Biology subjects

Bohlooly-Y, M.

Publications and source records attributed to Bohlooly-Y, M..

7 recordsLinked to original sources

bioRxiv2025

Integrative Analysis of Left Ventricle and Epicardial Adipose Tissue Identifies SDHA and OGDH as Candidate Targets for Ischemic Heart Disease

Ischemic heart disease (IHD) involves coordinated molecular changes across heart, yet their interplay remains poorly understood. Here, we investigated transcriptomic alterations in two heart tissue subtypes, left ventricle (LV) and epicardial adipose tissue (EAT), from age- and BMI-matched healthy and IHD individuals, including both diabetic and non-diabetic patients. We performed transcriptomic profiling and systems-level network analysis to identify disease-associated gene expression changes. Our analysis revealed: (1) stronger transcriptional responses in EAT compared to LV, particularly in diabetic individuals, and (2) widespread dysregulation of inflammatory and metabolic pathways, including oxidative phosphorylation, cytokine signaling, and fatty acid degradation, across both tissue subtypes. Co-expression network analysis uncovered shared gene modules, with SDHA (Succinate dehydrogenase complex, subunit A) and OGDH (Oxoglutarate Dehydrogenase) emerging as central, downregulated genes linked to mitochondrial function and inflammation, important processes in IHD pathophysiology. These findings were validated in independent human and mouse datasets. Overall, our integrative analysis identifies conserved molecular signatures across cardiac tissue subtypes, suggesting potential for therapeutic exploration in IHD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/676232v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@7e17eeorg.highwire.dtl.DTLVardef@552321org.highwire.dtl.DTLVardef@1755f30org.highwire.dtl.DTLVardef@bc5872_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase-and a nuclease-based PE to perform programmed short DNA substitution and insertion in various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA. Prime editing, CRISPR/Cas9, zebrafish, PE2, PEn

molecular biology↗

bioRxiv2021

Human rare variant and zebrafish CRISPR/Cas9-mediated mutant analyses reveal novel functions for API5, HSPB7, and LMO2 in heart failure

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

molecular biology↗

bioRxiv2021

Sphingosine 1-Phosphate Mediates Adiponectin Receptor Signaling Essential For Lipid Homeostasis And Embryogenesis

Cells and organisms require proper membrane composition to function and develop. Phospholipids are the major component of membranes and are primarily acquired through the diet. Given great variability in diet composition, cells must be able to deploy mechanisms that correct deviations from optimal membrane composition and properties. Here, using lipidomics and unbiased proteomics, we found that the embryonic lethality in mice lacking the fluidity regulators Adiponectin Receptors 1 and 2 (AdipoR1/2) is associated with aberrant high saturation of the membrane phospholipids. Using mouse embryonic fibroblasts (MEFs) derived from AdipoR1/2-KO embryos, human cell lines and the model organism C. elegans we found that, mechanistically, AdipoR1/2-derived sphingosine 1-phosphate (S1P) signals in parallel through S1PR3-SREBP1 and PPAR{gamma} to sustain the expression of the fatty acid desaturase SCD and maintain membrane properties. Thus, our work identifies an evolutionary conserved pathway by which cells and organism achieve membrane homeostasis and adapt to a variable environment.

cell biology↗

bioRxiv2020

AdipoR2 is Essential for Membrane Lipid Homeostasis in Response to Dietary Saturated Fats

Membrane lipid composition influences vital processes in all types of cells. The mechanisms by which cells maintain membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remain largely unknown. In an attempt to discover such mechanisms, we performed an unbiased forward genetic screen in Caenorhabditis elegans and conclude that the adiponectin receptor 2 (AdipoR2) pathway is essential to prevent saturated fat-mediated cellular toxicity. Transcriptomics, lipidomics and membrane property analyses in human HEK293 cells and primary human endothelial cells further support our conclusion that the essential function of AdipoR2 is to respond to membrane rigidification by promoting fatty acid desaturation. Our results demonstrate that AdipoR2-dependent regulation of membrane homeostasis is a fundamental mechanism conserved from nematodes to mammals that prevents saturated fat-mediated lipotoxicity. ONE SENTENCE SUMMARYThe AdipoR2 protein insures membrane homeostasis in response to dietary saturated fatty acids that promote membrane rigidification.

cell biology↗

bioRxiv2020

ObLiGaRe doxycycline Inducible (ODIn) Cas9 system driving pre-clinical drug discovery, from design to cancer treatment

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. We have generated a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene. Targeted ObLiGaRe resulted in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse can be utilized for robust and tunable genome editing allowing for flexibility, speed and uniformity at reduced cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

genomics↗

bioRxiv2018

In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1-5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8-12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

molecular biology↗