Simulated beetle defoliation on willow genotypes in mixture and monotype plantations

The effect of simulated beetle damage (0%, 25%, 50% and 75% mechanical defoliation) on 12 willow genotypes, grown in short‐rotation coppice, was studied in a modified criss‐cross experimental design. The design enabled the above‐ground effects of monoculture and mixed planting to be assessed. Repeated measurements were modelled to produce derived variables in terms of time or, more appropriately, in terms of accumulated day length (i.e. ‘developmental time’) units. These derived variables were then analysed using the REsidual Maximum Likelihood (REML) method implemented in GenStat? (2001) . No significant competition effect between the genotypes due to planting regime was detected. Genotypes Salix viminalis × Salix schwerinii‘Beagle’ and S. viminalis × S. schwerinii‘Torhild’ were found to have the greatest rate of increase in leaves regardless of defoliation and also the greatest height prior to defoliation. Genotype Salix dasyclados‘Loden’ showed the highest rate of growth under the stress of defoliation. When assessing height at the end of the growing season, S. viminalis × S. schwerinii‘Olof’ was the highest genotype for 25% and 75% levels of defoliation, but genotypes Salix aurita × Salix cinerea‘Delamere’, Loden and S. viminalis × Salix burjatica‘Ashton Parfitt’ appeared to be most tolerant by having consecutively lower base day lengths (i.e. increasing the accumulation of developmental units and the length of the growing season) for increasing defoliation. Shorter genotypes tended to be more tolerant, but of the higher genotypes reaching a control height of greater than 3 m by the end of the growing season, S. viminalis × S. schwerinii‘Tora’ and Beagle performed best to 50% defoliation.     

EFFECTS OF LIGHT ON PHOTOSYNTHESIS,GRAZING, AND POPULATION DYNAMICS OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

In studying how environmental factors control the population dynamics of Pfiesteria piscicida Steidinger et Burkholder, we examined the influence of light regime on kleptoplastidic photosynthesis, growth, and grazing. Prey (Rhodomonas sp.)‐saturated growth rate of P. piscicida increased (0.67 ± 0.03 d^?1 to 0.91 ± 0.11 d^?1) with light intensity varying from 0 to 200 μmol photons·m^?2·s^?1. No significant effect was observed on grazing, excluding the possibility that light enhanced P. piscicida growth through stimulating grazing. Light‐grown P. piscicida exhibited a higher gross growth efficiency (0.78 ± 0.10) than P. piscicida incubated in the dark (0.32 ± 0.16), and photosynthetic inhibitors significantly decreased growth of recently fed populations. These results demonstrate a role of kleptoplastidic photosynthesis in enhancing growth in P. piscicida. However, when the prey alga R. sp. was depleted, light's stimulating effect on P. piscicida growth diminished quickly, coinciding with rapid disappearance of Rhodomonas‐derived pigments and RUBISCO from P. piscicida cells. Furthermore, the effect of light on growth was reversed after extended starvation, and starved light‐grown P. piscicida declined at a rate significantly greater than dark‐incubated cultures. The observed difference in rates of decline appeared to be attributable to light‐dependent cannibalism. Using a 5‐chloromethylfluorescein diacetate staining technique, cannibalistic grazing was observed after 7 days of starvation, at a rate four times greater under illumination than in the dark. The results from this study suggest that kleptoplastidy enhances growth of P. piscicida only in the presence of algal prey. When prey is absent, P. piscicida populations may become vulnerable to light‐stimulated cannibalism.     

Ultrasonication of insulin-loaded microgel particles produced by internal gelation: Impact on particle's size and insulin bioactivity

Alginate-dextran sulfate (ADS) microgel has been used to protect insulin from gastrointestinal attack and as a carrier to promote insulin permeation through intestinal epithelium. The throughput of ADS submicron particles generation by emulsification/internal gelation is limited by its wide size distribution.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Cell structural changes in the needles of Norway spruce exposed to long-term ozone and drought

Effects of ozone and/or drought on Norway spruce needles werestudied using light microscopy and electron microscopy. Saplingswere exposed to ozone in open-top chambers during 1992–1995and also to drought in the late summers of 1993–1995.Samples from current and previous year needles were collectedfive times during 1995. Ozone increased the numbers of peroxisomesand mitochondria, which suggests that defence mechanisms againstoxidative stress were active. The results from peroxisomes suggestthat the oxidative stress was more pronounced in the upper sideof the needles, and those from mitochondria that defence wasmore active in the younger needle generation. Possibly due tothe good nitrogen status and the active defence, no ozone-specificchloroplast alterations were seen. At the end of the season,older needles from ozone treatments had smaller central vacuolescompared with other needles. Cytoplasmic vacuoles around thenucleus were increased by ozone in the beginning of the experiment,and did not increase towards the end of the season as in thecontrols. These results from vacuoles may indicate that ozoneaffected the osmotic properties of the cells. Decreased numberand underdevelopment of sclerenchyma cells and proliferationof tonoplast were related to nutrient imbalance, which was enhancedby drought. Larger vascular cylinders and more effective starchaccumulation before and after the drought periods compensatedfor the reduced water status. Numbers of peroxisomes and mitochondriawere increased in the drought-exposed needles before the onsetof drought treatments of the study year, i.e. these changeswere memory effects. Interactions between ozone and droughtwere few.     

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.      

Significance of chick quality score in broiler production

The quality of day old chicks is crucial for profitable broiler production, but a difficult trait to define. In research, both qualitative and quantitative measures are used with variable predictive value for subsequent performance. In hatchery practice, chick quality is judged on a binomial scale, as chicks are divided into first grade (Q1-saleable) and second grade (Q2) chicks right after hatch. Incidences and reasons for classifying chicks as Q2, and potential of these chicks for survival and post-hatch performance have hardly been investigated, but may provide information for flock performance. We conducted an experiment to investigate (1) the quality of a broiler flock and the relation with post-hatch flock performance based on a qualitative score (Pasgar©score) of Q1 chicks and based on the incidence of Q2 chicks and (2) the reasons for classifying chicks as Q2, and the potential of these chicks for survival and post-hatch growth. The performance was followed of Q1 and Q2 chicks obtained from two breeder flocks that hatched in two different hatching systems (a traditional hatcher or a combined hatching and brooding system, named Patio). Eggs were incubated until embryo day 18, when they were transferred to one of the two hatching systems. At embryo day 21/post-hatch day 0, all chicks from the hatcher (including Q2 chicks) were brought to Patio, where the hatchery manager marked the Q2 chicks from both flocks and hatching systems and registered apparent reasons for classifying these chicks as Q2. Chick quality was assessed of 100 Q1 chicks from each flock and hatching system. Weights of all chicks were determined at days 0, 7, 21 and 42. There were no correlations between mean Pasgar©score and post-hatch growth or mortality, and suboptimal navel quality was the only quality trait associated with lower post-hatch growth. Growth was clearly affected by breeder flock and hatching system, which could not be linked to mean Pasgar©score or incidence of Q2 chicks. Q2 chicks showed lower post-hatch growth compared to Q1 chicks but effects on flock performance at slaughter weight were limited because early mortality in Q2 chicks was high (62.50% at 7 days). We concluded that chick qualitative scores and the incidence of Q2 chicks may be informative for the quality of incubation, but are not predictive for post-hatch flock performance. Culling Q2 chicks after hatch is well-founded in terms of both animal welfare and profitability.