Area-based analyzing technique at cell array experiment using neuronal cell line

We propose a simple procedure for the identification and quantitative analysis of neurite outgrowth in neuronal cell lines that were uniformly differentiated. Upon stimulation most neuronal cell lines extend neurites in the differentiation process, resulting, according to our observation, in the increase of cell surface area. This increase is dependent on the length and the number of extended neurites. Furthermore, we use this method for the phenotype analysis of cell array experiments to perform large-scale functional evaluation of genes involved in the neurite outgrowth during neuronal differentiation.     

Body image and quality of life in post massive weight loss body contouring patients

Objective: Because post‐bariatric surgery patients undergo massive weight loss, the resulting skin excess can lead to both functional problems and profound dissatisfaction with appearance. Correcting skin excess could improve all these corollaries, including body image. Presently, few data are available documenting body image and weight‐related quality of life in this population. Research Methods and Procedures: Eighteen patients who underwent both bariatric surgery and body contouring completed our study. Both established surveys and new surveys designed specifically for the study were used to assess body perception and ideals, quality of life, and mood. Patients were surveyed at the following time‐points: pre‐body contouring (after massive weight loss) and both 3 and 6 month post‐body contouring. Statistical testing was performed using Student's t test and ANOVA. Results: The mean age of the patients was 46 ± 10 years (standard deviation). Quality of life improved after obesity surgery and was significantly enhanced after body contouring. Three months after body contouring, subjects ascribed thinner silhouettes to both current appearance and ideal body image. Body image also improved with body contouring surgery. Mood remained stable over 6 months. Discussion: Body contouring after surgical weight loss improved both quality‐of‐life measurements and body image. Initial body dissatisfaction did not correlate with mood. Body contouring improved body image but produced dissatisfaction with other parts of the body, suggesting that as patients become closer to their ideal, these ideals may shift. We further developed several new assessment methods that may prove useful in understanding these post‐surgical weight loss patients.     

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes. The enzyme under study forms a reaction product that is fluorescent or colored by conversion of either a fluorogenic or chromogenic substrate or a fluorescent substrate with different spectral characteristics. Most chromogenic staining methods were developed in the latter half of the twentieth century but still find new applications in modern cell biology and pathology. Fluorescence methods have rapidly evolved during the last decade. This review critically evaluates the methods that are available at present for metabolic mapping in living animals, unfixed cryostat sections of tissues, and living cells, and refers to protocols of the methods of choice. (J Histochem Cytochem 58:481–497, 2010)     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Compensatory growth of Phanerochaete velutina mycelial systems grazed by Folsomia candida (Collembola)

Phanerochaete velutina is a major agent of wood decomposition in temperate forests. It grows out of woody resources in search of other resources and is then vulnerable to grazing by invertebrates. The aim of this study was to determine how continuous grazing and grazing for only 2 days by different densities of collembola, Folsomia candida, affect mycelial development (radial extension, hyphal coverage and fractal dimension) of P. velutina growing across non-sterile soil. High density (80 collembola) continuous grazing resulted in different mycelial foraging patterns compared to controls and lower density (20 and 40 collembola) continuous grazing: radial extension rate was reduced from 8.4 mm day(-1) (control) to 6.9 mm day(-1) (80 collembola), hyphal coverage was reduced to 81% of controls and mass fractal dimension increased from 1.68 (control) to 1.72 (80 collembola). There was evidence of over-compensatory growth: when high density grazing ceased the new growth was considerably greater (38%) than in controls. Grazing also resulted in growth stimulation: at low density continuous grazing (20 collembola) hyphal coverage was 15.6% greater than in controls. The ecological implications of compensatory and stimulatory growth in fungal-invertebrate interactions are considered.     

