Biology subjects

Levine, E.

Publications and source records attributed to Levine, E..

10 recordsLinked to original sources

bioRxiv2026

Computational Profiling and Pathogenicity Assessment of VHL Missense Mutations

Mutations in the VHL tumor suppressor gene promote tumorigenesis by stabilizing HIF-1. In this study, we investigated the functional impact of VHL missense mutations using AlphaMissense pathogenicity scores, protein stability simulations, and molecular dynamics. AlphaMissense identified high pathogenicity scores for residues 60-200, including the {beta}-domain and HIF-binding domain, suggesting these regions are critical for VHL's tumor suppressor function. We categorized the mutations into two groups: those that primarily destabilize protein folding and those that likely disrupt VHL-HIF-1 binding. FoldX stability simulations revealed high {Delta}{Delta}G values for both groups, with proline substitutions exerting significant structural stress. Structural modeling confirmed that these mutations disrupt secondary structures, particularly {beta}-sheets. Molecular dynamics simulations showed that the VHL monomer is flexible and unstable in several regions, whereas the VHL-HIF complex, particularly the HIF-binding domain, exhibits enhanced stability. Mutational scanning revealed that bulky amino acid substitutions, such as tryptophan, cause notable destabilization. Interestingly, some mutations predicted to be structurally stable were still classified as pathogenic by AlphaMissense and validated in the literature, suggesting that pathogenicity may arise from subtle functional impairments rather than just structural instability. These findings underscore the value of combining machine learning, structural simulations, and experimental data to predict and interpret the pathogenicity of VHL mutations.

bioinformatics↗

bioRxiv2026

Enhancing Patient Lymphocyte Response to Peritoneal Malignancies Using a Personalized Immunocompetent Microfluidic Co-Culture Platform

Harnessing patient immune cells via adoptive cellular therapy is a promising cancer therapeutic strategy. However, major challenges remain for advanced solid malignancies, including difficulty isolating sufficient tumor infiltrating lymphocytes (TILs) and limited targeting of diverse neoantigens in heterogenous tumors. To address this, we have developed a tumor-on-a-chip platform with co-cultured patient-derived tumor cells, autologous peripheral blood mononuclear cells (PBMCs), and lymphoid tissue-derived antigen presenting cells. This approach generates organoid interacting lymphocytes (OILs) with enhanced anti-tumor activity. In peritoneal malignancies, OIL-induced cytotoxicity of patient-matched tumor cells surpasses both TILs and static-expanded PBMCs. We find that this improved performance was linked to increased CD8+ T and NK cells among OILs, and increased effector cytokine polyfunctionality, particularly Granzyme A. Our platform represents a versatile and scalable approach to generate patient-specific therapeutic lymphocytes even when TILs are insufficient, offering a promising avenue to treat diverse solid tumors associated with poor outcomes under current immunotherapies. TeaserA tumor-on-a-chip device primes patient immune cells with tumor recognition for personalized immunotherapy applications.

cancer biology↗

bioRxiv2025

Phosphorylation of a conserved intrinsically disordered region is necessary for activation of a bacterial Hanks-type Ser/Thr kinase signaling pathway

Intrinsically disordered regions (IDRs) are found throughout all domains of life, yet their contribution to bacterial signaling has remained unclear. Here we show that phosphorylation of a conserved juxtamembrane IDR is necessary for activation of the bacterial Hanks-type Ser/Thr kinase-phosphatase PrkC/PrpC signaling pathway in Bacillus subtilis. Phosphoablative mutation of a conserved IDR phosphosite has strong effect on kinase-activity-dependent phenotypes, including intrinsic {beta}-lactam resistance and stationary phase survival. Using a synthetic quantitative reporter for kinase activity, mutational analysis, and mathematical modeling, we show that phosphorylation of the IDR promotes trans autoactivation of the kinase, and that this modification is essential for amplifying kinase activity in response to a signal. Phylogenetic analysis demonstrates that this IDR and associated putative phosphosites proximal to the kinase domain are highly conserved across prokaryotic species that diverged at the last universal common ancestor. Together these findings suggest that kinase-domain proximal IDR phosphorylation has a critical role in bacterial Ser/Thr signaling, with direct implications for understanding antibiotic resistance mechanisms and developing kinase-targeting antimicrobials. Author SummaryBacteria use protein kinases to sense and respond to their environment. Despite decades of research, the activation process of an evolutionarily ancient group of bacterial Ser/Thr kinases that serve as master regulators of responses to antibiotics remains poorly understood. In this work we show that phosphorylation of an unstructured protein region, an intrinsically disordered region (IDR), is required for activating the prototypical Ser/Thr bacterial kinase in Bacillus subtilis. Without this phosphorylation, bacteria are more sensitive to {beta}-lactam antibiotics and show severe survival defects in stationary phase. Using genetic analysis and mathematical modeling, we show that IDR phosphorylation allows kinases to activate each other, generating signal amplification. This kinase IDR is conserved across bacteria and archaea that diverged billions of years ago, suggesting it arose early in evolution. Because closely related kinases in clinically important pathogens share these features, our findings suggest this IDR as a target for developing antimicrobials that could disrupt bacterial responses to antibiotic treatment.

