Evolutionary pressure | EPFL Graph Search

Any cause that reduces or increases reproductive success in a portion of a population potentially exerts evolutionary pressure, selective pressure or selection pressure, driving natural selection. It is a quantitative description of the amount of change occurring in processes investigated by evolutionary biology, but the formal concept is often extended to other areas of research. In population genetics, selective pressure is usually expressed as a selection coefficient. Amino acids selective pressure It has been shown that putting an amino acid bio-synthesizing gene like HIS4 gene under amino acid selective pressure in yeast causes enhancement of expression of adjacent genes which is due to the transcriptional co-regulation of two adjacent genes in Eukaryota. Antibiotic resistance Drug resistance in bacteria is an example of an outcome of natural selection. When a drug is used on a species of bacteria, those that cannot resist die and do not produce offspring

Design and Evaluation of New Quinazolin-4(3H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa

Eduard Vico Oton

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA P-pqsA promoter controlled by PqsR with an IC50 of 1 mu M. Using isothermal titration calorimetry, a K-d of 10 nM for the P-qsR ligand binding domain (PqsR(LBD)) was determined for 61. Furthermore, the crystal structure of 61 with PqsR(LBD) was attained with a resolution of 2.65 angstrom. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.

AMER CHEMICAL SOC2021

Multilevel regulation of the glass locus during Drosophila eye development

Bart Deplancke, Michael Vincent Frochaux, Abhishek Kumar Mishra

Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.

PUBLIC LIBRARY SCIENCE2019

Accelerating the development time of recombinant mammalian cell lines producing high titers of protein therapeutics

Sébastien Chenuet

The forthcoming arrival on the market of numerous protein therapeutics that require high clinical doses will foster the need for high-producing mammalian cell lines. Furthermore, pressures to reduce the development time for new therapeutics has meant more emphasis on efforts to accelerate the process that yields such cell lines. The objective of this thesis work was to develop methods to decrease the time needed to generate stable and high producing mammalian cell lines. This could be achieved by overcoming some of the major limitations that hamper the process of cell lines development. The host systems chosen for this study were Chinese hamster ovary (CHO DG44) and baby hamster kidney (BHK-21). The recombinant proteins used as reporters were the green fluorescent protein (GFP) and a human anti-Rhesus D IgG. One limitation hampering the development of cell lines producing high recombinant protein titers is the extensive variability of transgene expression within a transfected population as a result of the gene delivery process. This decreases the reproducibility of the generation of high producing cell lines. A direct comparison of two gene chemical delivery methods, polyethylenimine (PEI)- and calcium phosphate (CaPO4)-mediated transfection, with a mechanical method (microinjection) provided some insights into ways to overcome this limitation. Indeed, a correlation was observed between the amount of plasmid DNA delivered into cells and the specific productivity of the recovered recombinant cell lines. Hence, by increasing the amount of DNA delivered per cell, IgG-producing clones with specific productivities up to 22 pg/cell/day were recovered with a high efficiency. A second limitation is the time needed to assure the stability and clonality of cell lines by current analytical methods. This was overcome by using GFP as a reporter for the level of production of a co-expressed therapeutic protein. The co-transfection of plasmids individually carrying the GFP and IgG heavy (Hc)- and light chain (Lc) genes, resulted in integration of all three plasmids at the same site in the cell's genome. As a consequence, the stability of GFP expression correlated with that of IgG expression. This proved to be advantageous finding and eliminating unstable cell lines early in the process of selection. As a consequence, it was possible to derive in serum-free medium clones with a specific productivity almost 6 times higher than that of clones derived from transfection performed with the IgG Hc and Lc vectors only. A third limitation to the rapid recovery of stable cell lines is the time devoted to the cultivation of transfected cells in selective medium, normally one month. Here, the time of selection was shortened to one week by increasing the stringency of the selective pressure. The results from studies of plasmid integration kinetics by fluorescence in situ hybridization (FISH) showed that the increased stringency of selection decreased the time needed to enrich a transfected population with recombinant cells. Furthermore, stable clones were shown to be present in this population after one week of selection and could be uncovered by identifying patterns of GFP expression predictive of production stability. By limiting the screening to clones exhibiting these stable patterns of GFP expression, the number of clones needed in order to isolate stable cell lines producing high IgG titers could be dramatically reduced. In conclusion, these results combined together provided a new procedure to isolate within a week of the start of selection, stable and high-producing clones. 30-40% of the clones isolated using this procedure were stable for at least three months and their average specific productivity was 29 pg/cell/day.