Biology subjects

Woods, J.

Publications and source records attributed to Woods, J..

11 recordsLinked to original sources

bioRxiv2026

Pathway-wide base editing charts chemical-genetic interactions in MAPK signaling

CRISPR base editor (BE) scanning enables sequence-level interrogation of proteins at scale in their native cellular and genomic contexts. This approach opens new opportunities to dissect cellular pathways, where signaling depends on coordinated interactions among multiple pathway proteins. We apply BE scanning to the RAS-RAF-MEK-ERK (MAPK) cascade, a central oncogenic pathway and major therapeutic target. Compounds targeting the MAPK pathway have mechanisms of action and resistance that remain incompletely characterized. By base editing 22 MAPK genes under either hyperactivation or inhibition at various nodes, we present a chemical-genetic map of the signaling pathway. Pathway-wide analysis shows that drug resistance mutations frequently occur in proteins other than the direct drug target itself, and we exploit these chemical-genetic interactions to recapitulate key pathway connections. Our BE scanning also identified underappreciated functional hotspots, including an allosteric site in the KRAS N-terminus. We also identify catalytically impaired CRAF mutants that confer bidirectional resistance and sensitization to structurally similar MEK inhibitors, distinguishing functional differences between structurally related compounds. These findings establish BE scanning as a scalable approach for network-level chemical-genetic interaction mapping, revealing new mechanistic insights into one of the most studied oncogenic pathways.

cancer biology↗

bioRxiv2025

Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging

Fluorescent proteins and small molecule dyes have complementary strengths for biological imaging: the former are genetically manipulatable enabling tagging of specific proteins and detection of protein interactions, while the latter have greater photostability and brightness but are difficult to target. To combine these strengths, we used de novo protein design to generate binders to three bright, stable, cell-permeable dyes spanning the visible spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). For each dye, we obtain nanomolar binders with weak or no binding to the other two dyes; the accuracy of the design approach is confirmed by a crystal structure of one binder which is very close to the design model. Fusion of the JF567, JF596 and JF494 binders to three different targets followed by staining with the three dyes simultaneously enables multiplex imaging. We further expand functionality by incorporating an active site carrying out nucleophilic aromatic substitution to form a covalent linkage with the dye, and developing split versions which reconstitute fluorescence at subcellular locations where both halves are present, enabling both protein-protein interaction detection and chemically induced dimerization with fluorescence reporting. Our designs combine the advantages of fluorescent proteins and small molecule dyes and should be broadly useful for cellular imaging.

biochemistry↗

bioRxiv2025

Plakophilin-2 Coordinates Energy Metabolism and Contractility in Cardiomyocytes, Revealing Its Roles beyond Desmosomes

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Mutations in the desmosome gene Plakophilin-2, PKP2, lead to reduction in PKP2 protein and collapse of desmosomes that is known to compromise contractility and electrical stability of cardiomyocytes. Our previous studies demonstrated the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2 and revealed profound changes in mRNA signatures of metabolic enzymes that were reversed by the gene replacement approach. In this study, we used PKP2-deficient mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to identify changes in steady-state metabolite levels associated with impaired lipid homeostasis, glycolysis, and glucose oxidation. These metabolic phenotypes align with human ARVC metabolic data and reflect an intrinsic impairment of cellular energy metabolism. Here we showed for the first time that these intracellular metabolic defects were associated specifically with poor contractility of cardiomyocytes. AAV9:PKP2 restored contractility, improved electrophysiological properties and Ca2+ transients. In contrast, we observed that treating PKP2-deficient cardiomyocytes pharmacologically with small molecule metabolic enhancers improved contractility but not electrophysiological properties and Ca2+ transients, suggesting differential sensitivity of structure-mediated functions in response to metabolic perturbance. Our study modeled and revealed a direct intracellular connection between compromised PKP2 function and metabolic impairment. We proposed that an increased risk of decoupling energy-responsive contractility from less energy-responsive electrical activities can be a new arrhythmogenic mechanism, potentially responsible for exercise-triggered cardiac adversity in ARVC disease development and progression.

cell biology↗

bioRxiv2024

Dynamic B0 field shimming for improving pseudo-continuous arterial spin labeling at 7 Tesla

PurposeB0 inhomogeneity within the brain-feeding arteries is a major issue for pseudo-continuous arterial spin labeling (PCASL) at 7T because it reduces labeling efficiency and leads to a loss of perfusion signal. This study aimed to develop a vessel-specific dynamic B0 field shimming method for 7T PCASL to enhance labeling efficiency by correcting off-resonance in the arteries within the labeling region. MethodsWe implemented a PCASL sequence with dynamic B shimming at 7T that compensates for B0 field offsets at the brain-feeding arteries by updating linear shimming terms and adding a phase increment to the PCASL RF pulses. Rapidly acquired vessel-specific B field maps were used to calculate dynamic shimming parameters. We evaluated both 2D and 3D variants of our method, comparing their performance against established global frequency offset and optimal-encoding-scheme (OES)-based corrections. Cerebral blood flow (CBF) maps were quantified before and after corrections. CBF values from different methods in the whole brain, white matter, and grey matter regions were compared. ResultsAll off-resonance correction methods significantly enhanced perfusion signals across the brain. The proposed vessel-specific dynamic B shimming method improved labeling efficiency while maintaining optimal static shimming in the imaging region. Perfusion-weighted images demonstrated the superiority of 3D dynamic B shimming method compared to global or 2D-based correction approaches. CBF analysis revealed that 3D dynamic B shimming significantly increased CBF values relative to the other methods. ConclusionOur proposed dynamic B0 shimming method offers a significant advancement in PCASL robustness and effectiveness, enabling full utilization of 7T ASLs high sensitivity and spatial resolution.

