLV1 , ERL1–3 and TMM , were down-regulated in Ita but not in Sp2 in response to drought.

Description: Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase ( ispS ). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity ( R 2  = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.

Description: Clonal integration between ramets can be an ecological advantage of clonal plant species in environments where resources are patchily distributed. We investigated physiological integration among Populus balsamifera L. ramets under drought stress in order to demonstrate water sharing between connected ramets. Pairs of connected ramets were grown in separate pots in the greenhouse where half of ramets had the parental root connection severed and half were left intact. Drought stress was applied to one ramet, and growth, specific leaf area (SLA), net photosynthesis, stomatal conductance, leaf water potential and carbon isotopic composition ( 13 C) were measured after an 8-week growing period. Droughted ramets connected to watered ramets were able to maintain high gas exchange activity and water potential, similar to watered ramets. Leaf water potential and SLA results showed that the root connection was more beneficial for proximal compared with distal ramets. The parental root connection also allowed droughted ramets to discriminate more against 13 C compared with severed ramets. In conclusion, this study shows compelling evidence of physiological integration of connected P. balsamifera ramets through water sharing.

Description: A formula for the norm of a bilinear Schur multiplier acting from the Cartesian product $\mathcal S^2\times \mathcal S^2$ of two copies of the Hilbert–Schmidt classes into the trace class $\mathcal S^1$ is established in terms of linear Schur multipliers acting on the space $\mathcal S^\infty $ of all compact operators. Using this formula, we resolve Peller's problem on Koplienko–Neidhardt trace formulae. Namely, we prove that there exist a twice continuously differentiable function $f$ with a bounded second derivative, a self-adjoint (unbounded) operator $A$ and a self-adjoint operator $B\in \mathcal S^2$ such that \[ f(A+B)-f(A)-\left.\frac{d}{dt}(f(A+tB))\right\vert_{t=0}\notin \mathcal S^1. \]

Description: Let $\mu $ be a probability measure on $ \mathbb R^n$ with a bounded density $f$ . We prove that the marginals of $f$ on most subspaces are well-bounded. For product measures, studied recently by Rudelson and Vershynin, our results show there is a trade-off between the strength of such bounds and the probability with which they hold. Our proof rests on new affinely invariant extremal inequalities for certain averages of $f$ on the Grassmannian and affine Grassmannian. These are motivated by Lutwak's dual affine quermassintegrals for convex sets. We show that key invariance properties of the latter, due to Grinberg, extend to families of functions. The inequalities we obtain can be viewed as functional analogues of results due to Busemann–Straus, Grinberg and Schneider. As an application, we show that without any additional assumptions on $\mu $ , any marginal $\pi _E(\mu )$ , or a small perturbation thereof, satisfies a nearly optimal small-ball probability.

Description: Let $\pi :X\to \mathbb {P}^1_{\mathbb {Q}}$ be a non-singular conic bundle over $\mathbb {Q}$ having $n$ non-split fibres and denote by $N(\pi ,B)$ the cardinality of the fibres of Weil height at most $B$ that possess a rational point. Serre showed in 1990 that a direct application of the large sieve yields \[ N(\pi,B)\ll B^2(\log B)^{-n/2} \] and raised the problem of proving that this is the true order of magnitude of $N(\pi ,B)$ under the necessary assumption that there exists at least one smooth fibre with a rational point. We solve this problem for all non-singular conic bundles of rank at most 3. Our method comprises the use of Hooley neutralisers, estimating divisor sums over values of binary forms, and an application of the Rosser–Iwaniec sieve.

Description: We associate a dimer algebra $A$ to a Postnikov diagram $D$ (in a disc) corresponding to a cluster of minors in the cluster structure of the Grassmannian ${\rm Gr}(k,n)$ . We show that $A$ is isomorphic to the endomorphism algebra of a corresponding Cohen–Macaulay module $T$ over the algebra $B$ used to categorify the cluster structure of ${\rm Gr}(k,n)$ by Jensen–King–Su. It follows that $B$ can be realised as the boundary algebra of $A$ , that is, the subalgebra $eAe$ for an idempotent $e$ corresponding to the boundary of the disc. The construction and proof uses an interpretation of the diagram $D$ , with its associated plabic graph and dual quiver (with faces), as a dimer model with boundary. We also discuss the general surface case, in particular computing boundary algebras associated to the annulus.

Description: We describe a ring whose category of Cohen–Macaulay modules provides an additive categorification of the cluster algebra structure on the homogeneous coordinate ring of the Grassmannian of $k$ -planes in $n$ -space. More precisely, there is a cluster character defined on the category which maps the rigid indecomposable objects to the cluster variables and the maximal rigid objects to clusters. This is proved by showing that the quotient of this category by a single projective–injective object is Geiss–Leclerc–Schröer's category Sub $Q_k$ , which categorifies the coordinate ring of the big cell in this Grassmannian.

Description: We develop a way of seeing a complete orientable hyperbolic 4-manifold $ {\mathcal {M}}$ as an orbifold cover of a Coxeter polytope $ {\mathcal {P}} \subset \mathbb {H}^4$ that has a facet colouring. We also develop a way of finding a totally geodesic sub-manifold $ {\mathcal {N}}$ in $ {\mathcal {M}}$ , and describing the result of mutations along $ {\mathcal {N}}$ . As an application of our method, we construct an example of a complete orientable hyperbolic 4-manifold $ {\mathcal {X}}$ with a single non-toric cusp and a complete orientable hyperbolic 4-manifold ${\mathcal {Y}}$ with a single toric cusp. Both $ {\mathcal {X}}$ and $ {\mathcal {Y}}$ have twice the minimal volume among all complete orientable hyperbolic 4-manifolds.

Description: The covariogram $g_{K}$ of a convex body $K$ in $ \mathbb {R}^n$ is the function that associates to each $x\in \mathbb {R}^n$ the volume of the intersection of $K$ with $K+x$ . Determining $K$ from the knowledge of $g_K$ is known as the Covariogram Problem. It is equivalent to determining the characteristic function $1_K$ of $K$ from the modulus of its Fourier transform $\widehat {{1_K}}$ in $ \mathbb {R}^n$ , a particular instance of the Phase Retrieval Problem. We connect the Covariogram Problem to two aspects of the Fourier transform $\widehat {{1_K}}$ seen as a function in $\mathbb {C}^n$ . The first connection is with the problem of determining $K$ from the knowledge of the zero set of $\widehat {{1_K}}$ in $\mathbb {C}^n$ . To attack this problem T. Kobayashi studied the asymptotic behavior at infinity of this zero set. We obtain this asymptotic behavior assuming less regularity on $K$ and we use this result as an essential ingredient for proving that when $K$ is sufficiently smooth and in any dimension $n$ , $K$ is determined by $g_K$ in the class of sufficiently smooth bodies. The second connection is with the irreducibility of the entire function $\widehat {{1_K}}$ . This connection also shows a link between the Covariogram Problem and the Pompeiu Problem in integral geometry.

