Fragment d'Okazaki | EPFL Graph Search

Les fragments d’Okazaki sont des segments d'acide nucléique qui sont produits lors de la réplication des chromosomes. Leur existence fut mise en évidence pour la première fois en 1968 par et Tsuneko Okazaki ainsi que leurs collègues en étudiant la réplication de la bactérie Escherichia coli. Lors de la réplication, le brin « retardé » est synthétisé de manière discontinue, par petits fragments qui sont ensuite suturés les uns aux autres. Leur longueur est d’environ paires de bases chez Escherichia coli et de 100 à 200 pour les eucaryotes. On a baptisé ces segments « fragments d’Okazaki », du nom de leurs découvreurs. Ce phénomène s'explique par le fait que l'ADN polymérase ne peut polymériser l'ADN que dans le sens , la réplication étant faite au fur et à mesure du déroulement de l'ADN, un des deux brins qui sont antiparallèles ne peut être répliqué en continu. Pour démarrer la synthèse de l’ADN, l'ADN polymérase nécessite une amorce d'ARN qui est synthétisée par l'ARN Primase,

Micromechanically inspired investigation of cemented granular materials: part I-from X-ray micro tomography to measurable model variables

Edward Ando, Gioacchino Viggiani

Cemented granular materials are abundant in nature and are often artificially produced. Their macroscopic behaviour is driven by small-scale material processes, which are generally classified as: grain breakage, cement damage and fragment rearrangement. This paper presents an experimental analysis of the latter two processes as observed through in-situ X-ray tomography and quantified by a suite of novel image processing approaches. This allows for example all particles and the bonds between them to be identified and their evolution to be individually quantified on a statistically representative volume, in 3D, throughout a loading test. We reveal the high spatial correlation between cement damage and strain rate and their effect on the isotropy of the bonds. Being the second of a two-part contribution, the overarching aim of this paper is to propose a general framework for the micro-inspired study of cemented granular materials. This is developed here, in Part I, in terms of multi-scale experimental quantification at sample and grain-level, while in Part II in terms of a two-way interaction with micro-inspired constitutive modelling.

SPRINGER HEIDELBERG2022

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: What you see is not always what you get

Abdelrahman Mohammed Adel Elsaeed Mansour AlOkda, Bryan Frey, Hilal Lashuel, Nathan Alain Denis Riguet

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (alpha-syn) oligomerization in intact cells. Since then, several alpha-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased alpha-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with alpha-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of alpha-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of alpha-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to alpha-syn (alpha-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to alpha-syn (Vn-alpha-syn); (2) Vn-alpha-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of alpha-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-alpha-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of alpha-syn-Vc in Vn-alpha-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate alpha-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the alpha-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

2020

Generation of a Large Peptide Phage Display Library by Self-Ligation of Whole-Plasmid PCR Product

Kaycie Marie Butler, Vanessa Carle, Cristina Diaz Perlas, Christian Heinis, Xudong Kong

The success of phage display, used for developing target-specific binders based on peptides and proteins, depends on the size and diversity of the library screened, but generating large libraries of phage-encoded polypeptides remains challenging. New peptide phage display libraries developed in recent years rarely contained more than 1 billion clones, which appears to have become the upper size limit for libraries generated with reasonable effort. Here, we established a strategy based on whole-plasmid PCR and self-ligation to clone a library with more than 2 × 1010 members. The enormous library size could be obtained through amplifying the entire vector DNA by PCR, which omitted the step of vector isolation from bacterial cells, and through appending DNA coding for the peptide library via a PCR primer, which enabled efficient DNA circularization by end-ligation to facilitate the difficult step of vector-insertion of DNA fragments. Panning the peptide repertoires against a target yielded high-affinity ligands and validated the quality of the library and thus the new library cloning strategy. This simple and efficient strategy places larger libraries within reach for nonspecialist researchers to hopefully expand the possible targets of phage display applications.

2020

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: What you see is not always what you get

Abdelrahman Mohammed Adel Elsaeed Mansour AlOkda, Bryan Frey, Hilal Lashuel, Nathan Alain Denis Riguet

2020