Female Colour Polymorphism: Gender and the Eye of the Beholder in Damselflies

Damselflies provide a classic example of female colour polymorphism. Usually, one female morph resembles the blue male colour (andromorph) while one, or more, female morphs are seen as typically female (gynomorph). Damselfly species fall in two distinct groups with respect to recent developments in mimicry theory: in some species females are perfect, they match male colouration and black patterning, and in other species they are supposed to be imperfect mimics, only matching male colouration. However, the underlying assumption of one female morph looking male-like is mostly based on human vision. Therefore we investigated the black patterning and colour of the three female morphs in Coenagrion puella, an imperfect mimic, using image analysis. In C. puella the blue female morph is perceived as male-like. We found that the black patterning of such females cannot be distinguished from the other female morphs, and is clearly different from males. Furthermore, the blue colour of andromorph females differs from the blue colour of males. Intriguingly, however, the red content did not differ between blue males and females.     

Comparison of photosynthetic damage from arthropod herbivory and pathogen infection in understory hardwood saplings

Arthropods and pathogens damage leaves in natural ecosystems and may reduce photosynthesis at some distance away from directly injured tissue. We quantified the indirect effects of naturally occurring biotic damage on leaf-level photosystem II operating efficiency (Φ_PSII) of 11 understory hardwood tree species using chlorophyll fluorescence and thermal imaging. Maps of fluorescence parameters and leaf temperature were stacked for each leaf and analyzed using a multivariate method adapted from the field of quantitative remote sensing. Two tree species, Quercus velutina and Cercis canadensis, grew in plots exposed to ambient and elevated atmospheric CO₂ and were infected with Phyllosticta fungus, providing a limited opportunity to examine the potential interaction of this element of global change and biotic damage on photosynthesis. Areas surrounding damage had depressed Φ_PSII and increased down-regulation of PSII, and there was no evidence of compensation in the remaining tissue. The depression of Φ_PSII caused by fungal infections and galls extended >2.5 times further from the visible damage and was ∼40% more depressed than chewing damage. Areas of depressed Φ_PSII around fungal infections on oaks growing in elevated CO₂ were more than 5 times larger than those grown in ambient conditions, suggesting that this element of global change may influence the indirect effects of biotic damage on photosynthesis. For a single Q. velutina sapling, the area of reduced Φ_PSII was equal to the total area directly damaged by insects and fungi. Thus, estimates based only on the direct effect of biotic agents may greatly underestimate their actual impact on photosynthesis.     

Dynamics of root growth stimulation in Nicotiana tabacum in increasing light intensity

Light intensity is crucial for plant growth. In this study, the hypothesis was tested whether a sudden increase in light intensity leads to an immediate increase of root growth. Seedlings of Nicotiana tabacum grown in agar-filled Petri dishes were subjected to light intensities of 60 and 300 micromol m(-2) s(-1), respectively. Seedling biomass, sucrose, glucose and fructose concentration as well as primary root growth increased significantly with light intensity. The dynamics of the increase in root growth were analysed here in more detail. In transition experiments from low to high light intensities, root growth increased by a factor of four within 4 d, reaching the steady-state level measured in plants that were cultivated in high-light conditions. The distribution of relative elemental growth rates along the root growth zone retained a constant shape throughout this transition. During the first three hours after light increase, strong growth fluctuations were repeatedly observed with the velocity of the root tip cycling in a sinusoidal pattern between 120 and 180 microm h(-1). These dynamic patterns are discussed in the context of hydraulic and photosynthetic acclimation to the altered conditions. Experiments with externally applied sucrose and with transgenic plants having reduced capacities for sucrose synthesis indicated clearly that increasing light intensity rapidly enhanced root growth by elevating sucrose export from shoot to root.     

Molecular cloning and functional expression of a sodium bicarbonate cotransporter from guinea-pig parotid glands

We recently found that the concentration of HCO3- in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO3- transport has not yet been fully characterized. In order to investigate the mechanism of HCO3- transport, we identified, cloned, and characterized a sodium bicarbonate (Na(+)/HCO3- cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO3- or Na(+)-free solutions, which resulted in a marked change in membrane potentials (V(m)), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in V(m) and pH(i) were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.     

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.