microbiology↗

bioRxiv2025

Novel Roles of Sonic Hedgehog Signaling in Retinal Patterning and Neurogenesis During Mammalian Eye Development

The Sonic Hedgehog (Shh) signaling pathway is essential for the patterning, growth, and morphogenesis of many tissues. During early eye development, Shh is critical for the formation of the two optic vesicles, which give rise to the retina, retinal pigment epithelium (RPE), and optic stalk. It also regulates the balance between cell proliferation and differentiation during retinal histogenesis, a key process in shaping the cellular architecture of the mature retina. Despite these well-established roles, the temporal dynamics, region-specific functions, and downstream consequences of Shh signaling during retinal development remain poorly understood. Here, we present a comprehensive analysis of Shh signaling across multiple stages of retinal development using temporally and spatially controlled deletion of Smoothened (Smo), an essential transducer of the pathway. This approach reveals previously unrecognized requirements for Shh signaling in specifying optic nerve head identity and maintaining nasal-temporal polarity. We also show that Shh signaling coordinates neurogenesis by sustaining the retinal progenitor pool while also regulating progenitor competence, ensuring appropriate proportions of retinal cell types. Our data indicate that both proliferative capacity and the timing of cell fate specification are shaped by Shh pathway activity. Together, these findings establish new mechanistic links between Shh signaling, regional patterning, and temporal regulation of neurogenesis, providing novel insights into how morphogen signaling is repurposed across developmental time to orchestrate complex tissue architecture.

developmental biology↗

bioRxiv2024

Co-Therapy with S1P and Heparan Sulfate Derivatives to Restore Endothelial Glycocalyx and Combat Pro-Atherosclerotic Endothelial Dysfunction

Endothelial cell (EC) glycocalyx (GCX) shedding due to disturbed blood flow and chemical factors leads to low-density lipoprotein infiltration and reduced nitric oxide synthesis, causing vascular dysfunction and atherosclerosis. This study evaluates a novel therapy combining sphingosine-1-phosphate (S1P) and heparin (heparan sulfate derivative). We hypothesized that heparin/S1P would repair mechanically damaged EC GCX in disturbed flow (DF) regions and restore anti-atherosclerotic mechanotransduction function, addressing cardiovascular disease. We used a parallel-plate flow chamber to simulate flow conditions in vitro and a partial carotid ligation mouse model to mimic DF in vivo. Heparin and albumin-bound S1P were administered to assess their reparative effects on the endothelial GCX. Immunocytochemistry, fluorescent staining, confocal microscopy, cellular alignment studies, and ultrasound were performed to evaluate EC function and endothelial-dependent vascular function. Barrier functionality was assessed via macrophage uptake. Heparin/S1P mechanism-of-action insights were gained through fluid dynamics simulations and staining of GCX synthesis enzyme as well as S1P receptor. Statistical analyses validated results. In vitro data showed that heparin/S1P therapy improves the function of DF-conditioned ECs by restoring EC GCX and promoting EC alignment and elevated vasodilator eNOS (endothelial-type nitric oxide synthase) expression. The in vivo studies confirmed GCX degradation, increased vessel inflammation and hyperpermeability, and vessel wall thickening in the partially ligated left carotid artery. Heparin/S1P treatment restored GCX in the left carotid artery, enhancing GCX thickness and coverage of the blood vessel wall. This work advances a new approach to regenerating the EC GCX and restoring its function in ECs under DF conditions.

