Biology subjects

Khoury, M.

Publications and source records attributed to Khoury, M..

4 recordsLinked to original sources

bioRxiv2025

Exploring the deletion landscape of S. aureus Cas9 with SABER

Profiling tolerated amino acid deletions in proteins can elucidate structure-function relationships, reconstruct intermediate stages in protein evolution, and be used to engineer minimized versions of proteins with size-sensitive biotechnology applications. Despite advances in deletion library construction techniques over the past several decades, there are presently few methods available that are simultaneously efficient, precise, and easy to implement. Here we present SABER, a novel approach which utilizes SpRYCas9, a near-PAMless engineered SpCas9 variant, as a molecular biological tool for building deletion libraries with unprecedented speed and ease. We applied this technique to the small and structurally divergent Cas9 from Staphylococcus aureus (SaCas9) and mapped the set of deletions tolerated for DNA binding activity. We proceeded to use this information to design a set of minimal SaCas9-based effectors capable of CRISPRi transcriptional repression in bacterial cells. Our findings provide new insights into the function of certain structural elements in SaCas9, and we anticipate that our dSaCas9 deletion map may prove useful in further efforts to develop minimal Cas9-based effectors and gene editors.

molecular biology↗

bioRxiv2022

Partitioned Local Depth analysis of time course transcriptomic data reveals elaborate community structure

Transcriptome studies that provide temporal information are valuable for identifying groups of similarly-behaving transcripts, giving insight into overarching gene regulatory networks. Nevertheless, inferring transcriptional networks from time series data is challenging, in part because it is difficult to holistically consider both local relationships and global structure of these complex and overlapping transcriptional responses. To address this need, we employed the Partitioned Local Depth (PaLD) method to examine four time series transcriptomic datasets generated using the model plant Arabidopsis thaliana. Here, we provide a self-contained description of the method and demonstrate how it can be used to make predictions about gene regulatory networks based on time series data. The analysis provides a global network representation of the data from which graph partitioning methods and neighborhood analysis can reveal smaller, more well-defined groups of like-responding transcripts. These groups of transcripts that change in response to hormone treatment (e.g., auxin or ethylene) or high salinity were demonstrated to be enriched in common biological function and/or binding of transcription factors that were not identified with prior analyses of this data using other clustering and inference methodologies. These results reveal the ability of PaLD to generate predictions about gene regulatory networks using time series transcriptomic data, which can be of value to the systems biology community.

systems biology↗

bioRxiv2019

Active Notch Signaling is Required for Arm Regeneration in a Brittle Star

Cell signaling pathways play vital roles in coordinating cellular events in development. For example, the Notch signaling pathway is highly conserved across all multicellular animals and is known to coordinate a multitude of diverse cellular events, including proliferation, differentiation, fate specification, and cell death. Specific functions of the pathway are, however, highly context-dependent and are not well characterized in post-traumatic regeneration. Here, we use a small-molecule inhibitor of the pathway (DAPT) to demonstrate that Notch signaling is required for proper arm regeneration in the brittle star Ophioderma brevispina, a highly regenerative member of the phylum Echinodermata. We also employ a transcriptome-wide gene expression analysis to characterize the downstream genes controlled by the Notch pathway in the brittle star regeneration. We demonstrate that arm regeneration involves an extensive crosstalk between the Notch pathway and other cell signaling pathways. In the regrowing arm, Notch regulates the composition of the extracellular matrix, cell migration, proliferation, and apoptosis, as well as components of the innate immune response. We also show for the first time that Notch signaling regulates the activity of several transposable elements. Our data also suggests that one of the possible mechanisms through which Notch sustains its activity in the regenerating tissues is via suppression of Neuralized1.

developmental biology↗

bioRxiv2017

The Complex Simplicity of the Brittle Star Nervous System

Brittle stars (Ophiuroidea, Echinodermata) have been increasingly used in studies of animal behavior, locomotion, regeneration, physiology, and bioluminescence. The success of these studies directly depends on good working knowledge of ophiuroid nervous system. Here, we describe the arm nervous system at different levels of organization: microanatomy of the radial nerve cord and peripheral nerves, neural ultrastructure, and localization of different cell types using specific antibody markers. We standardize the nomenclature of nerves and ganglia and provide an anatomically accurate digital 3D model of the arm nervous system as a reference for future studies. Our results helped identify several general features characteristic to the adult echinoderm nervous system, including the extensive anatomical interconnections between the ectoneural and hyponeural components and neuroepithelial organization of the central nervous system with its supporting scaffold formed by radial glial cells. In addition, we provide further support to the notion that the echinoderm radial glia is a complex and diverse cell population. We also tested the suitability of a range of specific cell-type markers for studies of the brittle star nervous system and established that the radial glial cells are reliably labeled by the ERG1 antibodies, whereas the best neuronal markers are acetylated tubulin, ELAV and synaptotagmin B. The transcription factor Brn1/2/4, a marker of neuronal progenitors, is expressed not only in neurons, but also in a subpopulation of radial glia. For the first time, we describe putative ophiuroid proprioceptors associated with the hyponeural part of the central nervous system.

neuroscience↗