A key role for Mg(2+) in TRPM7's control of ROS levels during cell stress

The TRPM7 (transient receptor potential melastatin 7) channel has been shown to play a pivotal role in cell survival during brain ischaemia as well as in the survival of other cell types challenged with apoptotic stimuli. Ca(2+) is thought to be central to the channel's ability to regulate ROS (reactive oxygen species) production. However, channel-mediated entry of Mg(2+) and Zn(2+) have also been implicated in cell death. In the present study, we show that depletion of TRPM7 by RNA interference in fibroblasts increases cell resistance to apoptotic stimuli by decreasing ROS levels in an Mg(2+)-dependent manner. Depletion of TRPM7 lowered cellular Mg(2+), decreased the concentration of ROS and lessened p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) activation as well as decreased caspase 3 activation and PARP [poly(ADP-ribose) polymerase] cleavage in response to apoptotic stimuli. Re-expression of TRPM7 or of a kinase-inactive mutant of TRPM7 in TRPM7-knockdown cells increased cellular Mg(2+) and ROS levels, as did expression of the Mg(2+) transporter SLC41A2 (solute carrier family 41 member 2). In addition, expression of SLC41A2 increased the sensitivity of TRPM7-knockdown cells to apoptotic stimuli and boosted ROS generation in response to cell stress. Taken together, these data uncover an essential role for Mg(2+) in TRPM7's control of cell survival and in the regulation of cellular ROS levels.     

Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.     

Enemy at the Gates: Interaction-Specific Stomatal Responses to Pathogenic Challenge

Stomata regulate gas exchange and their closure in response to pathogens may, in some cases, contribute to resistance. However, in the cereal mildew and rust systems, stomatal closure follows establishment of compatible infections. In incompatible systems, expression of major (R) gene controlled hypersensitive responses (HR), causes drastic, permanent stomatal dysfunction: stomata become locked open following powdery mildew attack and locked shut following rust attack. Thus, stomatal locking can be a hitherto unsuspected negative consequence of R gene resistance that carries a physiological cost affecting plant performance.Key Words: stomata, rust, mildew, hypersensitive response, stomatal lock-up     

Development and Evaluation of ‘Briefing Notes’ as a Novel Knowledge Translation Tool to Aid the Implementation of Sex/Gender Analysis in Systematic Reviews: A Pilot Study

Background

There is increasing recognition of sex/gender differences in health and the importance of identifying differential effects of interventions for men and women. Yet, to whom the research evidence does or does not apply, with regard to sex/gender, is often insufficiently answered. This is also true for systematic reviews which synthesize results of primary studies. A lack of analysis and reporting of evidence on sex/gender raises concerns about the applicability of systematic reviews. To bridge this gap, this pilot study aimed to translate knowledge about sex/gender analysis (SGA) into a user-friendly ‘briefing note’ format and evaluate its potential in aiding the implementation of SGA in systematic reviews.

Methods

Our Sex/Gender Methods Group used an interactive process to translate knowledge about sex/gender into briefing notes, a concise communication tool used by policy and decision makers. The briefing notes were developed in collaboration with three Cochrane Collaboration review groups (HIV/AIDS, Hypertension, and Musculoskeletal) who were also the target knowledge users of the briefing notes. Briefing note development was informed by existing systematic review checklists, literature on sex/gender, in-person and virtual meetings, and consultation with topic experts. Finally, we held a workshop for potential users to evaluate the notes.

Results

Each briefing note provides tailored guidance on considering sex/gender to reviewers who are planning or conducting systematic reviews and includes the rationale for considering sex/gender, with examples specific to each review group’s focus. Review authors found that the briefing notes provided welcome guidance on implementing SGA that was clear and concise, but also identified conceptual and implementation challenges.

Conclusions

Sex/gender briefing notes are a promising knowledge translation tool. By encouraging sex/gender analysis and equity considerations in systematic reviews, the briefing notes can assist systematic reviewers in ensuring the applicability of research evidence, with the goal of improved health outcomes for diverse populations.     

Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4⁺ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.     

Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.     

Effect of training in minimalist footwear on oxygen consumption during walking and running

The present study sought to examine the effect of 5 weeks of training with minimalist footwear on oxygen consumption during walking and running. Thirteen college-aged students (male n = 7, female n = 6, age: 21.7±1.4 years, height: 168.9±8.8 cm, weight: 70.4±15.8 kg, VO₂max: 46.6±6.6 ml·kg⁻¹·min⁻¹) participated in the present investigation. The participants did not have experience with minimalist footwear. Participants underwent metabolic testing during walking (5.6 km·hr⁻¹), light running (7.2 km·hr⁻¹), and moderate running (9.6 km·hr⁻¹). The participants completed this assessment barefoot, in running shoes, and in minimalist footwear in a randomized order. The participants underwent 5 weeks of training with the minimalist footwear. Afterwards, participants repeated the metabolic testing. Data was analyzed via repeated measures ANOVA. The analysis revealed a significant (F_4,32= 7.576, ηp2=0.408, p ≤ 0.001) interaction effect (time × treatment × speed). During the initial assessment, the minimalist footwear condition resulted in greater oxygen consumption at 9.6 km·hr⁻¹ (p ≤ 0.05) compared to the barefoot condition, while the running shoe condition resulted in greater oxygen consumption than both the barefoot and minimalist condition at 7.2 and 9.6 km·hr⁻¹. At post-testing the minimalist footwear was not different at any speed compared to the barefoot condition (p> 0.12). This study suggests that initially minimalist footwear results in greater oxygen consumption than running barefoot, however; with utilization the oxygen consumption becomes similar.     

First multi-locus sequence typing scheme for Arcobacter spp.

Background  

Arcobacter spp. are a common contaminant of food and water, and some species, primarily A. butzleri and A. cryaerophilus, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first Arcobacter multilocus sequence typing (MLST) method for A. butzleri, A. cryaerophilus, A. skirrowii, A. cibarius and A. thereius.