biophysics↗

bioRxiv2024

Efficient 3D cone trajectory design for improved combined angiographic and perfusion imaging using arterial spin labeling

PurposeTo improve the spatial resolution and repeatability of a non-contrast MRI technique for simultaneous time resolved 3D angiography and perfusion imaging by developing an efficient 3D cone trajectory design. MethodsA novel parameterized 3D cone trajectory design incorporating the 3D Golden Angle was integrated into 4D combined angiography and perfusion using radial imaging and arterial spin labeling (CAPRIA) to achieve higher spatial resolution and sampling efficiency for both dynamic angiography and perfusion imaging with flexible spatiotemporal resolution. Numerical simulations and physical phantom scanning were used to optimize the cone design. Eight healthy volunteers were scanned to compare the original radial trajectory in 4D CAPRIA with our newly designed cone trajectory. A locally low rank reconstruction method was used to leverage the complementary k-space sampling across time. ResultsThe improved sampling in the periphery of k-space obtained with the optimized 3D cone trajectory resulted in improved spatial resolution compared with the radial trajectory in phantom scans. Improved vessel sharpness and perfusion visualization were also achieved in vivo. Less dephasing was observed in the angiograms due to the short echo time of our cone trajectory and the improved k-space sampling efficiency also resulted in higher repeatability compared to the original radial approach. ConclusionThe proposed 3D cone trajectory combined with 3D Golden Angle ordering resulted in improved spatial resolution and image quality for both angiography and perfusion imaging and could potentially benefit other applications that require an efficient sampling scheme with flexible spatial and temporal resolution.

neuroscience↗

bioRxiv2023

Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers

Allogeneic human pluripotent stem cell (hPSC)-derived cells and tissues for therapeutic transplantation must necessarily overcome immunological rejection by the recipient. To define these barriers and to create cells capable of evading rejection for preclinical testing in immunocompetent mouse models, we genetically ablated {beta}2m, Tap1, Ciita, Cd74, Mica, and Micb to limit expression of HLA-I, HLA-II, and natural killer cell activating ligands in hPSCs. Though these and even unedited hPSCs readily formed teratomas in cord blood-humanized immunodeficient mice, grafts were rapidly rejected by immunocompetent wild-type mice. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit natural killer cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Expression of additional inhibitory factors such as CD24, CD47, and/or PD-L1 had no discernible impact on teratoma growth or persistence. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of natural killer cells also led to persistent teratomas. Thus, T cell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to refine tissue- and cell type-specific immune barriers, and to conduct preclinical testing in immunocompetent mouse models.

immunology↗

bioRxiv2023

Base Editor Scanning Reveals Activating Mutations of DNMT3A

DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/536656v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1de1d3eorg.highwire.dtl.DTLVardef@151f12dorg.highwire.dtl.DTLVardef@b0e2dborg.highwire.dtl.DTLVardef@fdce96_HPS_FORMAT_FIGEXP M_FIG C_FIG Using base editor mutagenesis and a DNA methylation reporter optimized to find activating mutations, we identify novel hyperactivating mutations in DNMT3A that suggest new mechanisms of allosteric control.

biochemistry↗

bioRxiv2023

Functional genomics identifies extension of complex N-glycans as a mechanism to evade lysis by natural killer cells

Somatic mutations can lead to the transformation of healthy cells into malignant cells and allow their evasion from immune surveillance. To uncover genes that play a role in the detection and lysis of tumor cells by natural killer (NK) cells, a B lymphoblastoid cell line was subjected to a genome-wide CRISPR screen. Among the top hits that facilitated NK evasion was SPPL3, which encodes an intramembrane protease that cleaves transmembrane glycosyltransferases in the Golgi apparatus. SPPL3-deficient cells accumulated glycosyltransferases, such as acetylglucosaminyltransferase 5 (MGAT5), and displayed increased N-glycosylation. Binding of NK receptors NKG2D and CD2 to their corresponding ligands MICB and CD58, and binding of rituximab to CD20, was disrupted by SPPL3-deletion. Inhibition of N-glycan maturation restored receptor binding and sensitivity to NK cells. To elucidate the mechanism of this resistant phenotype, a secondary CRISPR screen was performed in SPPL3-deficient cells. This screen identified glycosyltransferases that catalyze the formation of highly branched N-glycans and N-acetyl-lactosamine (LacNAc) extensions as key regulators that prevent killing. A significant enrichment of poly-LacNAc-containing tetra-antennary species was confirmed by glycoproteomic analysis. These findings provide mechanistic insight into how SPPL3 deletions have been linked to cancer.

