Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Circular pools in the seagrass beds of the Banc d’Arguin,Mauritania, and their possible origin

The tidal flats of the Banc d’Arguin, Mauritania, are covered by vast beds of Zostera noltii. At low tide these seagrass beds appear to be interspersed with partly vegetated, circular pools of 5–25 m diameter. Between February and May 2001 we described these pools and studied their possible origin. Several hypotheses regarding the origin have been developed. The first group of hypotheses assumes that the pools result from erosion activity. Since human disturbance of seagrass beds at the Banc d’Arguin is virtually non-existent, causes should be found in natural bed disturbances and/or tide or wave action. Therefore, small gaps, simulating holes dug by the crab Callinectes marginatus, were made to see if they would further erode by tidal currents or waves. The experiments showed no erosion. Neither we found support for other hypotheses assuming erosion to be the cause of circular pools. The alternative group of hypotheses stated that sedimentation on the flats would be responsible. We conclude that accretion of creek remnants is the most likely process behind the development of the pools; this conclusion is based on both mapping of the pattern of pools, the sediment profile in and around the pools and the distribution of seagrass biomass. Also the disturbance experiments showed bed accretion rather than bed erosion and support this hypothesis.     

Single introductions of soil biota and plants generate long‐term legacies in soil and plant community assembly

Recent demonstrations of the role of plant–soil biota interactions have challenged the conventional view that vegetation changes are mainly driven by changing abiotic conditions. However, while this concept has been validated under natural conditions, our understanding of the long‐term consequences of plant–soil interactions for above‐belowground community assembly is restricted to mathematical and conceptual model projections. Here, we demonstrate experimentally that one‐time additions of soil biota and plant seeds alter soil‐borne nematode and plant community composition in semi‐natural grassland for 20 years. Over time, aboveground and belowground community composition became increasingly correlated, suggesting an increasing connectedness of soil biota and plants. We conclude that the initial composition of not only plant communities, but also soil communities has a long‐lasting impact on the trajectory of community assembly.     

The Investigation of Protein Diffusion via H-Cell Microfluidics

In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.     

Genomic landscape of rat strain and substrain variation

Background

Since the completion of the rat reference genome in 2003, whole-genome sequencing data from more than 40 rat strains have become available. These data represent the broad range of strains that are used in rat research including commonly used substrains. Currently, this wealth of information cannot be used to its full extent, because the variety of different variant calling algorithms employed by different groups impairs comparison between strains. In addition, all rat whole genome sequencing studies to date used an outdated reference genome for analysis (RGSC3.4 released in 2004).

Results

Here we present a comprehensive, multi-sample and uniformly called set of genetic variants in 40 rat strains, including 19 substrains. We reanalyzed all primary data using a recent version of the rat reference assembly (RGSC5.0 released in 2012) and identified over 12 million genomic variants (SNVs, indels and structural variants) among the 40 strains. 28,318 SNVs are specific to individual substrains, which may be explained by introgression from other unsequenced strains and ongoing evolution by genetic drift. Substrain SNVs may have a larger predicted functional impact compared to older shared SNVs.

Conclusions

In summary we present a comprehensive catalog of uniformly analyzed genetic variants among 40 widely used rat inbred strains based on the RGSC5.0 assembly. This represents a valuable resource, which will facilitate rat functional genomic research. In line with previous observations, our genome-wide analyses do not show evidence for contribution of multiple ancestral founder rat subspecies to the currently used rat inbred strains, as is the case for mouse. In addition, we find that the degree of substrain variation is highly variable between strains, which is of importance for the correct interpretation of experimental data from different labs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1594-1) contains supplementary material, which is available to authorized users.     

Measuring the Outcome of Biomedical Research: A Systematic Literature Review

Background

There is an increasing need to evaluate the production and impact of medical research produced by institutions. Many indicators exist, yet we do not have enough information about their relevance. The objective of this systematic review was (1) to identify all the indicators that could be used to measure the output and outcome of medical research carried out in institutions and (2) enlist their methodology, use, positive and negative points.

Methodology

We have searched 3 databases (Pubmed, Scopus, Web of Science) using the following keywords: [Research outcome* OR research output* OR bibliometric* OR scientometric* OR scientific production] AND [indicator* OR index* OR evaluation OR metrics]. We included articles presenting, discussing or evaluating indicators measuring the scientific production of an institution. The search was conducted by two independent authors using a standardised data extraction form. For each indicator we extracted its definition, calculation, its rationale and its positive and negative points. In order to reduce bias, data extraction and analysis was performed by two independent authors.

Findings

We included 76 articles. A total of 57 indicators were identified. We have classified those indicators into 6 categories: 9 indicators of research activity, 24 indicators of scientific production and impact, 5 indicators of collaboration, 7 indicators of industrial production, 4 indicators of dissemination, 8 indicators of health service impact. The most widely discussed and described is the h-index with 31 articles discussing it.

Discussion

The majority of indicators found are bibliometric indicators of scientific production and impact. Several indicators have been developed to improve the h-index. This indicator has also inspired the creation of two indicators to measure industrial production and collaboration. Several articles propose indicators measuring research impact without detailing a methodology for calculating them. Many bibliometric indicators identified have been created but have not been used or further discussed.     

Muscle Histidine-Containing Dipeptides Are Elevated by Glucose Intolerance in Both Rodents and Men

Objective

Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.

Design and Methods

Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured.

Results

Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects.

Conclusion

Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.