Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.     

Light availability influences root carbohydrates, and potentially vigor, in white oak advance regeneration

Oaks (Quercus spp.) are experiencing recurring regeneration failures associated with pervasive mid- and under-story strata of shade tolerant species in intact, undisturbed forests. Where oak regeneration occurs, inadequate vertical height and depleted root carbohydrate stores impede the ability of regenerating oaks to respond when light does become available. A variety of silvicultural techniques have been developed to increase the penetration of diffuse light, enhancing the light environment on the forest floor, and thereby increasing the likelihood of regenerating oaks to successfully respond to increased light transmittance. We measured shoot and root characteristics, and root soluble non-structural carbohydrate concentrations of white oak (Q. alba L.) advance regeneration exposed to enhanced light intensities associated with a mid-story removal and a clearcut, and compared white oak regeneration vigor to untreated controls.

Root diameter and soluble non-structural carbohydrates increased with increasing light availability. Our data suggest that white oak responds to increases in light transmittance by building below-ground biomass and carbohydrates in the root system prior to an above-ground response. Our study shows that white oak regeneration vigor increases with only modest increases in light. In the absence of other pressures, enhancing the light environment to the forest floor should contribute to successful regeneration of this species.     

Collembolan grazing affects the growth strategy of the cord-forming fungus Hypholoma fasciculare

Mycelia of cord-forming fungi show remarkable patterns of reallocation of biomass and nutrients indicating an important role of these, often extensive, organisms in the spatial translocation of energy and nutrients in forest soils. Despite the rich tradition of interaction studies between soil microarthropods and fungi, the spatial implications of these interactions, due to the potential growth responses of the fungi and to the translocation of energy and nutrients within the mycelial network, have been largely ignored. In this paper we analyse fungal growth responses in two-dimensional model systems composed of compressed soil, the cord-forming fungus Hypholoma fasciculare and three fungivorous Collembolan species. We hypothesised that (i) the highly co-ordinated nature of cord-forming fungi would lead to growth responses following collembolan grazing, and that, (ii) such changes are dependent on grazing intensity, and (iii) changes are dependent on the species grazing. Mycelial extent and hyphal cover decreased with increasing grazing density; at highest grazing density also the fractal dimension of the mycelial border decreased, indicating a less branched foraging front due to the regression of fine hyphae and the development of mycelial cords. Effects differed greatly between collembolan species although they exerted comparable grazing pressure (the smaller species were added in larger numbers according to their allometric size-metabolic rate relationships): while grazing by Folsomia candida resulted in less mycelial extension and hyphal cover, these variables were not affected when Proisotoma minuta and Hypogastrura cf. tullbergi grazed. The effects of a species mix suggested an additive effect of the component species. This shows that fungal mycelia may suffer from damage caused by few but large collembolans, affecting extension as well as coverage of the mycelium, but that fungi may compensate for the biomass loss caused by more but slightly smaller collembolans. In about 20% of the model systems H. fasciculare switched from a growth pattern with a broad contiguous foraging front and uniform growth in all directions to a pattern with fast growing sectors while other sectors stopped growth completely. The switch occurred in grazed systems exclusively; thus we interpret this observation as a fugitive response and as a strategy for quickly escaping from places where grazing pressure is experienced.     

Soil carbon release along a gradient of physical disturbance in a harvested northern hardwood forest

Changes in soil respiration associated with forest harvest could increase net loss of CO₂ to the atmosphere relative to pre-harvest values. By excavating quantitative soil pits across a gradient of physical disturbance in a harvested northern hardwood forest, this study examines C release from mineral soil. Mineral soil samples were analyzed for pH, percent organic matter (%OM), C and N concentration, δ¹³C, and total C per unit area. Results show a relationship between degree of disturbance and C concentration in soil 10-30 cm beneath the O-horizon. Highly disturbed sites show C depletion, with horizons from disturbed sites containing 25% less total C than the least disturbed sites. δ¹³C signatures of soil profiles at these sites show vertical mixing of plant-derived material into deeper mineral horizons. Mixing, as a result of physical disturbance, could have led to the observed C depletion by physical or chemical destabilization, or through the promotion of microbial respiration in deep mineral soil. Regardless of the mechanism, these results suggest elevated CO₂ emissions from soil following harvest, and, thus, have implications for the validity of wood biomass as a carbon neutral energy source.