Biology subjects

Knapp, J.

Publications and source records attributed to Knapp, J..

9 recordsLinked to original sources

bioRxiv2024

Gas1-Mediated Suppression of Hepatoblastoma Tumorigenesis

Background and AimsHepatoblastoma (HB), the most common pediatric liver cancer, often dysregulates the Wnt/{beta}-catenin, Hippo and NFE2L2/NRF2 pathways. Pairwise combinations of oncogenically active forms of the terminal transcription factor effectors of these pathways, namely {beta}-catenin (B), YAP (Y) and NRF2 (N) generate HBs in mice, with the triple combination (B+Y+N) being particularly potent. Each tumor group alters the expression of thousands of B-,Y- and N-driven unique and common target genes. Identifying those most responsible for transformation is thus an important question as it might reveal new mechanistic insights and therapeutic opportunities. Approach and ResultsTranscriptional profiling of >60 murine HBs driven by the above oncogenic combinations and different B mutants and in genetic backgrounds that impair tumor growth rates but not initiation has revealed a common set of 22 "BYN genes" that are similarly deregulated in all cases. Many are associated with multiple "Cancer Hallmarks" and their expression levels often correlate with survival in human HBs, hepatocellular carcinomas and other cancers. Among the most down-regulated of these is Gas1, which encodes a Glycosylphosphatidylinositol (GPI)-linked outer membrane protein. We show here that restoring Gas1 expression impairs B+Y+N-driven HB tumor growth in vivo and in HB-derived immortalized cell lines in vitro in a manner than requires membrane anchoring of the protein via its GPI moiety. ConclusionsOur findings implicate Gas1 as a proximal mediator of HB pathogenesis and validate the BYN gene set as one deserving of closer additional scrutiny in future studies.

cancer biology↗

bioRxiv2024

Efficient Derivation of Immortalized, Isogenic Cell Lines from Genetically Defined Murine Hepatoblastomas

Background & AimsMolecularly, hepatoblastoma (HB), the most common childhood liver cancer, is the simplest of all human neoplasms, with the vast majority deregulating the Wnt/{beta}-catenin, Hippo and/or NFE2/NRF2 signaling pathways. Murine HBs can be generated by over-expressing any pairwise or triple combination of mutant forms of these pathways terminal effectors, namely {beta}-catenin (B), YAP (Y) and NFE2L2/NRF (N). Each molecular subtypes displays distinct features resembling those of human HBs. However, research has been hampered by a paucity of established cell lines of any species. MethodsWe show here that immortalized cell lines can be routinely established from murine HBs that over-express B+Y and B+Y+N. This is facilitated by the concurrent in vivo, Crispr-mediated inactivation of the Cdkn2a tumor suppressor locus. ResultsEight BY and 3 BYN cell lines have been generated and characterized and are available to the HB research community. Ten of these lines can be regrown as subcutaneous and metastatic lung tumors in the immuno-competent mice from which they originated while retaining their original histologic features. During maintenance as spheroids in vitro, or during in vivo propagation, tumor cells express endothelial cell markers, particularly in regions that are hypoxic and/or in proximity to incipient blood vessels. ConclusionsThe ability to generate isogenic HB cell lines with defined oncogenic drivers should facilitate studies that are best performed in vitro. The approach may also be useful for deriving HB cell lines associated with less common molecular drivers and from human tumors. SynopsisThe derivation of multiple immortalized murine hepatoblastoma cell lines driven by defined oncogenes is described. These lines are isogenic, retain their tumorigenicity in immuno-competent mice, readily form spheroids and express endothelial markers in response to hypoxia. They will allow studies that have heretofore been difficult or impossible to perform in vivo.

cancer biology↗

bioRxiv2024

MYC Binding Near Transcriptional End Sites Regulates Basal Gene Expression, Read-Through Transcription and Intragenic Contacts

The MYC oncoprotein regulates numerous genes involved in cellular processes such as cell cycle and mitochondrial and ribosomal structure and function. This requires heterodimerization with its partner, MAX, and binding to specific promoter and enhancer elements. Here, we show that MYC and MAX also bind near transcriptional end sites (TESs) of over one-sixth of all annotated genes. These interactions are dose-dependent, evolutionarily conserved, stabilize the normally short-lived MYC protein and regulate expression both in concert with and independent of MYCs binding elsewhere. MYCs TES binding occurs in association with other transcription factors, alters the chromatin landscape, increases nuclease susceptibility and can alter transcriptional read-through, particularly in response to certain stresses. MYC-bound TESs can directly contact promoters and may fine-tune gene expression in response to both physiologic and pathologic stimuli. Collectively, these findings support a previously unrecognized role for MYC in regulating transcription and its read-through via direct intragenic contacts between TESs and promoters.

