Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines

Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out

Finding secluded places of special interest in graphs.

Finding a vertex subset in a graph that satisfies a certain property is one of the most-studied topics in algorithmic graph theory. The focus herein is often on minimizing or maximizing the size of the solution, that is, the size of the desired vertex set. In several applications, however, we also want to limit the “exposure” of the solution to the rest of the graph. This is the case, for example, when the solution represents persons that ought to deal with sensitive information or a segregated community. In this work, we thus explore the (parameterized) complexity of finding such secluded vertex subsets for a wide variety of properties that they shall fulfill. More precisely, we study the constraint that the (open or closed) neighborhood of the solution shall be bounded by a parameter and the influence of this constraint on the complexity of minimizing separators, feedback vertex sets, F-free vertex deletion sets, dominating sets, and the maximization of independent sets

Finding secluded places of special interest in graphs

Finding a vertex subset in a graph that satisfies a certain property is one of the most-studied topics in algorithmic graph theory. The focus herein is often on minimizing or maximizing the size of the solution, that is, the size of the desired vertex set. In several applications, however, we also want to limit the “exposure” of the solution to the rest of the graph. This is the case, for example, when the solution represents persons that ought to deal with sensitive information or a segregated community. In this work, we thus explore the (parameterized) complexity of finding such secluded vertex subsets for a wide variety of properties that they shall fulfill. More precisely, we study the constraint that the (open or closed) neighborhood of the solution shall be bounded by a parameter and the influence of this constraint on the complexity of minimizing separators, feedback vertex sets, F-free vertex deletion sets, dominating sets, and the maximization of independent sets

Effects of Age and Loading Velocity on the Delamination Strength of the Human Aorta

Delamination strength is the mechanical property which plays a key role in the pathological process referred to as Arterial Dissection. This dissection, known especially for its occurrence in the thoracic aorta, is manifested by a separation of the layers of an artery wall, and may end with total rupture and internal haemorrhaging. Although its incidence is relatively rare, from 3 to 6 cases per 100 000 per year, it is a life-threating disease with a significant lethality [1-3]. The exact conditions under which the dissection is initiated, and as a crack propagates through the arterial wall, remain an open topic in computational as well as experimental mechanics. The aim of our study is to contribute to the deepening of our knowledge of Arterial Dissection, by collecting experimental data which is suitable for the purpose of showing how the delamination strength measured in the peeling experiments depends on age and anatomical location. In addition to the effects of age and location, our study also focuses on the effect of loading rate. The experimental branch of our research is complemented by a computational modelling of the delamination interface, in which we are looking for a numerical characterization of the material parameters describing discontinuity propagation. An XFEM model of the peeling experiment is built in Abaqus, which in our approach plays the role of the regression analysis, incorporating the cohesive zone (CZ) in order to model the delaminating arterial layers. The main objective is to obtain a detailed description of a set of constitutive parameters, which would be age- and location-specific. Our present data suggest that delamination strength strongly depends on age, and furthermore, the anatomical site also seems to be a significant factor. On the other hand, the loading velocity does not cause significant changes in results

Lipolytic and Hypolipidemic Properties of Newly Synthesized Aryloxypropanolamine Derivatives

In this study, the lipolytic effect of two newly synthesized potential β3-adrenergic agonists A482 and B496 in active acid forms was tested using isolated sliced epididymal adipose tissue of Wistar rats, and compared with Isoprenaline and BRL37344. Furthermore, effects of an eight-week oral administration of the newly synthesized substances on serum cholesterol, triglycerides, glucose, adiponectin, resistin and weight gain were studied in C57Bl/6J mice that were fed high energy diet. The newly synthesized substance A482 (4-(2-{[2-hydroxy-3-(4-methyl-carbamoylphenoxy) propyl]amino}ethyl)phenoxy-acetic acid hydrochloride) was able to produce almost full lipolysis at a 1 × 10-7 M concentration, and its effect on the rat epididymal adipose tissue was similar to the specific β3-adrenergic agonist BRL37344. Ethyl ester of this substance significantly lowered plasma total cholesterol (p p p p p 3-adrenergic agonists. Our results suggest that the newly synthesised substance A482 may represent a potent β3-adrenergic agonist

Porous composites based on siloxane resin for hard tissue replacements

A composite material based on polysiloxane and first on glass fabrics was prepared. The composite samples were prepared with controlled open porosity, the sizes of which were in the range: i) 0,2-0,4 mm, ii) 0,4-0,6 mm, and iii) >0,6 mm. For further improvement of osseointegration, a powder hydroxyapatite, particle size of 0,005 mm in the amount of 10 wgt %. The effects of surface treatment and hydroxyapatite matrix additives on osseointegration were studied by methods in vitro and in vivo

Some surface improvement and osseointegration

As a further perspective material to be used in orthopedics, glass fibers (R-glass, 21055 satin-woven fabric, VETROTEX, Saint Gobain, France) and polysiloxane matrix (M130, Lučební závody Kolín, Czech Republic) based composite materials have been investigated

The parameterized complexity of finding secluded solutions to some classical optimization problems on graphs

This work studies the parameterized complexity of finding secluded solutions to classical combinatorial optimization problems on graphs such as finding minimum - separators, feedback vertex sets, dominating sets, maximum independent sets, and vertex Herein, one searches not only to minimize or maximize the size of the solution, but also to minimize the size of its neighborhood. This restriction has applications in secure routing and community detection