Description: The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional ‘small molecule’ drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo , as this would ensure that the relevant ‘free’ linker forms stemming from in vivo catabolism are tested.

Description: Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

Description: Galectin-4 is a member of the galectin family which consists of 15 galactoside-binding proteins. Previously, galectin-4 has been shown to have a role in cancer progression and metastasis and it is found upregulated in many solid tumours, including colorectal cancer (CRC). Recently, the role in the metastatic process was suggested to be via promoting cancer cells to adhere to blood vascular endothelium. In the present study, the regulatory region of LGALS4 (galectin-4) in seven colon cell lines was investigated with respect to genetic variation that could be linked to expression levels and therefore a tumourigenic effect. Interestingly, qRT-PCR and sequencing results revealed that galectin-4 upregulation is associated with SNPs rs116896264 and rs73933062. By use of luciferase reporter- and pull-down assays, we confirmed the association between the gene upregulation and the two SNPs. Also, using pull-down assay followed by mass spectrometry, we found that the presence rs116896264 and rs73933062 is changing transcription factors binding sites. In order to assess the frequencies of the two SNPs among colon cancer patients and healthy individuals, we genotyped 75 colon cancer patients, 12 patients with adenomatous polyposis and 17 patients with ulcerative colitis and we performed data mining in the 1000 genomes databank. We found the two SNPs co-occuring in 21% of 75 CRC patients, 0 out of 12 patients of adenomatous polyposis, and 6 out of 17 patients (35%) with ulcerative colitis. Both in the patient samples and in the 1000 genomes project, the two SNPs were found to co-occur whenever present (D' = 1).

Description: The dose effect between nucleoplasmic bridges (NPB) and relatively low doses of ionising radiation remains unknown. Accordingly, this study investigated the NPB frequencies in human peripheral blood lymphocytes exposed to low-dose 60 Co -rays. Complex anomalies, including fused nuclei (FUS), horse-shoe nuclei (HS) and circular nuclei (CIR), which possibly originated from multiple NPBs, were also scored. Human peripheral blood samples were collected from three healthy males and irradiated with 0–1 and 0–0.4 Gy 60 Co -rays. A cytokinesis-block micronucleus cytome assay was then conducted to analyse NPB, PFHC (NPB plus three complex nuclear anomalies) and micronucleus (MN) in binucleated cells. All dose–response curves followed the linear model for both NPB frequency and PFHC cell frequency. The dose–response curves between NPB frequency and absorbed dose at 0–1 and 0–0.4 Gy were y = 0.0037 x + 0.0005 ( R 2 = 0.979, P 〈 0.05) and y = 0.0043 x + 0.0004 ( R 2 = 0.941, P 〈 0.05), respectively. The dose–response curves between PFHC cell frequency and absorbed dose at 0–1 and 0–0.4 Gy were y = 0.0044 x + 0.0007 ( R 2 = 0.982, P 〈 0.05) and y = 0.0059 x + 0.0005 ( R 2 = 0.969, P 〈 0.05), respectively. The statistical significance of differences between the irradiated groups (0–0.4 Gy) and background levels of NPB, PFHC and MN were also analysed. The lowest analysable doses of NPB, PFHC and MN were 0.12, 0.08 and 0.08 Gy, respectively. In conclusion, NPBs and PFHC positively correlated with the absorbed radiation at a relatively low dose.

Description: The aim of the study was to investigate how coadministration of resveratrol (RSV) at different time after the start of irradiation influences the frequency of micronuclei (MN) in reticulocytes of bone marrow and peripheral blood, and if the RSV supplementation after termination of irradiation may influence the recovery process of damaged cells. Coadministration of RSV with 1-day delay after 1 Gy irradiation significantly decreased the levels of MN in bone marrow and in peripheral blood, whereas with 1-week delay, only in bone marrow reticulocytes. Above combined treatment did not improve the process of recovery. RSV supplementation with 1-day delay relatively to 0.5 Gy irradiation, significantly decreased the frequencies of MN, especially after coadministration with 28mg/kg bw of RSV. Coadministration of RSV since eighth day did not influence the frequencies of MN compared to irradiated cells. The recovery process in the presence of RSV proceeded faster. Supplementation of RSV following initiation of irradiation is beneficial in case of irradiation with lower doses. RSV should be supplemented as soon as possible. Supplementation of RSV after termination of irradiation significantly speed up the recovery. Current results confirmed the ability of RSV to mitigate the effect of irradiation.

Description: Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups ( trans -3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups ( trans -3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se . Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.

Description: α-, β- and -asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae , Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and β-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and β-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic -asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and β-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and β-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic -asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH--asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic -asarone.

Description: Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.

Description: Pioglitazone (PTZ) is an oral antidiabetic agent whose anti-cancer properties have been described recently. Since PTZ increases the production of reactive oxygen species in mammalian cells, the aim of current study was to evaluate the cytotoxic, mutagenic and recombinogenic effects of PTZ using respectively the in vitro mitotic index assay and the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans , which detects the loss of heterozygosity due to somatic recombination. Although the lowest PTZ concentrations (4–36 μM) did not show any significant rise in the micronucleus production, the higher PTZ concentration (108 μM) produced a statistically higher number of micronuclei than the negative control and significantly altered the cell-proliferation kinetics, demonstrating the mutagenic and antiproliferative effects of PTZ, respectively. The recombinogenic activity of PTZ, demonstrated here for the first time, was observed at the highest tested concentration (400 μM) through the homozygotization index rates significantly different from the negative control. Taken together, our results show that PTZ is genotoxic at a concentration higher than the therapeutic plasma concentration. This PTZ genotoxicity may be a potential benefit to its previously described antitumour activity.

Description: Environmental pollutants are complex mixtures in which metals are ubiquitous. Metal mixtures of arsenic, cadmium and lead are present in the occupational environment and generate health effects such as cardiovascular, renal and cancer diseases. Cell transformation induced by metal mixtures that depend on reactive oxygen species (ROS) generation, cell viability maintenance and avoidance of senescence was previously reported by our group. The aim of the present study was to explore the role of a Obg-like ATPase1 (OLA1) in the cell transformation of BALB/c 3T3 A31-1-1 clonal cells induced by a metal mixture (2 µM NaAsO 2 , 2 µM CdCl 2 and 5 µM Pb(C 2 H 3 O 2 ) 2 . 3H 2 O) through ROS generation. The interest in OLA1 is justified because this protein has been proposed to be a negative regulator of the cellular antioxidant response. Small interfering RNA (siRNA) was used to knockdown OLA1 before the initiation stage of the transformation assay. We evaluated (ROS) and OLA1 protein expression throughout the initiation and promotion stages of transformation. OLA1 knockdown modulated metal mixture-induced cell transformation more strongly when the metal mixture was an initiator stimulus than when it was a promoter. The ability of the metal mixture to initiate cell transformation was diminished by OLA1 knockdown, an effect that depended on intracellular ROS levels. The effect of OLA1 was synergistic with N -Acetyl- l -cysteine (NAC) co-treatment. Oxidative stress-associated transcription factors Egr1 and Smad were also down-regulated by the OLA1 knockdown, contributing to the rescue of metal mixture cell transformation.

