1,354,387 research outputs found
Assessing Neuronogenic Versus Astrogenic Bias of Neural Stem Cells Via In Vitro Clonal Assay
No full textWithin the developing cerebral cortex, neural stem cells (NSCs) give rise to neurons and glial cells, according to complex spatio-temporal trajectories. In this respect, a key issue is how NSCs are committed to different neural lineages in time and space. Clonal assays are a powerful tool to address this issue. Here we describe an easy clonal assay protocol employable to dissect NSCs lineage commitment and molecular mechanisms underlying it. NSCs of distinctive spatio-temporal origin, and/or having undergone different molecular manipulations, are plated at low density and allowed to differentiate for a few days. Then, systematic immunoprofiling of the resulting clones allows to quantify commitment of their NSC ancestors to neuronal and astroglial fates
Late Control of Cortical Histogenesis by Transcription Factors Patterning the Early Pallial Field
No full textA number of transcription factor genes implicated in primary regional patterning of the pallial primordium continue to be active or are specifically reactivated upon completion of such patterning. This late expression may be required for proper tuning of advanced cerebro- cortical histogenesis. It may account for key neurological aberrancies associated to mutations affecting such transcription factor genes.
We have previously shown that a transcription factor implicated in primary tangential patterning of the pallial primordium, Foxg1, shapes the temporal progression of the rates at which pallial stem cells give rise to astrogenic committed progenitors and the latter progress to astrocytes. Inspired by these findings, we hypothesized that these two rates might be also differentially regulated in space, in distinctive regions of the murine developing pallium, and that factors patterning pallium might be implicated in such regulation. This prediction turned out to be correct. We found that, compared to their age-matched rostro-lateral counterparts, early caudo-medial pallial stem cells are more biased to astrogenesis, and generate astroblasts less prone to proliferation and more keen to early differentiation. We got evidence that Emx2, a transcription factor gene mastering caudo-medial cortical specification, drives regionally patterned articulation of astrogliogenesis. Moreover, we demonstrated that three established pro-astrogenic factors, Sox9, Nfia and Couptf1, mediate Emx2 impact on neural stem cells commitment to astrogenesis.
On the other hand, the two mutant FOXG1 alleles FOXG1G224S and FOXG1W308X have been found in children affected by severe varieties of the FOXG1 Syndrome. Previous functional characterization of these alleles in murine wild type cells showed that they act as gain- and loss-of-function variants of their healthy counterpart, respectively. Before any possible therapeutic exploitation, results of this study require a stringent validation in human neural cells, harboring gene configurations matching the patients' ones. To this aim, we obtained human pluripotent stem cells heterozygous for these mutations and their co-isogenic healthy controls, and we set up a protocol suitable to reproducibly generate dorsal telencephalic tissue starting from healthy human pluripotent stem cells. We discovered that, applying such protocol to heterozygous FOXG1W308X mutants, the generation of pallial neuroepithelium is defective. To allow robust interrogation of more advanced derivatives of FOXG1W308X/+ neuroepithelium, we are now managing to mitigate this issue
In Silico Engineering of Enzyme Access Tunnels
No full textEnzyme engineering is a tailoring process that allows the modification of naturally occurring enzymes to provide them with improved catalytic efficiency, stability, or specificity. By introducing partial modifications to their sequence and to their structural features, enzyme engineering can transform natural enzymes into more efficient, specific and resistant biocatalysts and render them suitable for virtually countless industrial processes. Current enzyme engineering methods mostly target the active site of the enzyme, where the catalytic reaction takes place. Nonetheless, the tunnel that often connects the surface of an enzyme with its buried active site plays a key role in the activity of the enzyme as it acts as a gatekeeper and regulates the access of the substrate to the catalytic pocket. Hence, there is an increasing interest in targeting the sequence and the structure of substrate entrance tunnels in order to fine-tune enzymatic activity, regulate substrate specificity, or control reaction promiscuity. In this chapter, we describe the use of a rational in silico design and screening method to engineer the access tunnel of a fructosyl peptide oxidase with the aim to facilitate access to its catalytic site and to expand its substrate range. Our goal is to engineer this class of enzymes in order to utilize them for the direct detection of glycated proteins in diabetes monitoring devices. The design strategy involves remodeling of the backbone structure of the enzyme, a feature that is not possible with conventional enzyme engineering techniques such as single-point mutagenesis and that is highly unlikely to occur using a directed evolution approach. The proposed strategy, which results in a significant reduction in cost and time for the experimental production and characterization of candidate enzyme variants, represents a promising approach to the expedited identification of novel and improved enzymes. Rational enzyme design aims to provide in silico strategies for the fast, accurate, and inexpensive development of biocatalysts that can meet the needs of multiple industrial sectors, thus ultimately promoting the use of green chemistry and improving the efficiency of chemical processes
Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel
No full textFructosyl peptide oxidases (FPOXs) are enzymes currently used in enzymatic assays to measure the concentration of glycated hemoglobin and albumin in blood samples, which serve as biomarkers of diabetes. However, since FPOX are unable to work directly on glycated proteins, current enzymatic assays are based on a preliminary proteolytic digestion of the target proteins. Herein, to improve the speed and costs of the enzymatic assays for diabetes testing, we applied a rational design approach to engineer a novel enzyme with a wider access tunnel to the catalytic site, using a combination of Rosetta design and molecular dynamics simulations. Our final design, L3_35A, shows a significantly wider and shorter access tunnel, resulting from the deletion of five-amino acids lining the gate structures and from a total of 35 point mutations relative to the wild-type (WT) enzyme. Indeed, upon experimental testing, our engineered enzyme shows good structural stability and maintains significant activity relative to the WT
Linear correlation between fractal dimension of surface EMG signal from Rectus Femoris and height of vertical jump
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Experimental evidence of two distinct charge carriers in underdoped cuprate superconductors
No full textWe present the results on heavily underdoped Y(1-x)Ca(x)Ba(2)Cu(3)O(6+y), which provide the evidence that the doping mechanism (cation substitution or oxygen loading) directly determines whether the corresponding injected mobile holes contribute to superconductivity or only to high-temperature transport. We argue that this hole tagging is a signature of the complexities of single-hole doping in Mott insulators, and it calls for a subtler description of the correlated bands than the usual one. We also map in great detail the underdoped superconducting phase diagram T(c) vs hole doping, which shows that the total number of mobile holes is not the driving parameter for superconductivity
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