Biology subjects

Gutierrez, J.

Publications and source records attributed to Gutierrez, J..

21 records · Page 2Linked to original sources

bioRxiv2020

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for Autism Spectrum Disorder (ASD) and Developmental Delay (DD). To investigate how Cul3 mutations impact brain development, we generated haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of the brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified neuronal cytoskeleton and Rho signaling as primary targets of Cul3 mutation during early brain development.

neuroscience↗

bioRxiv2019

Characterization, modelling and mitigation of gene expression burden in mammalian cells

Despite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

synthetic biology↗

bioRxiv2019

Tracking of antibiotic resistance transfer and rapid plasmid evolution in a hospital setting by Nanopore sequencing

BackgroundInfection of patients with multidrug-resistant (MDR) bacteria often leave very limited or no treatment options. The transfer of antimicrobial resistance genes (ARG) carrying plasmids between bacterial species by horizontal gene transfer represents an important mode of expansion of ARGs. Here, we evaluated the application of Nanopore sequencing technology in a hospital setting for monitoring the transfer and rapid evolution of antibiotic resistance plasmids within and across multiple species.\n\nResultsIn 2009 we experienced an outbreak with an extensively multidrug resistant P. aeruginosa harboring the carbapenemase enzyme blaIMP-8, and in 2012 the first Citrobacter freundii and Citrobacter werkmanii harboring the same enzyme were detected. Using Nanopore and Illumina sequencing we conducted a comparative analysis of all blaIMP-8 bacteria isolated in our hospital over a 6-year period (n = 54). We developed the computational platforms pathoLogic and plasmIDent for Nanopore-based characterization of clinical isolates and monitoring of ARG transfer, comprising de-novo assembly of genomes and plasmids, polishing, QC, plasmid circularization, ARG annotation, comparative genome analysis of multiple isolates and visualization of results. Using plasmIDent we identified a 40 kb plasmid carrying blaIMP-8 in P. aeruginosa and C. freundii, verifying that plasmid transfer had occurred. Within C. freundii the plasmid underwent further evolution and plasmid fusion, resulting in a 164 kb mega-plasmid, which was transferred to C. werkmanii. Moreover, multiple rearrangements of the multidrug resistance gene cassette were detected in P. aeruginosa, including deletions and translocations of complete ARGs.\n\nConclusionPlasmid transfer, plasmid fusion and rearrangement of the multidrug resistance gene cassette mediated the rapid evolution of opportunistic pathogens in our hospital. We demonstrated the feasibility of tracking plasmid evolution dynamics and ARG transfer in clinical settings in a timely manner. The approach will allow for successful countermeasures to contain not only clonal, but also plasmid mediated outbreaks.

genomics↗