molecular biology↗

bioRxiv2024

Comparison of conservation strategies for California Channel Island Oak (Quercus tomentella) using climate suitability predicted from genomic data

Management strategies, such as assisted gene flow, could increase resilience to climate change in tree populations. Knowledge of evolutionary history and genetic structure of species is needed to assess the risks and benefits of different strategies. Quercus tomentella, or Island Oak, is a rare oak restricted to six Channel Islands in California, USA, and Baja California, Mexico. Previous work has shown that Island Oaks on each island are genetically differentiated, but it is unclear whether assisted gene flow could enable populations to tolerate future climates. We performed whole-genome sequencing on Island Oak individuals and Q. chrysolepis, a closely related species that hybridizes with Island Oak (127 total), to characterize genetic structure and introgression across its range and assess the relationship between genomic variation and climate. We introduce and assess three potential management strategies with different trade-offs between conserving historic genetic structure and enabling populations to survive changing climates: the status quo approach; ecosystem preservation approach, which conserves the trees and their associated biodiversity; and species preservation approach, which conserves the species. We compare the impact of these approaches on predicted maladaptation to climate using Gradient Forest. We also introduce a climate suitability index to identify optimal pairs of seed sources and planting sites for approaches involving assisted gene flow. We found one island (Santa Rosa) that could benefit from the ecosystem preservation approach and also serve as a species preservation site. Overall, we find that both the ecosystem and species preservation approaches will do better than the status quo approach. If preserving Island Oak ecosystems is the goal, assisted migration into multiple sites could produce adapted populations. If the goal is to preserve a species, the Santa Rosa population would be suitable. This case study both illustrates viable conservation strategies for Island Oak and introduces a framework for tree conservation.

evolutionary biology↗

bioRxiv2023

DDHD2 is necessary for activity-driven fatty acid fueling of nerve terminal function

HSP54, a hereditary spastic paraplegia associated with cognitive impairment, is caused by mutations in the neuron-specific triglyceride (TG) lipase DDHD2. Loss of DDHD2 function results in lipid accumulation in human brains1 and lipid droplets (LDs) in mouse neurons2. In metabolically demanding tissues, TG lipases generate a fatty acid (FA) flux from LDs to fuel mitochondrial ATP production, but neurons are considered unable to use fat as an energy source. Thus, the basis for cognitive impairment driven by DDHD2 loss remains enigmatic. To resolve this paradox, we took advantage of presynaptic sensitivity to metabolic perturbations to determine if FAs derived from LDs could power local {beta}-oxidation to support synaptic functions and whether DDHD2 activity would be required in the process. We demonstrate that nerve terminals are enriched with DDHD2 and blocking its activity leads to presynaptic accumulation of LDs. Moreover, we show that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion and drive local mitochondrial ATP production allowing nerve terminals to sustain function in the complete absence of glucose. Our data demonstrate that neurons and their nerve terminals can make use of LDs during electrical activity to provide metabolic support when glucose is in short supply.

neuroscience↗

bioRxiv2023

Isolation and Lipidomic Profiling of Neuronal Lipid Droplets: Unveiling the Lipid Landscape for insights into Neurodegenerative Disorders

Recent advances have expanded the role of lipid droplets (LDs) beyond passive lipid storage, implicating their involvement in various metabolic processes across mammalian tissues. Neuronal LDs, whose existence has long been debated, have now been identified in several neural structures, raising questions about their contribution to neurodegenerative disorders. Elucidating the specific chemical makeup of these organelles within neurons is critical for understanding their role in neural pathologies. However, the inherent submicron size and low abundance in neurons have posed challenges in their isolation; and physical and biochemical characterization. Here, we present our protocol which overcomes this obstacle: we blocked neuron-specific triglyceride lipase to enlarge LDs, rendering them amenable to floatation in a sucrose gradient. We further employed an improved methodology of lipidomic analysis on the LDs purified from cultured primary neurons, offering insights into their unique lipid composition. This protocol can be implemented by researchers with basic expertise in neuronal culture, imaging, ultracentrifuge handling, and LC-MS analysis. The complete workflow requires 3-4 weeks, from neuronal culture to lipidomics analysis. Future integration of this method with high-throughput techniques holds promises for uncovering disease-specific alterations in lipid metabolism, providing new avenues for potential therapeutic interventions. Key pointsO_LIThis protocol describes a robust workflow for pharmacological induction, purification, and characterization of mature lipid droplets from primary cortical neurons without any detectable cytosolic membrane carryover. C_LIO_LILC-MS analysis of neuronal lipid droplets can reveal lipids previously uncharacterized by other methods, providing a platform to integrate high-throughput proteomics and other techniques for probing neurodegenerative disease linked metabolic alterations in neurons. C_LI Key referenceKumar, M. et al. Triglycerides are an important fuel reserve for synapse function in the brain. Nat Metab 7, 1392-1403 (2025). https://doi.org:10.1038/s42255-025-01321-x Editorial summaryThis protocol describes a robust workflow for pharmacological induction, purification, and characterization of mature lipid droplets from primary cortical neurons without any detectable cytosolic membrane carryover. Proposed teaserIsolation and lipidomic profiling of neuronal lipid droplets