Description: G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20nM folate medium compared with 2000nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza-2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by H2AX immunofluorescence ( r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level.

Description: Metal oxide nanoparticles (NPs), including zinc oxide (ZnO) NPs have shown success for use as vehicles for drug delivery and targeting gene delivery in many diseases like cancer. Current anticancer chemotherapeutics fail to effectively differentiate between cancerous and normal cells. There is an urgent need to develop novel drug delivery system that can better target cancer cells while sparing normal cells and tissues. Particularly, ZnO NPs exhibit a high degree of cancer cell selectivity and induce cell death, oxidative stress, interference with the cell cycle progression and genotoxicity in cancerous cells. In this scenario, effective cellular uptake of NP seems to be crucial, which is shown to be affected by cell cycle progression. In the present study, the cytotoxic potential of ZnO NPs and the effect of different cell cycle phases on the uptake of ZnO NPs were examined in A431 cells. It is shown that the ZnO NPs led to cell death and reactive oxygen species generation and were able to induce cell cycle arrest in S and G 2 /M phase with the higher uptake in G 2 /M phase compared with other phases.

Description: Malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, is a mutagenic and carcinogenic compound that can react with DNA to form several types of DNA adducts including the deoxyguanosine adduct (M 1 dG). The aim of this cross-sectional study was to evaluate the association between individual dietary and lifestyle habits and M 1 dG levels, measured in peripheral leukocytes in a large representative sample of the general population of Florence City (Italy). Selected anthropometric measurements, detailed information on dietary and lifestyle habits and blood samples were available for 313 adults of the Florence City Sample enrolled in the frame of European Prospective Investigation into Cancer and nutrition (EPIC) study. A multivariate regression analysis adjusted for selected individual characteristics possibly related to M 1 dG levels (sex, age, BMI, smoke, physical activity level, education level, total caloric intake and a Mediterranean dietary score) was performed to estimate the association between these parameters and M 1 dG levels. M 1 dG levels were significantly higher in women ( P = 0.014) and lower in moderately active or active subjects ( P = 0.037).We also found a significant inverse association with the Modified Mediterranean dietary score ( P for trend = 0.049), particularly evident for the highest categories of adherence. Our results indicate that M 1 dG levels can be modulated by selected individual characteristics such as gender, physical activity and a Mediterranean dietary pattern.

Description: Let $\varphi :X\to S$ be a morphism between smooth complex analytic spaces and let $f=0$ define a free divisor on $S$ . We prove that if the deformation space $T^1_{X/S}$ of $\varphi $ is a Cohen–Macaulay $\mathcal {O}_X$ -module of codimension 2, and all of the logarithmic vector fields for $f=0$ lift via $\varphi $ , then $f\circ \varphi =0$ defines a free divisor on $X$ ; this is generalized in several directions. Among applications we recover a result of Mond–van Straten, generalize a construction of Buchweitz–Conca, and show that a map $\varphi :\mathbb {C}^{n+1}\to \mathbb {C}^n$ with critical set of codimension 2 has a $T^1_{X/S}$ with the desired properties. Finally, if $X$ is a representation of a reductive complex algebraic group $G$ and $\varphi $ is the algebraic quotient $X\to S=X\!{/\!/} G$ with $X\!{/\!/} G$ smooth, then we describe sufficient conditions for $T^1_{X/S}$ to be Cohen–Macaulay of codimension 2. In one such case, a free divisor on $\mathbb {C}^{n+1}$ lifts under the operation of ‘castling’ to a free divisor on $\mathbb {C}^{n(n+1)}$ , partially generalizing work of Granger–Mond–Schulze on linear free divisors. We give several other examples of such representations.

Description: Let $M^n$ be a compact manifold of dimension $n$ with free $T^k$ -action. We consider collapsings of $M$ on $N=M/T^k$ such that the sectional curvature and diameter of $M$ satisfy $|K(M)|\leq a$ and $ {\rm diam}(M) 〈 d$ , and give examples of collapsings for all $k$ such that the first non-zero eigenvalue of Laplacian acting on 1-forms and 2-forms of $M$ are bounded above by $c(M)\cdot \hbox {inj}(M)^{2k}$ . Moreover, we prove that the first non-zero eigenvalue of Laplacian acting on 1-forms of all principal $T^k$ -bundle $M$ over $N$ is bounded below by $c(n,a,d,N)\cdot {\rm Vol}(M)^2$ and $c\cdot \hbox {inj}(M)^{2k}$ when $M$ collapses on $N$ .

Description: We give explicit atomic bases of arbitrary coefficient-free cluster algebras of types A and à . This entails showing that the minimal elements of the positive semiring of these cluster algebras form a linear basis over the integers for the cluster algebra.

Description: We prove that strongly F -regular and F -pure singularities satisfy Bertini-type theorems (including in the context of pairs) by building upon a framework of Cumino, Greco and Manaresi (compare with the work of Jouanolou and Spreafico). We also prove that F -injective singularities fail to satisfy even the most basic Bertini-type results.

Description: This is the second of a pair of papers on the Delta-group structure on the braid and mapping class groups of a surface. We obtain a description of the homotopy groups of these Delta-groups and generalize to an arbitrary surface the Berrick–Cohen–Wong–Wu exact sequence relating the Brunnian braid groups of the 2-sphere to its homotopy groups. We prove a similar result for Brunnian mapping class groups.

Description: We construct a geometric realization of the Khovanov–Lauda–Rouquier algebra R associated with a symmetric Borcherds–Cartan matrix A = ( a ij ) i , j I via quiver varieties. As an application, if a ii != 0 for any i I , we prove that there exists a one-to-one correspondence between Kashiwara's lower global basis (or Lusztig's canonical basis) of U A – (g) (respectively, V A ( )) and the set of isomorphism classes of indecomposable projective graded modules over R (respectively, R ).

Description: The purpose of this paper is to study the nature of quasi-invariant measures for finitely generated non-discrete subgroups of Diff ( S 1 ). For this, we apply ideas involving the closure of these groups to find out that the regularity of the measure depends on a ‘measurable version’ of well-known problems concerning stable self-intersection of Cantor sets. As applications, we prove that every d -quasiconformal probability measure for a non-solvable and non-discrete group must be absolutely continuous. Concerning singular quasi-invariant measures, it is also proved that their associated Hausdorff measures must either be zero or of infinite mass, a result contrasting with the case of dynamically defined Cantor sets and also applicable to the examples of singular stationary measures constructed by Kaimanovich and Le Prince. As a further application of our methods, a theorem of rigidity for measurable conjugations between groups as above is obtained.

