Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants
A calculator mutagenesis technique has been used to characterize the structural effects associated with more than 46,000 amino acid variants simple and multiple green fluorescent proteins Aequorea Victoria (GFP), whose functional effects (fluoresce levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall variation of the compatibility of the sequence structure relative to the Aboriginal GFP, as well as a vector of environmental disturbance scores (EP) characterizing the impact of all GFP residue positions. A significant relationship of the GFP structure function (p <0.0001) has been elucidated by comparing the compatibility scores of the sequence structure with functional data.
Then, the vectors calculated for the GFP mutants were used to form predictive fluorescence models by implementing the random forest classification and tree regression learning algorithms. The performance of the classification reach 0.93 for the sensitivity, 0.91 for precision and 0.90 for a balanced accuracy and regression models led to the pearson correlation as high as r = 0.83 between the fluorescence Experimental mutant and planned by the GFP.
An RF model formed on a subset of more than 1000 experimental mutants of the experimental residue GFP with measured fluorescence has been used to predict the remaining 3300 mutants remaining simple unjustified residues, with results supplementing biochemical and biophysical properties of Known GFP. In addition, models formed at the subset of the experimental mutants of the GFP host multiple residue substitutes have predicted the fluorescence of the mutants of the FPS of simple residues. The models developed for this study were accurate and effective, and their forecasts outperformed those of several state-of-the-art methods.
A new protocol for detecting the green fluorescent protein in a non-fixed tissue
The green fluorescent protein (GFP) is a powerful journalist protein that allows the labeling of specific proteins or entire cells. However, as GFP is a small soluble protein, it easily passes the membranes if the integrity of the cells is disturbed and the GFP signal is lost or diffuse if the sample is not fixed beforehand. Although pre-fixation is often feasible for histological analyzes, numerous molecular biology procedures and new imaging techniques, such as imaging mass spectrometry, require undetermined specimens. In order to use GFP labeling in tissues prepared for such applications, we have tested various protocols to minimize GFP signal loss.
We show here that, in the cryocut sections of frozen cerebral fabrics from two mouse lines of the GFP journalist, a leak of the GFP signal is prevented by omitting the drying commonly performed cryostections and by direct post-fixation with 4% of Paraformaldehyde warmed at 30-37 ° C. Although GFP coloring does not reach the same quality as that obtained with pre-fixed tissues, the location of the GFP in the cells that express it is preserved with this method. This protocol can therefore be used to identify GFP positive cells on sections from non-fixed cryosted fabric.Lactobacillus plantarum is an important probiotic with a variety of physiological functions.
Studies focused on the effects of L. plantarum on the physiology of the host and the microbiota, but studies on the fate of strains after their entry into the intestine are lacking. In this study, L. Plantam ST-III was genetically designed to express a green fluorescent protein (GFP). The mice were administered by ST-III-GFP and fluorescence imaging was used to study the distribution, location and quantity of strains within 8 hours after entering the intestine.
T-lymphopoiesis is seriously compromised in fluorescent fluorescent transgenic mice of ubiquitin-green
The marking of the cells of experimental organisms with genetic markers is commonly used in biomedical research. The insertion of artificial gene constructions can be very beneficial for research as long as this marking is functionally neutral and does not change the fabric function. The transgenic UBC-GFP mouse has recently been found to be debatable in this regard, due to a fault of latent stem cells compromising its lymphopiesis and significantly influencing the results of competitive transplant tests.
In this study, we show that the fault of the stem cells present in the UBC-GFP mice negatively affects the significantly more than b-lymphopoyal t-lymphopoiesis. The production of granulocytes is not negatively affected. The lack of T-lymphopoiesis causes a low number of white blood cells in the UBC-GFP mouse peripheral blood which, associated with the lower lymphoid / myeloid ratio in nucleated blood cells, are the only untreated UBCGFP mouse abnormal phenotype for been found so far. Faulty lymphopoiesis in UBC-GFP mice can be repaired by the transplant of congenic wild-type bone marrow cells, which then compensate for insufficient production of T cells. Interestingly, the wild-type branch of hematopoiesis in mice UBC-GFP / wild-type chemaeric was more active in lymphopoiesis, and in particular towards the production of T cells, compared to lymphopoiesis in normal wild-type donors.
However, the mRNA for GRP is widely disseminated among superficial backborn excitatory interneurmen. We show here that, although the GRP arname is present in several transcriptomically defined populations, EGFP is limited to a discreet subset of cells in the mouse GRP :: EGFP, some of which express the receiver of neuromedrique 2 and belong. Probably at a cluster defined as glutant8. We show that these cells receive a large portion of their MRGA3 / MRGD-expressing synaptic entry of non-nociceptive / pruritory relating and low threshold mechanics.