An image-based model of calcium waves in differentiated neuroblastoma cells

Calcium waves produced by bradykinin-induced inositol-1,4, 5-trisphosphate (InsP(3))-mediated release from endoplasmic reticulum (ER) have been imaged in N1E-115 neuroblastoma cells. A model of this process was built using the "virtual cell," a general computational system for integrating experimental image, biochemical, and electrophysiological data. The model geometry was based on a cell for which the calcium wave had been experimentally recorded. The distributions of the relevant cellular components [InsP(3) receptor (InsP(3)R)], sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pumps, bradykinin receptors, and ER] were based on 3D confocal immunofluorescence images. Wherever possible, known biochemical and electrophysiological data were used to constrain the model. The simulation closely matched the spatial and temporal characteristics of the experimental calcium wave. Predictions on different patterns of calcium signals after InsP(3) uncaging or for different cell geometries were confirmed experimentally, thus helping to validate the model. Models in which the spatial distributions of key components are altered suggest that initiation of the wave in the center of the neurite derives from an interplay of soma-biased ER distribution and InsP(3) generation biased toward the neurite. Simulations demonstrate that mobile buffers (like the indicator fura-2) significantly delay initiation and lower the amplitude of the wave. Analysis of the role played by calcium diffusion indicated that the speed of the wave is only slightly dependent on the ability of calcium to diffuse to and activate neighboring InsP(3) receptor sites.     

Cytoskeletal interactions regulate inducible L-selectin clustering

L-selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of L-selectin and serves to increase its adhesiveness, signaling, and colocalization with ₂-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin’s lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin’s effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton. membrane domains; adhesion; leukocyte; inflammation     

The Virtual Cell: a software environment for computational cell biology

The newly emerging field of computational cell biology requires software tools that address the needs of a broad community of scientists. Cell biological processes are controlled by an interacting set of biochemical and electrophysiological events that are distributed within complex cellular structures. Computational modeling is familiar to researchers in fields such as molecular structure, neurobiology and metabolic pathway engineering, and is rapidly emerging in the area of gene expression. Although some of these established modeling approaches can be adapted to address problems of interest to cell biologists, relatively few software development efforts have been directed at the field as a whole. The Virtual Cell is a computational environment designed for cell biologists as well as for mathematical biologists and bioengineers. It serves to aid the construction of cell biological models and the generation of simulations from them. The system enables the formulation of both compartmental and spatial models, the latter with either idealized or experimentally derived geometries of one, two or three dimensions.     

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg^-1 and a further eight samples contained fumonisin at a level below 1 mgkg^-1 but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg^-1.     

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O₂/5% CO₂, pH = 7.4, 37°C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F_2α (2 × 10^-6 M). Intraluminal perfusion of adenosine diphosphate (10^-5 M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10^-5 M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10^-5 M) can be inhibited by site-specific administration of atropine (10^-5 M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.     

Surgery for hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a genetically determined cardiac disease characterised by otherwise unexplained myocardial hypertrophy of the left ventricle, and may result in left ventricular outflow tract obstruction. It is the most common cause of sudden cardiac death in young adults due to arrhythmias. Septal myectomy is a surgical treatment for HCM with moderate to severe outflow tract obstruction, and is indicated for patients with severe symptoms refractory to medical therapy. The surgical approach involves obtaining access to the interventricular septum via transaortic, transapical or transmitral approaches, and excising a portion of the hypertrophied myocardium to relieve the outflow tract obstruction. Large, contemporary series from centres experienced in septal myectomy patients have demonstrated a low early mortality of <2 %, excellent long-term survival that matches the general population, and durable relief of symptoms.     

Partial protein domains: evolutionary insights and bioinformatics challenges

Protein domains are generally thought to correspond to units of evolution. New research raises questions about how such domains are defined with bioinformatics tools and sheds light on how evolution has enabled partial domains to be viable.With the rapid expansion in the number of determined protein sequences - over 92 million in UniProt in March 2015 - an ever-increasing number of biologists are using bioinformatics tools for annotation of these sequences. One widely used strategy is to identify occurrences of Pfam families within the sequence of interest [1]. A Pfam family is a multiple sequence alignment of the occurrences of a particular domain both in different species and in different regions of the same protein. The concept underpinning Pfam is that proteins typically comprise one or more domains (regions), each of which is an evolutionary unit that generally has a well-defined biological function. A significant sequence similarity between a query protein and a Pfam family provides the basis for annotations. Two recent articles [2,3] in Genome Biology evaluate the implications of having the query sequence only matching part of a Pfam family, which is an intriguing finding, given that a Pfam family is considered to be an evolutionary unit.     

Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis

Introduction

Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model.

Methods

DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression.

Results

Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1β, IL-6, and IL-22 in the CFA/CII group and IL-1β, tumor necrosis factor-α, transforming growth factor-β, IL-6 and IL-23 in the IFA/CII group.

Conclusions

Chronic P. gingivalis oral infection prior to arthritis induction increases the immune system activation favoring Th17 cell responses, and ultimately accelerating arthritis development. These results suggest that chronic oral infection may influence RA development mainly through activation of Th17-related pathways.     

The evolutionary emergence of stochastic phenotype switching in bacteria

Stochastic phenotype switching - or bet hedging - is a pervasive feature of living systems and common in bacteria that experience fluctuating (unpredictable) environmental conditions. Under such conditions, the capacity to generate variable offspring spreads the risk of being maladapted in the present environment, against offspring likely to have some chance of survival in the future. While a rich subject for theoretical studies, little is known about the selective causes responsible for the evolutionary emergence of stochastic phenotype switching. Here we review recent work - both theoretical and experimental - that sheds light on ecological factors that favour switching types over non-switching types. Of particular relevance is an experiment that provided evidence for an adaptive origin of stochastic phenotype switching by subjecting bacterial populations to a selective regime that mimicked essential features of the host immune response. Central to the emergence of switching types was frequent imposition of 'exclusion rules' and 'population bottlenecks' - two complementary faces of frequency dependent selection. While features of the immune response, exclusion rules and bottlenecks are likely to operate in many natural environments. Together these factors define a set of selective conditions relevant to the evolution of stochastic switching, including antigenic variation and bacterial persistence.     

Scats and den contents as indicators of the diet of stoats (Mustela erminea) in the Tasman Valley,South Canterbury,New Zealand

Stoats are significant predators of native fauna in New Zealand. They occur in many habitat types and consume a wide range of prey. The diet of stoats in the Tasman River, South Canterbury, was studied by analysis of scats and den contents. Analysis of 206 scats showed that stoats ate mainly lagomorphs, birds and invertebrates. Minor components included mice, lizards, fish and hedgehogs. Stoats ate more birds in spring than in autumn, and female stoats ate more invertebrates than did males. The contents of 219 dens collected in the same area at the same time provided further information. Birds and lagomorphs occurred at high frequency in dens, and other components were minor. Remains in dens were larger than in scats and allowed identification of many more prey items to species level. Den contents revealed a potentially substantial impact of stoats on threatened shorebirds locally; this impact was not detected by analysis of scats.