immunology↗

bioRxiv2023

Human Dermal Fibroblast Subpopulations and Epithelial Mesenchymal Transition Signals in Hidradenitis Suppurativa Tunnels are Normalized by Spleen Tyrosine Kinase Antagonism in Vivo

Hidradenitis Suppurativa is a chronic inflammatory disease of which the pathogenesis is incompletely understood. Dermal fibroblasts have been previously identified as a major source of inflammatory cytokines, however information pertaining to the characteristics of subpopulations of fibroblasts in HS remains unexplored. Using in silico-deconvolution of whole-tissue RNAseq, Nanostring gene expression panels and confirmatory immunohistochemistry we identified fibroblast subpopulations in HS tissue and their relationship to disease severity and lesion morphology. Gene signatures of SFRP2+ fibroblast subsets were increased in lesional tissue, with gene signatures of SFRP1+ fibroblast subsets decreased. SFRP2+ and CXCL12+ fibroblast numbers, measured by IHC, were increased in HS tissue, with greater numbers associated with epithelialized tunnels and Hurley Stage 3 disease. Pro-inflammatory CXCL12+ fibroblasts were also increased, with reductions in SFRP1+ fibroblasts compared to healthy controls. Evidence of Epithelial Mesenchymal Transition was seen via altered gene expression of SNAI2 and altered protein expression of ZEB1, TWIST1, Snail/Slug, E-Cadherin and N-Cadherin in HS lesional tissue. The greatest dysregulation of EMT associated proteins was seen in biopsies containing epithelialized tunnels. The use of the oral Spleen tyrosine Kinase inhibitor Fostamatinib significantly reduced expression of genes associated with chronic inflammation, fibroblast proliferation and migration suggesting a potential role for targeting fibroblast activity in HS.

immunology↗

bioRxiv2023

Highly sensitive in vivo detection of dynamic changes in enkephalins following acute stress

Enkephalins are opioid peptides that modulate analgesia, reward, and stress. In vivo detection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani, 2018, eLife). Here we show improved detection resolution and stabilization of enkephalin detection, which allowed us to investigate enkephalin release during acute stress. We present an analytical method for real-time, simultaneous detection of Met- and Leu-Enkephalin (Met-Enk & Leu-Enk) in the mouse Nucleus Accumbens shell (NAcSh) after acute stress. We confirm that acute stress activates enkephalinergic neurons in the NAcSh using fiber photometry and that this leads to the release of Met- and Leu-Enk. We also demonstrate the dynamics of Met- and Leu-Enk release as well as how they correlate to one another in the ventral NAc shell, which was previously difficult due to the use of approaches that relied on mRNA transcript levels rather than post-translational products. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enkephalin through methionine oxidation, and provides novel insight into the relationship between Met- and Leu-Enkephalin following stress.

neuroscience↗

bioRxiv2018

Trafficking of Cholesterol from Lipid Droplets to Mitochondria in Bovine Luteal Cells: Acute Control of Progesterone Synthesis

The corpus luteum (CL) is a transient endocrine gland that synthesizes and secretes the steroid hormone, progesterone. Progesterone biosynthesis is a complex process, converting cholesterol via a series of enzymatic reactions, into progesterone. Lipid droplets in luteal cells store cholesterol in the form of cholesterol esters, which can be utilized for steroidogenesis. In small luteal cells, luteinizing hormone (LH) increases intracellular cAMP concentrations leading to activation of protein kinase A (PKA), which phosphorylates downstream proteins, such as hormone sensitive lipase (HSL). Phosphorylation of HSL at Ser563 leads to increased HSL activation and association with lipid droplets, events which theoretically release cholesterol, which can be used for progesterone synthesis. Bovine CL were obtained from a local abattoir, dispersed, and luteal cells were enriched for SLC via centrifugal elutriation. Our results reveal that LH, forskolin, and cAMP induce HSL phosphorylation at Ser563and Ser660. Moreover, inhibiting HSL activity attenuates LH-induced P4 synthesis. Confocal analysis revealed that LH stimulates translocation of HSL to lipid droplets and mitochondria. Furthermore, LH increased trafficking of cholesterol from the lipid droplets to the mitochondria which was dependent on both PKA and HSL activation. These results demonstrate cholesterol stored in lipid droplets are utilized for LH-induced progesterone biosynthesis. Likewise, PKA-induced activation of HSL is required for release and trafficking of cholesterol from the lipid droplets to the mitochondria. Taken together, these findings support a role for a PKA/HSL signaling pathway in response to LH and demonstrate the dynamic relationship between PKA, HSL, and the lipid droplets in the synthesis of progesterone.\n\nHighlightsO_LILH and PKA induce HSL phosphorylation at Ser563and Ser660\nC_LIO_LIHSL is required for optimal LH-induced P4 synthesis\nC_LIO_LILH stimulates translocation of HSL to lipid droplets and mitochondria\nC_LIO_LILH stimulated trafficking of cholesterol from lipid droplets to mitochondria\nC_LI

cell biology↗