biochemistry↗

bioRxiv2023

Regulation of Lung Immune Tone by the Gut-Lung Axis via Dietary Fiber, Gut Microbiota, and Short-Chain Fatty Acids

Lung immune tone, i.e. the immune state of the lung, can vary between individuals and over a single individuals lifetime, and its basis and regulation in the context of inflammatory responses to injury is poorly understood. The gut microbiome, through the gut-lung axis, can influence lung injury outcomes but how the diet and microbiota affect lung immune tone is also unclear. We hypothesized that lung immune tone would be influenced by the presence of fiber-fermenting short-chain fatty acid (SCFA)-producing gut bacteria. To test this hypothesis, we conducted a fiber diet intervention study followed by lung injury in mice and profiled gut microbiota using 16S sequencing, metabolomics, and lung immune tone. We also studied germ-free mice to evaluate lung immune tone in the absence of microbiota and performed in vitro mechanistic studies on immune tone and metabolic programming of alveolar macrophages exposed to the SCFA propionate (C3). Mice on high-fiber diet were protected from sterile lung injury compared to mice on a fiber-free diet. This protection strongly correlated with lower lung immune tone, elevated propionate levels and enrichment of specific fecal microbiota taxa; conversely, lower levels of SCFAs and an increase in other fatty acid metabolites and bacterial taxa correlated with increased lung immune tone and increased lung injury in the fiber-free group. In vitro, C3 reduced lung alveolar macrophage immune tone (through suppression of IL-1{beta} and IL-18) and metabolically reprogrammed them (switching from glycolysis to oxidative phosphorylation after LPS challenge). Overall, our findings reveal that the gut-lung axis, through dietary fiber intake and enrichment of SCFA-producing gut bacteria, can regulate innate lung immune tone via IL-1{beta} and IL-18 pathways. These results provide a rationale for the therapeutic development of dietary interventions to preserve or enhance specific aspects of host lung immunity.

immunology↗

bioRxiv2023

Prelimbic neuron calcium activity predicts perceived hedonic value across drinking solutions and ethanol dependent states in mice

AO_SCPLOWBSTRACTC_SCPLOWThe medial prefrontal cortex integrates information about salience and valence of stimuli, including rewarding solutions like alcohol and sucrose, and regulates aspects of alcohol seeking and consumption. However, our understanding of how cortical outputs encode alcohol consumption is limited. Using fiber photometry to measure calcium activity in putative prelimbic (PrL) glutamatergic projection neurons, we show similar but distinct patterns of activity during the peri-consummatory phase in response to consumption of water (non-deprived conditions), ethanol (20% v/v), or sucrose (1% w/v). PrL population activity appears to track hedonic value, as GCaMP6f signals ramped immediately preceding bouts for water, ethanol, and sucrose, and the signal scaled with presumed hedonic value, i.e., water<ethanol<sucrose. Further, using machine learning, population activity of PrL neurons prior to consumption was sufficient to predict both consumption of and distinguish between these different solutions. To assess valence encoding, we adulterated the ethanol solution with quinine, a bitter tastant. In non-dependent mice, calcium activity surrounding drinking bouts was reduced, paralleling the decreased consumption of quinine-adulterated ethanol. This effect was not present in ethanol dependent mice, suggesting altered hedonic value of the adulterated solution either due to reduced sensitivity to the aversiveness of quinine or increased sensitivity to the reinforcing value of ethanol. The global population of PrL glutamatergic neurons also display sustained GCaMP6f "up-states" that last tens to hundreds of seconds, which were longer and larger when consummatory bouts occurred. Overall, our results demonstrate a functional signature in PrL neurons that differs across solutions and is disrupted by ethanol dependence.

neuroscience↗

bioRxiv2017

Expanding the Atlas of Functional Missense Variation for Human Genes

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon-mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features, and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

molecular biology↗