Description: We study the space of period polynomials associated with modular forms of integral weight for finite-index subgroups of the modular group. For the modular group, this space is endowed with a pairing, corresponding to the Petersson inner product on modular forms via a formula of Haberland, and with an action of Hecke operators, defined algebraically by Zagier. We generalize Haberland's formula to (not necessarily cuspidal) modular forms for finite-index subgroups, and we show that it conceals two stronger formulas. We extend the action of Hecke operators to period polynomials of modular forms, we show that the pairing on period polynomials appearing in Haberland's formula is nondegenerate, and we determine the adjoints of Hecke operators with respect to it. We give a few applications for 1 ( N ): an extension of the Eichler–Shimura isomorphism to the entire space of modular forms; the determination of the relations satisfied by the even and odd parts of period polynomials associated with cusp forms, which are independent of the period relations; and an explicit formula for Fourier coefficients of Hecke eigenforms in terms of their period polynomials, generalizing the Coefficient theorem of Manin.

Description: We develop theorems which produce a multitude of hyperbolic triples for the finite classical groups. We apply these theorems to prove that every quasisimple group except Alt (5) and SL 2 (5) is a Beauville group. In particular, we settle a conjecture of Bauer, Catanese and Grunewald which asserts that all non-abelian finite quasisimple groups except for the alternating group Alt (5) are Beauville groups.

Description: Let U R d be open and convex. We prove that every (not necessarily Lipschitz or strongly) convex function f : U -〉 R can be approximated by real analytic convex functions, uniformly on all of U . We also show that C 0 -fine approximation of convex functions by smooth (or real analytic) convex functions on R d is possible in general if and only if d = 1. Nevertheless, for d ≥ 2, we give a characterization of the class of convex functions on R d which can be approximated by real analytic (or just smoother) convex functions in the C 0 -fine topology. It turns out that the possibility of performing this kind of approximation is not determined by the degree of local convexity or smoothness of the given function, but by its global geometrical behaviour. We also show that every C 1 convex and proper function on U can be approximated by C convex functions in the C 1 -fine topology, and we provide some applications of these results, concerning prescription of (sub-)differential boundary data to convex real analytic functions, and smooth surgery of convex bodies.

Description: Wood biophysical properties and the dynamics of water storage discharge and refilling were studied in the trunk of canopy tree species with diverse life history and functional traits in subtropical forests of northeast Argentina. Multiple techniques assessing capacitance and storage capacity were used simultaneously to improve our understanding of the functional significance of internal water sources in trunks of large trees. Sapwood capacitances of 10 tree species were characterized using pressure–volume relationships of sapwood samples obtained from the trunk. Frequency domain reflectometry was used to continuously monitor the volumetric water content in the main stems. Simultaneous sap flow measurements on branches and at the base of the tree trunk, as well as diurnal variations in trunk contraction and expansion, were used as additional measures of stem water storage use and refilling dynamics. All evidence indicates that tree trunk internal water storage contributes from 6 to 28% of the daily water budget of large trees depending on the species. The contribution of stored water in stems of trees to total daily transpiration was greater for deciduous species, which exhibited higher capacitance and lower sapwood density. A linear relationship across species was observed between wood density and growth rates with the higher wood density species (mostly evergreen) associated with lower growth rates and the lower wood density species (mostly deciduous) associated with higher growth rates. The large sapwood capacitance in deciduous species may help to avoid catastrophic embolism in xylem conduits. This may be a low-cost adaptation to avoid water deficits during peak water use at midday and under temporary drought periods and will contribute to higher growth rates in deciduous tree species compared with evergreen ones. Large capacitance appears to have a central role in the rapid growth patterns of deciduous species facilitating rapid canopy access as these species are less shade tolerant than evergreen species.

Description: Nuclear magnetic resonance (NMR) and NMR imaging (magnetic resonance imaging) offer the possibility to quantitatively and non-invasively measure the presence and movement of water. Unfortunately, traditional NMR hardware is expensive, poorly suited for plants, and because of its bulk and complexity, not suitable for use in the field. But does it need to be? We here explore how novel, small-scale portable NMR devices can be used as a flow sensor to directly measure xylem sap flow in a poplar tree ( Populus nigra L.), or in a dendrometer-like fashion to measure dynamic changes in the absolute water content of fruit or stems. For the latter purpose we monitored the diurnal pattern of growth, expansion and shrinkage in a model fruit (bean pod, Phaseolus vulgaris L.) and in the stem of an oak tree ( Quercus robur L.). We compared changes in absolute stem water content, as measured by the NMR sensor, against stem diameter variations as measured by a set of conventional point dendrometers, to test how well the sensitivities of the two methods compare and to investigate how well diurnal changes in trunk absolute water content correlate with the concomitant diurnal variations in stem diameter. Our results confirm the existence of a strong correlation between the two parameters, but also suggest that dynamic changes in oak stem water content could be larger than is apparent on the basis of the stem diameter variation alone.

Description: While natural spatial temperature gradients between measurement needles have been thoroughly investigated for continuous heat-based sap flow methods, little attention has been given to how natural changes in stem temperature impact heat pulse-based methods through temporal rather than spatial effects. By modelling the theoretical equation for both an ideal instantaneous pulse and a step pulse and applying a finite element model which included actual needle dimensions and wound effects, the influence of a varying stem temperature on heat pulse-based methods was investigated. It was shown that the heat ratio (HR) method was influenced, while for the compensation heat pulse and T max methods changes in stem temperatures of up to 0.002 °C s –1 did not lead to significantly different results. For the HR method, rising stem temperatures during measurements led to lower heat pulse velocity values, while decreasing stem temperatures led to both higher and lower heat pulse velocities, and to imaginary results for high flows. These errors of up to 40% can easily be prevented by including a temperature correction in the data analysis procedure, calculating the slope of the natural temperature change based on the measured temperatures before application of the heat pulse. Results of a greenhouse and outdoor experiment on Pinus pinea L. show the influence of this correction on low and average sap flux densities.

