Biology subjects

Bukari, A.-R. A.

Publications and source records attributed to Bukari, A.-R. A..

3 recordsLinked to original sources

bioRxiv2022

Acquisition of cross-azole tolerance and aneuploidy in Candida albicans strains evolved to posaconazole

A number of in vitro studies have examined the acquisition of drug resistance to the triazole fluconazole, a first-line treatment for many Candida infections. Much less is known about posaconazole, a newer triazole. We conducted the first in vitro experimental evolution of replicates from eight diverse strains of C. albicans in a high level of the fungistatic drug posaconazole. Approximately half of the 132 evolved replicates survived 50 generations of evolution, biased towards some of the strain backgrounds. We found that although increases in drug resistance were rare, increases in drug tolerance (the slow growth of a subpopulation of cells in a level of drug above the resistance level) were common across strains. We also found that adaptation to posaconazole resulted in widespread cross-tolerance to other azole drugs. Widespread aneuploidy variation was also observed in evolved replicates from some strain backgrounds. Trisomy of chromosomes 3, 6, and R was identified in 11 of 12 whole-genome sequenced evolved SC5314 replicates. These findings document rampant evolved cross-tolerance among triazoles and highlight that increases in drug tolerance can evolve independently of drug resistance in a diversity of C. albicans strain backgrounds.

evolutionary biology↗

bioRxiv2021

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Billions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-{beta}-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to {beta}-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to "calm down" its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo. SIGNIFICANCE STATEMENTThe (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1-3, little is known about the underlying mechanisms. We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth. We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution. Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.

synthetic biology↗

bioRxiv2020

RNA-seq analyses: Benchmarking differential expression analyses tools reveals the effect of higher number of replicates on performance.

The introduction of several differential gene expression analysis tools has made it difficult for researchers to settle on a particular tool for RNA-seq analysis. This coupled with the appropriate determination of biological replicates to give an optimum representation of the study population and make biological sense. To address these challenges, we performed a survey of 8 tools used for differential expression in RNA-seq analysis. We simulated 39 different datasets (from 10 to 200 replicates, at an interval of 5) using compcodeR with a maximum of 100 replicates. Our goal was to determine the effect of varying the number of replicates on the performance (F1-score, recall and precision) of the tools. EBSeq and edgeR-glmRT recorded the highest (0.9385) and lowest (0.6505) average F1-score across all replicates, respectively. We also performed a pairwise comparison of all the tools to determine their concordance with each other in identifying differentially expressed genes. We found the greatest concordance to be between limma voom treat and limma voom ebayes. Finally, we recommend employing edgeR-glmRT for RNA-seq experiments involving 10-50 replicates and edgeR-glmQLF for studies with 55 to 200 replicates. Author summaryDownstream analysis of RNA-seq data in R often poses several challenges to researchers as it is a daunting task to choose a specific differential expression analysis tool over another. Researchers also find it challenging to determine the number (replicates) of samples to use in order to give comparable and accurate results. In this paper, we surveyed eight differential expression analysis tools using different number of replicates of simulated RNA-seq count data. We measured the performance of each tool and based on the recorded F1-scores, recall and precision, we made the following recommendations; consider edgeR-glmRT and edgeR-glmQLF for replicates of 10-50 and 55-200 respectively.

bioinformatics↗