Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO₂ wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO₂ regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.     

Assessing internal epicormic dynamics in Quercus alba L. using CT scanning: the strong effects of shoot development and tree growth relative to progeny level genetic variation

Epicormic branches can be a serious silvicultural problem in many Quercus species because of the potential reduction in log value associated with their occurrence. The phenomenon is also problematic for tree improvement since the genetic component of epicormic branching has not been well quantified. The strong influence of ontogeny on epicormic development in Quercus is well established; however, the long-standing assumption that genetic variation also influences epicormics has not been rigorously tested. With trees from two, 25-year-old Quercus alba L. progeny tests in IN, USA, we used computed tomography scanning to characterize internal epicormic development. We sampled trees from upper and lower crown classes of families that had been classified as having low, medium and high numbers of epicormic sprouts. We also measured an array of variables related to growth and competition with the objective of assessing the relative impacts of genetics and vigor on epicormic development. Using generalized linear and linear mixed models, we found that ontogenetic and vigor variables were strongly associated with epicormic structure and development, and that the genetic effect was negligible. The total number of epicormics was most significantly influenced by the number of sequential branches that bore epicormics (p < 0.001) and the proportion of undeveloped epicormics was most significantly influenced by diameter increment (p < 0.001). We propose that a strong focus on individual tree vigor and form in tree improvement could minimize the impact of epicormic branching in Q. alba trees.     

Distribution and Numbers of Pygmies in Central African Forests

Pygmy populations occupy a vast territory extending west-to-east along the central African belt from the Congo Basin to Lake Victoria. However, their numbers and actual distribution is not known precisely. Here, we undertake this task by using locational data and population sizes for an unprecedented number of known Pygmy camps and settlements (n = 654) in five of the nine countries where currently distributed. With these data we develop spatial distribution models based on the favourability function, which distinguish areas with favourable environmental conditions from those less suitable for Pygmy presence. Highly favourable areas were significantly explained by presence of tropical forests, and by lower human pressure variables. For documented Pygmy settlements, we use the relationship between observed population sizes and predicted favourability values to estimate the total Pygmy population throughout Central Africa. We estimate that around 920,000 Pygmies (over 60% in DRC) is possible within favourable forest areas in Central Africa. We argue that fragmentation of the existing Pygmy populations, alongside pressure from extractive industries and sometimes conflict with conservation areas, endanger their future. There is an urgent need to inform policies that can mitigate against future external threats to these indigenous peoples’ culture and lifestyles.     

Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a “molecular cytogenetics” approach to solving problems in genomic analysis.     

Historical Ecology on Sandoy, Faroe Islands: Palaeoenvironmental and Archaeological Perspectives

We present palaeoenvironmental, geomorphological, archaeological, and place-name data which allow a holistic assessment of the history of landscape change on Sandoy, Faroe Islands, especially in terms of the changes that occurred in response to the colonization of the island by humans. In contrast to other situations in the North Atlantic region, there is considerable continuity in the patterns and processes of landscape evolution across the initial settlement horizon. Many of the characteristic features of post-settlement North Atlantic landscapes—absence of trees, widespread blanket mires, high rates of soil erosion—were already in place when the first people arrived. Although human impact on Sandoy appears to have been light, conversely, the unusual environment forced major alterations of the subsistence economy imported by the colonists. Settlement-era archaeological records suggest that, from the start, patterns of resource use differed substantially from the regional norm, and these differences became amplified over time as the Faroese economy created a locally sustainable cultural landscape.     

Detection of ecological change using multiple organism groups: metrics and uncertainty

A number of biological approaches are commonly used to assess the ecological integrity of stream ecosystems. Recently, it is becoming increasingly common to use multiple organism groups in bioassessment. Advocates of the multiple organism approach argue that the use of different organism groups should strengthen inference-based models and ultimately result in lower assessment error, while opponents argue that organism groups often respond similarly to stress implying a high degree of redundancy. Using fish, macroinvertebrate, macrophyte and benthic diatom data, site-specific parameters (e.g., water chemistry and substratum) and catchment variables from European mountain (n = 77) and lowland (n = 85) streams we evaluated the discriminatory power and uncertainty associated with the use of a number of biological metrics commonly used in stream assessment. The primary environmental gradient for both streams types was land use and nutrient enrichment. Secondary and tertiary gradients were related to habitat quality or alterations in hydromorphology. Benthic diatom and macroinvertebrate metrics showed high discriminatory power (R² values often >0.50) and low error (<30%) with the primary (nutrient) gradient, while both fish and macrophyte metrics performed relatively poorly. Conversely, both fish and macrophyte metrics showed higher response (high coefficients of determination) than either benthic diatom or macroinvertebrate metrics to the second (e.g., alteration in habitat/hydromorphology) gradient. However, the discriminatory power and error associated with individual metrics varied markedly, indicating that caution should be exercised when selecting the ‘best’ organism group or metric to monitor stress.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.