Description: The control of plant transpiration by stomata under water stress and recovery conditions is of paramount importance for plant performance and survival. Although both chemical and hydraulic signals emitted within a plant are considered to play a major role in controlling stomatal dynamics, they have rarely been assessed together. The aims of this study were to evaluate (i) the dynamics of chemical and hydraulic signals at leaf, stem and root level, and (ii) their effect on the regulation of stomatal conductance ( g s ) during water stress and recovery. Measurements of g s , water potential, abscisic acid (ABA) content and loss of hydraulic functioning at leaf, stem and root level were conducted during a water stress and recovery period imposed on 1-year-old olive plants ( Olea europaea L.). Results showed a strong hydraulic segmentation in olive plants, with higher hydraulic functioning losses in roots and leaves than in stems. The dynamics of hydraulic conductance of roots and leaves observed as water stress developed could explain both a protection of the hydraulic functionality of larger organs of the plant (i.e., branches, etc.) and a role in the down-regulation of g s . On the other hand, ABA also increased, showing a similar pattern to g s dynamics, and thus its effect on g s in response to water stress cannot be ruled out. However, neither hydraulic nor non-hydraulic factors were able to explain the delay in the full recovery of g s after soil water availability was restored.

Description: For isohydric trees mid-day water uptake is stable and depends on soil water status, reflected in pre-dawn leaf water potential ( pd ) and mid-day stem water potential ( md ), tree hydraulic conductance and a more-or-less constant leaf water potential ( l ) for much of the day, maintained by the stomata. Stabilization of l can be represented by a linear relationship between canopy resistance ( R c ) and vapor pressure deficit ( D ), and the slope ( B D ) is proportional to the steady-state water uptake. By analyzing sap flow (SF), meteorological and md measurements during a series of wetting and drying ( D / W ) cycles in a nectarine orchard, we found that for the range of md relevant for irrigated orchards the slope of the relationship of R c to D , B D is a linear function of md . R c was simulated using the above relationships, and its changes in the morning and evening were simulated using a rectangular hyperbolic relationship between leaf conductance and photosynthetic irradiance, fitted to leaf-level measurements. The latter was integrated with one-leaf, two-leaf and integrative radiation models, and the latter gave the best results. Simulated R c was used in the Penman–Monteith equation to simulate tree transpiration, which was validated by comparing with SF from a separate data set. The model gave accurate estimates of diurnal and daily total tree transpiration for the range of md s used in regular and deficit irrigation. Diurnal changes in tree water content were determined from the difference between simulated transpiration and measured SF . Changes in water content caused a time lag of 90–105 min between transpiration and SF for md between –0.8 and –1.55 MPa, and water depletion reached 3 l h –1 before noon. Estimated mean diurnal changes in water content were 5.5 l day –1  tree –1 at md of –0.9 MPa and increased to 12.5 l day –1  tree –1 at –1.45 MPa, equivalent to 6.5 and 16.5% of daily tree water use, respectively. Sixteen percent of the dynamic water volume was in the leaves. Inversion of the model shows that md can be predicted from D and R c , which may have some importance for irrigation management to maintain target values of md . That relationship will be explored in future research.

Description: Let $G$ be a compact connected Lie group, or more generally a path connected topological group of the homotopy type of a finite CW-complex, and let $X$ be a rational nilpotent $G$ -space. In this paper, we analyze the homotopy type of the homotopy fixed point set $X^{hG}$ , and the natural injection $k\colon X^G\hookrightarrow X^{hG}$ . We show that if $X$ is elliptic, that is, it has finite-dimensional rational homotopy and cohomology, then each path component of $X^{hG}$ is also elliptic. We also give an explicit algebraic model of the inclusion $k$ based on which we can prove, for instance, that for $G$ a torus, $\pi _* (k)$ is injective in rational homotopy but, often, far from being a rational homotopy equivalence.

Description: We employ the ergodic-theoretic machinery of scenery flows to address classical geometric measure-theoretic problems on Euclidean spaces. Our main results include a sharp version of the conical density theorem, which we show to be closely linked to rectifiability. Moreover, we show that the dimension theory of measure-theoretical porosity can be reduced back to its set-theoretic version, that Hausdorff and packing dimensions yield the same maximal dimension for porous and even mean porous measures, and that extremal measures exist and can be chosen to satisfy a generalized notion of self-similarity. These are sharp general formulations of phenomena that had been earlier found to hold in a number of special cases.

Description: A notion of tangential thickness of a manifold is introduced. An extensive calculation within the class of lens and fake lens spaces leads to a classification of such manifolds with thickness 1, 3 or 2 $k$ , for $k\geq 1$ . On the other hand, calculations of tangential thickness in terms of the dimension of the manifold and the rank of the fundamental group show very interesting and quite surprising correlations between these invariants.

Description: Extending a classical result of Widom from 1969, polynomials with small supremum norms are constructed for a large family of compact sets $\Gamma$ : their norm is at most a constant times the theoretical lower limit ${{\rm cap}}(\Gamma )^n$ , where ${{\rm cap}}(\Gamma )$ denotes logarithmic capacity. The construction is based on a discretization of the equilibrium measure, and the polynomials have the additional property that outside the given set $\Gamma$ they increase as fast as possible, namely as ${{\rm cap}}(\Gamma )^n\exp (ng_{ \overline {{{}C}}\setminus \Gamma }(z))$ , with the Green's function with pole at infinity in the exponent. This latter fact allows us to use these polynomials as building blocks in constructing Dirac delta-type polynomials around corners: if a compact set $K$ has a corner at some point $z_0$ , then Dirac delta-type polynomials (fast decreasing polynomials) peaking at $z_0$ are polynomials $P_n(z)$ with $P_n(z_0)=1$ that decrease as $|P_n(z)|\prec \exp (-n^ \beta |z-z_0|^ \gamma )$ on the set $K$ as $z$ moves away from $z_0$ . The possible $(\beta , \gamma )$ pairs are completely described in turn of the angle $\alpha \pi$ at $z_0$ ( $\beta \lt 1$ and $\gamma \ge \beta /(2- \alpha )$ or $\beta =1$ and $\gamma 〉 \beta /(2- \alpha )$ ). As application of these fast decreasing polynomials sharp Nikolskii- and Markov-type inequalities are proved for Jordan domains with corners. The paper uses distortion properties of conformal maps, potential theoretic techniques as well as the theory of weighted logarithmic potentials.

Description: We consider for every $n\in \mathbb {N}$ an algebra $\mathcal {A}_{n}$ of germs at $0\in \mathbb {R}^{n}$ of continuous real-valued functions, such that we can associate to every germ $f\in \mathcal {A}_{n}$ a (divergent) series $\mathcal {T}(f)$ with non-negative real exponents, which can be thought of as an asymptotic expansion of $f$ . We require that the $\mathbb {R}$ -algebra homomorphism $f\mapsto \mathcal {T}(f)$ be injective (quasianalyticity property). In this setting, we prove analogue results to Denef and van den Dries’ quantifier elimination theorem and Hironaka's rectilinearization theorem for subanalytic sets.

