Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Cutting edge: involvement of the type I IFN production and signaling pathway in lipopolysaccharide-induced IL-10 production

Macrophages respond to LPS by the rapid activation of proinflammatory cytokines that serve to initiate host defense against microbial invasion. To prevent injury to the host from excess production of these cytokines, IL-10 is up-regulated to feedback inhibit the proinflammatory response. However, the molecular events responsible for LPS-induced up-regulation of IL-10 remain to be elucidated. In this study, we provide evidence that production of and signaling by type I IFN is required for LPS-induced IL-10 up-regulation. In addition, we demonstrate that defect in type I IFN production and signaling results in a trend toward LPS-mediated superinduction of proinflammatory genes and cytokines in bone marrow-derived macrophages. Our findings suggest a novel anti-inflammatory role for the type I IFN production and signaling pathway in regulating LPS response in bone marrow-derived macrophages.     

A low-cost instrumented spatial linkage accurately determines ASIS position during cycle ergometry

The purpose of this study was to develop and evaluate an alternative method for determining the position of the anterior superior iliac spine (ASIS) during cycling. The approach used in this study employed an instrumented spatial linkage (ISL) system to determine the position of the ASIS in the parasagittal plane. A two-segment ISL constructed using aluminum segments, bearings, and digital encoders was tested statically against a calibration plate and dynamically against a video-based motion capture system. Four well-trained cyclists provided data at three pedaling rates. Statically, the ISL had a mean horizontal error of 0.03 +/- 0.21 mm and a mean vertical error of -0.13 +/- 0.59 mm. Compared with the video-based motion capture system, the agreement of the location of the ASIS had a mean error of 0.30 +/- 0.55 mm for the horizontal dimension and -0.27 +/- 0.60 mm for the vertical dimension. The ISL system is a cost-effective, accurate, and valid measure for two-dimensional kinematic data within a range of motion typical for cycling.     

Texture of locomotor path: a replicable characterization of a complex behavioral phenotype

A database of mouse locomotor path in spatial tests can be used to search in silico for behavioral measures that better discriminate between genotypes and are more replicable across laboratories. In this study, software for the exploration of exploration (SEE) was used to search a large database for a novel behavioral measure that would characterize complex movement paths. The database included mouse open-field behavior assessed in 3 laboratories, 7 inbred strains, several pharmacological treatments and hundreds of animals. The new behavioral measure, "path texture", was characterized using the local curvature of the path (the change of direction per unit distance, in degrees/cm) across several spatial scales, starting from scales smaller than the animal's body length and up to the scale of the arena size. Path texture analysis differs from fractal dimension analysis in that it does not assume self-similarity across scales. Path texture was found to discriminate inbred strains with relatively high broad-sense heritability (43%-71%) and high replicability across laboratories. Even genotypes that had similar path curvatures in some scales usually differed in other scales, and self-similarity across scales was not displayed by all genotypes. Amphetamine decreased the path curvature of C57BL/6 mice in small and medium scales, while having no effect on DBA/2J mice. Diazepam dose-dependently decreased the curvature of C57BL/6 mice across all scales, while 2 anxiogenic drugs, FG-7142 and pentylenetetrazole, increased it. Path texture thus has high potential for behavioral phenotyping and the study of drug effects in the mouse.     

A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2?% reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70?% identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3′-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.     

Community Assembly of Terrestrial Testate Amoebae: How is the Very First Beginning Characterized?

Testate amoebae play an important role at the very first beginning of succession on land. We used litterbags buried into four different soils to study the early colonization (which occurred within less than 55 days) and establishment of testate amoebae. The litterbag cellulose exposed at the youngest mining site poor in nitrogen and phosphorus was colonized firstly in high abundances, whereas the substrate introduced into the reference sites of undisturbed soil was colonized slowly and in low densities. Besides the (expected) small-sized r-strategists (e.g., Euglypha rotunda, Tracheleuglypha dentata, and Trinema lineare), large-sized K-strategists (e.g., Centropyxis spp., Phryganella acropodia) occurred in remarkably high densities on all sites. Species that colonized the cellulose in high densities (e.g., P. acropodia and T. dentata) were found extremely rarely in the adjacent source substrate and vice versa, stressing the importance of the target substrate quality. In the course of the experiment, the influencing environmental factors became more complex, as shown by redundancy analysis (RDA). Concerning the amoebal community, there was a change from variability to stability, as visualized by cluster analysis. Adjacent litterbags within an investigation site revealed amoebal species and abundances with an increasing similarity during exposition time, whereas the litterbags between the four investigation sites were colonized differently. These observations point to a stochastic (variable) beginning of community assembly, changing to a more deterministic (stable) course. No species replacement has been observed, which is an essential part of most successional theories. Thus, the more flexible concept of "community assembly" should be considered instead of "succession" for protozoa. The stochastic beginning of community assembly and the lack of species replacement are explained by a neutral community model.     

Seeds as Vehicles for Pathogen Importation

Since the 1900s, consumer demand for new plant products gave opportunity for many plant pathogens to disseminate to new areas on imported seeds. New markets for plant commodities encouraged plant breeders to begin collecting seed stocks from abroad. The birth of new seed companies extend their markets to new area. These events began the global dissemination of many seedborne pathogens. Many seedborne pathogens gained entry and escaped detection by specific traits that favored their dissemination. Three recent case scenarios are presented that illustrate how plant pathogens that passively employ the seed coats of their host achieved global dissemination and permanence in each patho-system. Evidence is presented to show that asparagus (Asparagus officinalis) seed produced in the US acted as a vehicle for disseminating one vegetatively compatible group (VCG) of a pathogenic fungus on asparagus called Fusarium proliferatum throughout new plantings in Australia. Similarly, public demand for Mediterranean cuisine in the US and abroad during the last 20 years led to an increase in the importation of basil (Ocimum basilicum) seed along with an inconspicuous fungus called Fusarium oxysporum. The fungus caused a destructive disease called Fusarium wilt of basil that appeared in over 25 separate locals spanning three continents. The third example demonstrated how new developments in lupine (Lupinus spp.) cultivars and increased public demand led to the global dispersal of a seedborne pathogen called Colletotrichum gloeosporioides. Each case highlights how these pathogens use seeds, humans, and particular traits to disperse globally in short period of time.