Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes

1. Download high-res image (209KB)
2. Download full-size image

     

Propagation of fluorescent viruses in growing plaques

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.     

Leaf functional traits and monodominance in Southern Amazonia tropical forests

Tropical monodominant forests are rare communities with low tree species diversity. Species monodominance is not the product of a single mechanism, but the result of a set of not yet fully understood integrated ecological factors acting together. We compared populations of Brosimum rubescens in monodominant and mixed forests in Southern Amazonia to test whether leaf functional traits are ecological factors related to monodominance. Individuals of B. rubescens in the mixed forest invest in conservative strategies, while those in the monodominant forest invest in acquisitive strategies. Leaf functional traits, such as petiole length and adaxial cuticle thickness, could be associated with the monodominance of B. rubescens. Our study highlights for the first time the power of integrating leaf functional traits as a component of the set of ecological conditions to explain species monodominance. B. rubescens showed different functional strategies to establish and maintain its population in different forests, which makes it a strong competitor for resources, such as water and light, through variation in its leaf functional traits. We also suggest that such high plasticity can be an important condition for the persistence of the species over time.

     

Optical Limnology of a Hypertrophic Gravel-Pit Lake

The underwater light field has been studied in a hypertrophic, gravel-pit lake close to Madrid (Spain) during a one year cycle. Both the inherent and the apparent properties of the underwater light field have been weekly surveyed. As theoretically expected, there is a link between inherent and apparent properties in this lake. Evidence is given suggesting that a seasonal trend in the underwater light field seems to occur. The main factor attenuating light in the vertical column is phytoplankton chlorophyll “a” but humic substances also appear to play an importtant role in light attenuation.     

Changes in tropomyosin during primary culture of embryonic myocardiocytes.

We chose the Hamburger and Hamilton's stage 29 (HH 29) to investigate the expression of tropomyosin in chick myocardiocytes during 14 days on culture. Throughout 14 days of cell culture, changes in cell morphology were accompanied by a redistribution of tropomyosin in different cell compartments. We used FACScan, SDS-PAGE and densitometric analysis to quantify total cell tropomyosin and concentrations of this protein in different cell fractions. Tropomyosin was found mostly in the cytoskeletal fraction than in the cytoplasmic. When we compared the densitometric values from SDS-PAGE of cells in different stages of development we found that in HH 19, tropomyosin was more abundant in the cytoplasmic than in the cytoskeletal fraction. By HH 29, the two fractions had become inverted, and in HH 39, tropomyosin was clearly more abundant in the cytoskeletal than in the cytoplasmic fraction. In the IFI analysis, tropomyosin was found to label the Stress fiber-like structures (SFL) in different patterns depending on the area of the cell which expressed this protein.     

Field and in vitro assay of three methods for freezing ram semen

Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25 ml straws (final cellular concentration: 100 x 10(6) spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35 degrees C. The sample was cooled to 5 degrees C (-0.30 degrees C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5 degrees C for 2h. It was then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Method M2: The sample was diluted with a specific solution at 35 degrees C (final concentration of glycerol 3%), cooled to 5 degrees C (-0.20 degrees C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 degrees C (-0.25 degrees C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1h at 5 degrees C and then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values (P<0.05) for kinetic parameters: average path velocity (VAP) (81.3 and 85.2 microm/s), straight-line velocity (VSL) (72.8 and 77.3 microm/s) and linearity (LIN) (66.6 and 68.8%). Method M2 showed the lowest kinetic parameters of motility (VAP 74.4, VSL 67.3 and LIN 62.5) and the highest percentage of cells with damaged plasma membrane (53.8%). Method M1 gave the worst results in viability and acrosome status assessed using fluorescence probes (31.3%-dead cells with damaged acrosomes-versus 25.4% in M2 and 23.3% in M3). A field trial carried out on fertility showed a significantly higher percentage of pregnant or lambing ewes (P<0.05) with Method M3 (67.3% versus 51.1% for M1 and 58.8% for M2). We concluded that the use of a simple dilution medium (test-fructose-glycerol-egg yolk) with the addition of glycerol (to 2% at 35 degrees C and to 4% at 5 degrees C) in two steps together with a programmable biofreezer was a productive method for freezing ram semen.