Identifying Candidate Biomarkers of Ionizing Radiation in Human Pulmonary Microvascular Lumens Using Microfluidics-A Pilot Study.

The microvasculature system is critical for the delivery and removal of key nutrients and waste products and is significantly damaged by ionizing radiation. Single-cell capillaries and microvasculature structures are the primary cause of circulatory dysfunction, one that results in morbidities leading to progressive tissue and organ failure and premature death. Identifying tissue-specific biomarkers that are predictive of the extent of tissue and organ damage will aid in developing medical countermeasures for treating individuals exposed to ionizing radiation. In this pilot study, we developed and tested a 17 µL human-derived microvascular microfluidic lumen for identifying candidate biomarkers of ionizing radiation exposure. Through mass-spectrometry-based proteomics, we detected 35 proteins that may be candidate early biomarkers of ionizing radiation exposure. This pilot study demonstrates the feasibility of using humanized microfluidic and organ-on-a-chip systems for biomarker discovery studies. A more elaborate study of sufficient statistical power is needed to identify candidate biomarkers and test medical countermeasures of ionizing radiation.

Separating the effects of tree size and meristem maturation on shoot development of grafted scions of red spruce (Picea rubens Sarg.).

In order to separate the effects of size and meristem maturation on age-related changes in shoot growth behaviour, a reciprocal grafting experiment was conducted involving juvenile (J), mid-age (MA) and old-growth (OG) red spruce (Picea rubens Sarg.) trees as both scion donors and rootstock. The effects of rootstock and scion age on vegetative growth, foliar morphology and reproductive development were assessed over 7 years after grafting. Vegetative growth potential declines with meristem maturation, but the high growth potential of J meristems on J rootstock cannot be expressed when J scions are grafted on MA and OG rootstock. Branch density decreases with meristem maturation. The tendency for high J branch density is expressed across all rootstock ages, but is minimally expressed on MA rootstock where elongation of terminal shoots is significantly greater than on OG rootstock. Both needle width and leaf mass area increase with meristem maturation and increasing tree size. Reproductive competence is mainly a function of meristem maturity, and rootstock had little effect on strobilus development, although the more fecund rootstocks did promote some flowering on J scions. Developmental decline in height growth does not appear to be a function of decreased meristem vigour, but reduced terminal long shoot elongation and decreased apical control in OG trees will reduce height growth.

Age-related trends in red spruce foliar plasticity in relation to declining productivity.

Phenotypic plasticity in needle morphology with increasing tree size and age was investigated by comparing four age classes of red spruce (Picea rubens Sarg.) ranging from juvenile (3-12 years old) to mature (over 100 years old). With increase in tree age there were significant increases in leaf mass per unit area (LMA), mesophyll and vascular bundle area as a percentage of total needle cross-sectional area, and stomatal density. Within the vascular bundle, both xylem cross-sectional area and tracheid lumen area increased significantly, whereas air space as a percentage of total cross-sectional area decreased. These morphological changes were associated with a significant decrease in photosynthetic capacity and stomatal conductance, and an increase in (13)C enrichment. Although both photosynthetic capacity and whole-tree conductance decreased significantly between age classes 3 and 12 years, they did not differ between age classes 53 and 127 years, even though needle (13)C enrichment was significantly greater in the 127-year age class. Thus there appear to be compensatory mechanisms that maintain photosynthetic capacity as trees increase in size and vascular complexity, which in red spruce and other species, may affect leaf hydraulic conductance. Although increased LMA may contribute to reduced photosynthetic capacity in red spruce, similar relationships are not seen in other conifers.

Developmental decline in height growth in Douglas-fir.

The characteristic decline in height growth that occurs over a tree's lifespan is often called "age-related decline." But is the reduction in height growth in aging trees a function of age or of size? We grafted shoot tips across different ages and sizes of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees to determine whether the decline in height growth is mediated by tree size or by the age of the apical meristem. We also evaluated whether reduced carbon assimilation plays an important role in height growth decline. In one experiment we cut shoot tips from old-growth, young-mature and seedling trees and grafted them onto 2-year-old graft-compatible rootstock in a seed orchard in Lebanon, Oregon. In another experiment we performed reciprocal grafts between lateral branches of old-growth trees accessible from the canopy crane at Wind River, Washington and young-mature trees in a nearby plantation. We measured growth (diameter and elongation of the dominant new stem) and mortality annually for three years in the Seed Orchard experiment and for two years in the Reciprocal Graft experiment. In the Seed Orchard experiment we also measured photosynthetic capacity (determined from the response of net carbon assimilation to the intercellular CO(2) concentration of the leaf, or A/C(i) curves), leaf mass per area (LMA) and carbon isotope composition (delta(13)C) of cellulose in 1-year-old foliage. Grafting caused changes in both growth and physiology of the grafted stems. Within two years after grafting, growth and physiology of all combinations of scions and rootstock exhibited characteristics of the rootstock. In some cases, the change in growth was dramatic-cuttings from old-growth trees showed a 10-fold increase in stem elongation rate within 2 years of grafting onto seedling rootstock. Similarly, carbon isotope discrimination of new foliage on shoots from old-growth trees increased by nearly 3 per thousand and 2 per thousand after grafting onto young-mature and seedling rootstock, respectively, whereas discrimination decreased by a similar magnitude in scions from young-mature trees after grafting on old- growth trees. Furthermore, differences in carbon assimilation estimated from carbon isotope discrimination and A/C(i )relationships were small relative to growth differences. Our results confirm that size, not age, drives developmental changes in height growth in Douglas-fir. Reduced carbon assimilation does not play an important role in height growth decline.

Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (Pinus taeda) stem cuttings.

Hypocotyl cuttings (from 20- and 50-day-old Pinus taeda L. seedlings) rooted readily within 30 days in response to exogenous auxin, while epicotyl cuttings (from 50-day-old seedlings) rarely formed roots within 60 days. Responses to auxin during adventitious rooting included the induction of cell reorganization and cell division, followed by the organization of the root meristem. Explants from the bases of both epicotyl and hypocotyl cuttings readily formed callus tissue in response to a variety of auxins, but did not organize root meristems. Auxin-induced cell division was observed in the cambial region within 4 days, and later spread to the outer cortex at the same rate in both tissues. Cells at locations that would normally form roots in foliated hypocotyl cuttings did not produce callus any differently than those in other parts of the cortex. Therefore, auxin-induced root meristem organization appeared to occur independently of auxin-induced cell reorganization/division. The observation that N-(1-naphthyl)phthalamic acid (NPA) promoted cellular reorganization and callus formation but delayed rooting implies the existence of an auxin signal transduction pathway that is specific to root meristem organization. Attempts to induce root formation in callus or explants without foliage were unsuccessful. Both the cotyledon and epicotyl foliage provided a light-dependent product other than auxin that promoted root meristem formation in hypocotyl cuttings.

Variation in lateral shoot elongation patterns and hybrid vigor in full-sib families and interspecific hybrids of larch.

Time of shoot initiation and cessation, and rate and duration of lateral shoot elongation of full-sib families of Larix decidua Mill., L. laricina (Du Roi) C. Koch, and interspecific hybrids between L. decidua and L. kaempferi (Lamb.) Carrière, L. kaempferi x decidua, L. laricina x decidua, and L. laricina x kaempferi were explored in relation to hybrid vigor determined by height growth after 5 years. Height growth of L. laricina and L. decidua families was significantly less than height growth of the interspecific hybrids. Differences were observed for all lateral shoot elongation traits among L. laricina, L. decidua, and the interspecific hybrids. Lateral shoots of L. laricina families were the first to both initiate and cease elongation, and grew for the shortest period of time. Overall, the length of the shoot elongation period and date of cessation of shoot growth were significantly correlated with total height growth after 5 years, but the time of shoot initiation was not. Rate of elongation of lateral shoots was strongly correlated with growth increment in 1996, but was weakly correlated with total height growth after 5 years. Hybrid vigor in Larix seems to be partly a function of late cessation or increased duration of shoot elongation, or both.

Flowering on long and short shoots of Larix laricina in response to differential timing of GA(4/7) applications.

In Larix, reproductive buds most often occur terminally on short shoots, but they can also differentiate in lateral positions on long shoots. The phenology of long and short shoots differs considerably, with short shoots breaking bud and expanding about 5 weeks before the start of long shoot extension. Foliar sprays of GA(4/7) were applied to 160 branches on 10 greenhouse-grown Larix laricina (Du Roi) K. Koch grafts either before (early) of after (late) the start of long shoot extension, or during both periods, to test whether the timing of GA(4/7) application affects flowering on short and long shoots. All three treatments induced flowering on both long and short shoots. The early GA(4/7) treatment led to slightly, but not significantly, more flowering on short shoots than on long shoots, whereas the late GA(4/7) treatment resulted in increased flowering on both types of shoots, but primarily on long shoots. Application of GA(4/7) during both periods did not result in increased flowering over the early or late treatment alone. Based on the fact that gibberellins are metabolized rapidly in conifers and our finding that GA(4/7) applied before shoot elongation, when the bud primordia were at a very early stage of development (detectable bud differentiation only occurred several weeks later), induced flowering on long shoots, we conclude that the early GA(4/7) treatment did not affect differentiation as it was occurring, but somehow predisposed the bud primordia to differentiate reproductively.

Juvenility and maturation in conifers: current concepts.

Maturation in conifers includes several distinct and persistent changes in the growth habits of apical meristems. Despite many studies on maturation in conifers, there are still many aspects of the process that have not been elucidated. For example, it is not known why maturation of cotyledon-derived tissue culture plantlets is rapid, whereas the natural maturation process is gradual. Also, it is not known whether rejuvenation occurs as a result of mature cells reverting to the juvenile state, or whether rejuvenation results from selective multiplication of cells that have never matured. In this paper, I review the primary causes of maturation in woody plants, with emphasis on gene expression and its role in cell determination during the maturation process. Recent experiments demonstrating both accelerated maturation and apparent rejuvenation in several woody species are discussed with reference to several maturation models.