Description: Suppose that $F(x)\in \mathbb {Z}[\![x]\!]$ is a Mahler function and that $1/b$ is in the radius of convergence of $F(x)$ for an integer $b\geq 2$ . In this paper, we consider the approximation of $F(1/b)$ by algebraic numbers. In particular, we prove that $F(1/b)$ cannot be a Liouville number. If, in addition, $F(x)$ is regular, we show that $F(1/b)$ is either rational or transcendental, and in the latter case that $F(1/b)$ is an $S$ -number or a $T$ -number in Mahler's classification of real numbers.

Description: We develop techniques for computing zeta functions associated with nilpotent groups, not necessarily associative algebras, and modules, as well as Igusa-type zeta functions. At the heart of our method lies an explicit convex-geometric formula for a class of $p$ -adic integrals under non-degeneracy conditions with respect to associated Newton polytopes. Our techniques prove to be especially useful for the computation of topological zeta functions associated with algebras, resulting in the first systematic investigation of their properties.

Description: Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500mg/kg/day) or EC (250mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9 x 10 –6 on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2 x 10 –6 on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action.

Description: As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET CD59– and RBC CD59– , respectively) in peripheral blood of male Sprague Dawley ® rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET CD59– and RBC CD59– (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies.

Description: The Pig-a assay has shown promise as a regulatory assay for evaluating in vivo gene mutation. A recent International Workshop on Genotoxicity Testing workgroup discussed the state of the assay and identified several knowledge gaps in assay development. This Mutagenesis Special Topic includes a collection of reports that addresses some of these knowledge gaps, including identifying the mutations responsible for the Pig-a mutant phenotype, the effect of sex on the response, probing the robustness of the assay and expanding the number of agents tested in the assay, especially agents expected to yield negative responses.

Description: The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo . Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies. To address this concern, Wistar-Han rats were dosed by oral gavage with a non-genotoxic haemolytic agent, 2-butoxyethanol (2-BE). Dose levels ranging from 0 to 450mg/kg were tested using both single administration and 28-day treatment regimens. Haematology parameters were assessed at minimum within the first 24h of treatment and 8 days after the final administration. Pig-a mutant frequencies were assessed on Days 15 and ~30 for both treatment protocols and also on Days 43 and 57 for the 28-day protocol. Even at doses of 2-BE that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical vehicle control distribution. 2-BE therefore showed no evidence of in vivo mutagenicity in these studies. The data suggest that perturbations in haematopoiesis alone do not lead to an observation of increased mutant frequency in the Pig-a assay.

Description: The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N -ethyl- N -nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect—gene mutation in the Pig-a gene.

Description: Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, 7- or 14-week old male and female Sprague Dawley rats were exposed to N -ethyl- N -nitrosourea (ENU). In the study with the 7-week old rats, exposure was to 0, 1, 5 or 25mg ENU/kg/day for three consecutive days (study Days 1–3). Pig-a mutant phenotype reticulocyte (RET CD59– ) and mutant phenotype erythrocyte (RBC CD59– ) frequencies were determined on study Days –4, 15, 29 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis ( In Vivo MutaFlow ® ). Additionally, blood samples collected on Day 4 were analysed for micronucleated reticulocyte (MN-RET) frequency ( In Vivo MicroFlow ® ). The percentage of reticulocytes (%RET) was markedly higher in the 7-week old males compared to females through Day 15 (2.39-fold higher on Day –4). At 25mg/kg/day, ENU reduced Day 4 RET frequencies in both sexes, and the two highest dose levels resulted in elevated MN-RET frequencies, with no sex or treatment x sex interaction. The two highest dose levels significantly elevated the frequencies of mean RET CD59– and RBC CD59– in both sexes from Day 15 onward. RET CD59– and RBC CD59– frequencies were somewhat lower for females compared to males at the highest dose level studied, and differences in RET CD59– resulted in a statistically significant interaction effect of treatment x sex. In the study with 14-week old rats, treatment was for 3 days with 0 or 25mg ENU/kg/day. RET frequencies differed to a lesser degree between the sexes, and in this case there was no evidence of a treatment x sex interaction. These results suggest that the slightly higher response in younger males than in the younger females may be related to differences in erythropoiesis function at that age. In conclusion, while some quantitative differences were noted, there were no qualitative differences in how males and females responded to a prototypical mutagen, and support the contention that both sexes are equally acceptable for Pig-a gene mutation studies.

Description: Isothiocyanates are plant-derived compounds that may be beneficial in the prevention of certain chronic diseases. Yet, by stimulating the production of reactive oxygen species (ROS), isothiocyanates can be genotoxic. Whether antioxidants influence isothiocyanate-induced genotoxicity is unclear, but this situation was clarified appreciably herein. In HCT116 cells, phenethyl isothiocyanate (PEITC) increased ROS production, which was inhibited by N -acetylcysteine (NAC) and deferoxamine (DFO) but not by ascorbic acid (ASC) and trolox (TRX) that were found to be more potent radical scavengers. Surprisingly, ASC and TRX each intensified the DNA damage that was caused by PEITC, but neither ASC nor TRX by themselves caused any DNA damage. In contrast, NAC and DFO each not only attenuated PEITC-induced DNA damage but also attenuated the antioxidant-intensified, PEITC-induced DNA damage. To determine if the DNA damage could be related to possible changes in the major antioxidant defence system, glutathione (GSH) was investigated. PEITC lowered GSH levels, which was prevented by NAC, whereas ASC, TRX and DFO neither inhibited nor enhanced the GSH-lowering effect of PEITC. The GSH synthesis inhibitor, buthionine sulphoxime, intensified PEITC-induced DNA damage, although by itself buthionine sulphoxime did not directly cause DNA damage. The principal findings suggest that ASC and TRX make PEITC more genotoxic, which might be exploited in killing cancer cells as one approach in killing cancer cells is to extensively damage their DNA so as to initiate apoptosis.

Description: The mycotoxin aflatoxin B 1 (AFB 1 ) may initiate cancer by causing oxidatively damaged DNA, specifically by causing 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) lesions. Base excision repair removes these lesions, with 8-oxoguanine glycosylase (OGG1) being the rate-limiting enzyme. The aim of this study was to determine the effect of ogg1 deficiency on AFB 1 -induced oxidatively damaged DNA and tumourigenesis. Female wild-type, heterozygous and homozygous ogg1 null mice were given a single dose of 50mg/kg AFB 1 or 40 µl dimethyl sulfoxide (DMSO) ip. Neither ogg1 genotype nor AFB 1 treatment affected levels of oxidised guanine in lung or liver 2h post-treatment. AFB 1 -treated ogg1 null mice showed exacerbated weight loss and mortality relative to DMSO-treated ogg1 null mice, but AFB 1 treatment did not significantly increase lung or liver tumour incidence compared with controls, regardless of ogg1 genotype. Suspect lung masses from three of the AFB 1 -treated mice were adenomas, and masses from two of the mice were osteosarcomas. No osteosarcomas were observed in DMSO-treated mice. All liver masses from AFB 1 -treated mice were adenomas, and one also contained a hepatocellular carcinoma. In DNA from the lung tumours, the K- ras mutation pattern was inconsistent with initiation by AFB 1 . In conclusion, ogg1 status did not have a significant effect on AFB 1 -induced oxidatively damaged DNA or tumourigenesis, but deletion of one or both alleles of ogg1 did increase susceptibility to other aspects of AFB 1 toxicity.

