Development of simple sequence repeat (SSR) markers for the assessment of gene flow and genetic diversity in pigeonpea (Cajanus cajan)

Pigeonpea (Cajanus cajan) is an important subsistence crop in India where traditional landraces and improved hybrids are grown alongside each other. Gene flow may result in genetic erosion of these landraces and their wild relatives, whilst transgene escape from future genetically engineered varieties is another potential hazard. To assess the impact of these factors gene flow needs to be measured. A set of 10 simple sequence repeat markers have been developed, which exhibit polymorphism across a range of pigeonpea varieties. Use of these markers also offers an efficient system for the assessment of genetic diversity within populations of pigeonpea.     

Two-dimensional and epitaxial crystallization of a mutant form of yeast RNA polymerase II

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.     

High-throughput protein crystallization

High-throughput structural biology is a focus of a number of academic and pharmaceutical laboratories around the world. The use of X-ray crystallography in these efforts is critically dependent on high-throughput protein crystallization. The application of current protocols yields crystal leads for approximately 30% of the input proteins and well-diffracting crystals for a smaller fraction. Increasing the success rate will require a multidisciplinary approach that must invoke techniques from molecular biology, protein biochemistry, biophysics, artificial intelligence, and automation.     

Quantitative analysis of Escherichia coli metabolic phenotypes within the context of phenotypic phase planes

In silico models of Escherichia coli metabolism have been developed to predict metabolic behavior and propose experimentally testable hypotheses. However, a thorough assessment of the metabolic phenotype requires well-designed experimentation and reproducible experimental techniques. A method for the quantitative analysis of E. coli metabolism in vivo within the framework of in silico phenotypic phase plane analysis is presented. Using this approach, we have quantitatively studied E. coli metabolism in various environmental conditions and nutritional media. Our experimental methodology, in combination with steady-state metabolic models, can be used to study biological properties and evaluate the metabolic capabilities of microbes.     

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Preemption of space can lead to intransitive coexistence of competitors

Intransitive competition has the potential to be a powerful contributor to species coexistence, but there are few proposed biological mechanisms that could create intransitivities in natural communities. Using a three‐species model of competition for space, we demonstrate a mechanism for coexistence that combines a colonization–competition tradeoff between two species with the ability of a third species to preempt space from the other competitors. The combination of differential abilities to colonize, preempt, and overtake space creates a community where no single species can exclude both of its competitors. The dynamics of this kind of community are analogous to rock‐paper‐scissors competition, and the three‐species community can persist even though not all pairs of species can coexist in isolation. In distinction to prior results, this is a mechanism of intransitivity that does not require nonhierarchical local interference competition. We present parameter estimates from a subtidal marine community illustrating how documented competitive traits can lead to preemption‐based intransitivities in natural communities, and we describe methods for an empirical test of the occurrence of this mechanism.     

Sputum neutrophils as a biomarker in COPD: findings from the ECLIPSE study

Introduction

The percentage of neutrophils in sputum are increased in COPD patients, and may therefore be a biomarker of airway inflammation. We studied the relationships between sputum neutrophils and FEV₁, health status, exacerbation rates, systemic inflammation and emphysema, and long term variability at 1 year.

Methods

Sputum samples were obtained from 488 COPD patients within the ECLIPSE cohort. 359 samples were obtained at baseline, and 297 after 1 year. 168 subjects provided samples at both visits. Serum interleukin-6 (IL-6), IL-8, surfactant protein D and C-reactive protein levels were measured by immunoassays. Low-dose CT scans evaluated emphysema.

Results

Sputum neutrophil % increased with GOLD stage. There was a weak association between % sputum neutrophils and FEV₁ % predicted (univariate r² = 0.025 and 0.094 at baseline and year 1 respectively, p < 0.05 after multivariate regression). Similar weak but significant associations were observed between neutrophil % and health status measured using the St Georges Respiratory Questionairre. There were no associations between neutrophils and exacerbation rates or emphysema. Associations between sputum neutrophils and systemic biomarkers were non-significant or similarly weak. The mean change over 1 year in neutrophil % was an increase of 3.5%.

Conclusions

Sputum neutrophil measurements in COPD are associated weakly with FEV₁ % predicted and health status. Sputum neutrophil measurements were dissociated from exacerbation rates, emphysema and systemic inflammation.