Description: Integration of in vivo genotoxicity testing into standard toxicology studies presents multiple advantages as it reduces animal use and costs, accelerates data generation and provides concurrent data that are useful for interpreting results. The in vivo Pig-a assay is a mammalian gene mutation assay that utilises peripheral blood and thus has a high integration potential. Although inter-laboratory reproducibility has been well demonstrated, further characterisation is required for this assay. In this study, we evaluated intra-laboratory reproducibility of the in vivo Pig-a gene mutation assay (MutaFlow® kit) in rats through the conduct of an assay characterisation prior to subsequent use in Good Laboratory Practices (GLP) toxicology studies. To evaluate intra-laboratory reproducibility, intra-assay and inter-assay variability, ruggedness, robustness and blood storage stability were assessed. These assessments were performed using blood obtained from male Sprague–Dawley rats exposed to 0, 20 or 40mg/kg/day N -ethyl- N -nitrosourea via oral gavage for three consecutive days. Blood was collected from these rats on multiple occasions from Day 29 to Day 71 and samples were analysed for Pig-a mutation using the rat MutaFlow kit. Frequencies of reticulocytes (RET), mutant phenotype erythrocytes (RBC CD59– ) and mutant phenotype RET (RET CD59– ) were determined. Overall, the proportion of RET and frequencies of RBC CD59– and of RET CD59– were consistent throughout the different assessments. The assay demonstrated acceptable intra-run and inter-run variability with coefficients of variation of ≤4.8 and 20.6%, respectively. The method was shown to be independent of the analyst performing the assay and unaffected by small changes in assay conditions. Comparable results were obtained from freshly collected samples and samples refrigerated for up to 4 days. Although technically challenging, the rat Pig-a gene mutation assay using a high-throughput automated method was shown to be reliable. The different assay parameters evaluated during the conduct of this study yielded acceptable results. Thus, the method was considered suitable for use in GLP toxicology studies.

Description: Research over the years has generated enough evidence to implicate areca nut, as a carcinogen in humans. Besides oral, significant rise in the incidence of cancers of the oesophagus, liver and stomach was seen among areca nut chewers. Early diagnosis seems key to understand the initial processes of carcinogenesis which is highly curable. In North-East India, betel quid contains raw areca nut (RAN), lime and small portion of betel leaf without any other constituents. This study was not intended to isolate any active ingredients from the RAN and to look its action. The present objective is to validate the screening of precocious anaphase and analysis of expression of Securin and p53 in non-target cells like human peripheral blood lymphocytes (PBLs) and mouse bone marrow cells (BMCs) as early indicative parameters of RAN + lime-induced cancers. A total of 35 mice were examined at different time points for following ad libitum administration of RAN extract in drinking water with lime. Peripheral blood was collected from 32 human donors of which, 24 were RAN + lime heavy chewers. Expression of genes was assessed by immunoblotting and/or by immunohistochemistry. Histological preparation of stomach tissue of mice revealed that RAN + lime induced stomach cancer. A gradual increase in the frequency of precocious anaphases and aneuploid cells was observed in both RAN + lime-treated mouse BMC and human PBL of RAN heavy chewers. Levels of p53 and Securin were increased in these cells during early days of RAN + lime exposure. The level of Securin was significantly higher in human tumour samples than their adjacent normal counterpart. The expression of Securin was increased significantly in RAN + lime-administered mice as well as in stomach tumour. Present study revealed that precocious anaphase and expression of p53 and Securin in non-target cells are significantly associated with an increased risk of RAN-induced cancer and thus these parameters can be of early diagnostic value.

Description: Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials due to their antibacterial properties. Owing to the recent boost in the usage of AgNPs-containing products, human exposure to AgNPs is increasing, highlighting the need for careful evaluation of AgNPs toxicity in humans. We used two cellular models, hepatic HepG2 and epithelial A549 cell lines, to study the mechanism of AgNPs-induced toxicity at the cellular level. These two cell lines differ significantly in their response to AgNPs treatment. In the case of A549 cells, a minor decrease in viability and increase in the extent of DNA breakage were observed. A markedly different response to AgNPs was observed in HepG2 cells. In short term, a massive induction of DNA breakage was observed, suggesting that the basal activity of antioxidant defence in these cells was not sufficient to effectively protect them from the nanoparticle-induced oxidative stress. After prolonged exposure, the extent of DNA breakage decreased to the level observed in the control cells proving that a successful adaptation to the new conditions had taken place. The cells that were unable to adapt must have died, as revealed by the Neutral Red assay that indicated less than half viable cells after 24-h treatment with 100 µg/ml of 20nm AgNPs. The gene expression analysis revealed that the observed adaptation was underlain by a pro-proliferative, anti-apoptotic signal leading to up-regulation of the genes promoting proliferation and inflammatory response ( EGR1 , FOS , JUN , HK2 , IL4 , MMP10 , VEGFA , WISP1 , CEBPB , IL8 , SELPLG ), genes coding the anti-apoptotic proteins ( BCL2A1 , CCL2 ) and factors involved in the response to stress ( HSPB1 , GADD45A ). Such a selection of highly resistant population of cells should be taken into account in the case of medical applications of nanoparticles since the sustained proliferative signalling and resistance to cell death are hallmarks of cancer, acquired by the cells in the process of carcinogenesis.

Description: Ionising radiation induces single-strand breaks, double-strand breaks (DSB) and base damages in human cell. DSBs are the most deleterious and if not repaired may lead to genomic instability and cell death. DSB can be repaired through non-homologous end joining (NHEJ) pathway in resting lymphocytes. In this study, NHEJ genes and proteins were studied in irradiated human peripheral blood mononuclear cells (PBMC) at resting stage. Dose-response, time point kinetics and adaptive-response studies were conducted in irradiated PBMC at various end points such as DNA damage quantitation, transcription and protein expression profile. Venous blood samples were collected from 20 random, normal and healthy donors with written informed consent. PBMC was separated and irradiated with various doses between 0.1 and 2.0 Gy ( 60 CO- source) for dose-response study. Repair kinetics of DNA damage and time point changes in expression of genes and proteins were studied in post-irradiated PBMC at 2.0 Gy at various time points up to 240min. Adaptive-response study was conducted with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4-h incubation. Our results revealed that Ku70, Ku80, XLF and Ligase IV were significantly upregulated ( P 〈 0.05) at 4-h post-irradiation at transcript and protein level. Adaptive-response study showed significantly increased expression of the proteins involved in NHEJ, suggesting their role in adaptive response in human PBMC at G 0 /G 1, which has important implications to human health.

Description: Excision repair cross complementing group 1 (ERCC1) and X-ray repair cross-complementing groups 1 (XRCC1) are DNA repair enzymes. Polymorphisms in DNA repair genes may be important factors affecting cancer susceptibility, prognosis and therapy outcome. The purpose of this study was to investigate the correlation of ERCC1 and XRCC1 polymorphisms with colorectal cancer (CRC) risk, and explore the effect of polymorphisms on event-free, overall survival and oxaliplatin-based therapy in CRC patients. Genotyping was examined with the iMLDR technique. An unconditional logistic regression model was used to estimate the association of certain polymorphisms with CRC risk. The Kaplan–Meier method, log-rank test and Cox regression model were employed to evaluate the effects of polymorphisms on survival analysis. Results showed that Trp/Trp genotype of XRCC1 Arg194Trp and AA genotype of ERCC1 rs2336219 have a significantly increased risk of CRC; Trp allele of XRCC1 Arg194Trp and CC genotype of ERCC1 rs735482 were associated with lower response to oxaliplatin-based chemotherapy, a shorter survival and a higher risk of relapse or metastasis. 194Trp/280Arg/399Arg haplotype was associated with a significant resistance, and the ERCC1 protein expression was statistically higher in tumours with rs735482 CC genotype than with AA genotype. Our studies indicate that XRCC1 and ERCC1 polymorphisms probably affect susceptibility, chemotherapy response and survival of CRC patients.

Description: Epigenetic control of gene expression in children remains poorly understood, but new technologies can help elucidate the relationship between expression and DNA methylation. Here, we utilized the nCounter Analysis System to characterise the expression of 60 genes in 69 9-year-old children from a cohort with a high prevalence of obesity. nCounter expression levels ranged broadly (from 3 to over 10000 messenger RNA counts) and were divided into four categories: high (〉2000 counts), moderate (200–1000 counts), low (100–200 counts) and marginal (〈100 counts). For a subset of five genes ( ADIPOR1 , PPARG1 , GSTM1 , PON1 and ACACA ) from different expression level categories, we validated nCounter data using reverse transcription-polymerase chain reaction (RT-PCR), and expanded RT-PCR analysis of ADIPOR1 to include 180 children. Expression data from the two methodologies were correlated for all five genes included in the validation experiment, with estimates ranging from r s = 0.26 ( P = 0.02) to r s = 0.88 ( P 〈 5 x 10 –6 ). ADIPOR1 and PPARG1 nCounter expression levels were negatively correlated ( r = –0.60, P 〈 5 x 10 –5 ), and this relationship was stronger in overweight children ( r = –0.73, P 〈 5 x 10 –5 ) than in normal weight children ( r = –0.42, P = 0.016). Using methylation data from the Infinium HumanMethylation450 BeadChip ( n = 180), we found eight CpG sites in ADIPOR1 and PPARG where methylation level was associated with expression by RT-PCR ( P 〈 0.05). Hypomethylation of PPARG gene body site cg10499651 was associated with increased expression as measured by both RT-PCR and nCounter ( P 〈 0.05). We found no statistically significant relationships between either expression or methylation of ADIPOR1 and PPARG and body mass index or waist circumference. In addition to demonstrating the validity of expression data derived from nCounter, our results illustrate the use of new technologies in assessing epigenetic effects on expression in children.

Description: The in vitro micronucleus test is a well-known test for the screening of genotoxic compounds. However until now, most studies have been performed on either human peripheral lymphocytes or established cancer cell lines. This study provides human mesenchymal stem cells as an alternative to the conventional micronucleus test. We grew umbilical cord mesenchymal stem cells (UC-MSCs) on coverslips eliminating the cumbersome technique involving hypotonic treatment, fixation and preparing smears required for suspension culture (lymphocytes). The background frequency of nuclear blebs and micronuclei in UC-MSCs was found to be 7±5, in lymphocytes 16±3.5 and 9±3 and that for A549 cell line was 65±5 and 15±5 per 1000 cells, respectively, suggesting differences in the repair mechanism of normal and cancer cell lines. We inspected the cytotoxic and genotoxic effects of two known mutagens, mitomycin-C and hydrogen peroxide (H 2 O 2 ), on UC-MSCs, lymphocytes and A549 cells. Treatment with mitomycin-C and H 2 O 2 demonstrated drastic differences in the degree of cytotoxicity and genotoxicity suggesting a constitutional difference between normal and cancer cells. In addition we tested two solvents, dimethyl sulfoxide (DMSO) and ethanol, and two drugs, metformin and rapamycin. DMSO above 1% was found to be cytotoxic and genotoxic, whereas ethanol at same concentration was neither cytotoxic nor genotoxic indicating the minimal non-toxic level of the solvents. This study thus offers UC-MSCs as a better substitute to peripheral lymphocytes and cancer cell lines for high throughput screening of compounds and reducing the animal studies.

Description: Cells exhibiting radiation-induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during n ormal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesis that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.

Description: We give a bordism-theoretic characterization of those closed almost contact $(2q{+ }1)$ -manifolds (with $q\geq 2$ ) that admit a Stein fillable contact structure. Our method is to apply Eliashberg's $h$ -principle for Stein manifolds in the setting of Kreck's modified surgery. As an application, we show that any simply connected almost contact 7-manifold with torsion-free second homotopy group is Stein fillable. We also discuss the Stein fillability of exotic spheres and examine subcritical Stein fillability.

Description: Casson-type invariants emerging from Donaldson theory over certain negative-definite four-manifolds were recently suggested by Teleman. These are defined by an algebraic count of points in a zero-dimensional moduli space of flat instantons. Motivated by the cobordism programme of proving Witten's conjecture, we use a moduli space of ${\rm PU}(2)$ Seiberg–Witten monopoles to exhibit an oriented one-dimensional cobordism of the instanton moduli space to the empty space. The Casson-type invariant must therefore vanish.

Description: The dynamical and stationary behaviors of a fourth-order equation in the unit ball with clamped boundary conditions and a singular reaction term are investigated. The equation arises in the modeling of microelectromechanical systems and includes a positive voltage parameter $\lambda$ . It is shown that there is a threshold value $\lambda _* 〉 0$ of the voltage parameter such that no radially symmetric stationary solution exists for $\lambda 〉 \lambda _* $ , while at least two such solutions exist for $\lambda \in (0,\lambda _* )$ . Local and global well-posedness results are obtained for the corresponding hyperbolic and parabolic evolution problems as well as the occurrence of finite time singularities when $\lambda 〉 \lambda _* $ .