bioengineering↗

bioRxiv2022

Within-species control reveals novel immune response genes in Caenorhabditis elegans

The nematode Caenorhabditis elegans is a simple model host for studying the interaction between bacterial pathogens and the metazoan innate immune system. In the last two decades, much focus has been given to studying intestinal infection in the worm by the clinical strain Pseudomonas aeruginosa PA14. Powerful genetic and molecular tools in both species facilitate the identification and analysis of bacterial virulence factors as well as host defense factors. However, findings from these studies are confounded by the use of the genetically, metabolically, and physiologically divergent E. coli OP50 as a food source and as the non-virulent control. Here we report the use of P. aeruginosa z11 strain as a preferable control for PA14 infection studies. We demonstrate that many aspects of worm behavior, health-span, longevity, food attraction, brood size, lifespan, and pathogen avoidance and survival are not affected in z11. We show that the use of z11 as a control for transcriptomics analysis can increase the discovery power. In particular, we identified three novel pathogenesis-related genes in C. elegans. Our findings accentuate the importance of choosing an appropriate control environment for host-pathogen studies.

genomics↗

bioRxiv2019

Dynamics of Heat Shock Detection and Response in theIntestine of Caenorhabditis elegans

The heat shock response is the organized molecular response to stressors which disrupt proteostasis, potentially leading to protein misfolding and aggregation. While the regulation of the heat shock response is well-studied in single cells, its coordination at the cell, tissue, and systemic levels of a multicellular organism is poorly understood. To probe the interplay between systemic and cell-autonomous responses, we studied the upregulation of HSP-16.2, a molecular chaperone induced throughout the intestine of Caenorhabditis elegans following a heat shock, by taking longitudinal measurements in a microfluidic environment. Based on the dynamics of HSP-16.2 accumulation, we showed that a combination of heat shock temperature and duration define the intensity of stress inflicted on the worm and identified two regimes of low and high intensity stress. Modeling the underlying regulatory dynamics implicated the saturation of heat shock protein mRNA production in defining these two regimes and emphasized the importance of time separation between transcription and translation in establishing these dynamics. By applying a heat shock and measuring the response in separate parts of the animals, we implicated thermosensory neurons in accelerating the response and transducing information within the animal. We discuss possible implications of the systemic and cell level aspects and how they coordinate to facilitate the organismal response.

biophysics↗

bioRxiv2018

A heterochromatic histone methyltransferase lowers nucleosome occupancy at euchromatic promoters

H3K9me3 (histone H3 modified with tri-methylation at lysine 9) is a hallmark of transcriptional silencing and heterochromatin. However, its global effects on the genome, including euchromatin, are less well understood. Here we develop Formaldehyde-Assisted Identification of Regulatory Elements (FAIRE) for C. elegans to examine the chromatin configuration of mutants that lack virtually all H3K9me3, while leaving H3K9me1 and H3K9me2 intact. We find that nucleosomes are mildly disrupted, and levels of H3K9me2 and H3K27me3 rise in mutant embryos. In addition to these expected changes, the most dramatic change occurs in euchromatin: many regions encompassing transcription start sites (TSSs) gain an average of two nucleosomes in mutants. The affected regions normally lack H3K9me3, revealing a locus non-autonomous role for H3K9me3. Affected TSSs are associated with genes that are active in epithelia and muscles, and implicated in development, locomotion, morphogenesis and transcription. Mutant embryos develop normally under ideal laboratory conditions but die when challenged, with defects in morphogenesis and development. Our findings reveal that H3K9me3 protects transcription start sites within euchromatin from nucleosome deposition. These results may be relevant to mammals, where diseases that disrupt the nuclear lamina and heterochromatin can alter epithelial and muscle gene expression.

developmental biology↗

bioRxiv2018

Hyperexcitable phenotypes in iPSC-derived neurons from patients with 15q11-q13 duplication syndrome, a genetic form of autism

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, Dup15q patients commonly meet the criteria for autism spectrum disorder (ASD) and have a high prevalence of seizures. Here, we explored mechanisms of hyperexcitability in neurons derived from induced pluripotent stem cell (iPSC) lines from Dup15q patients. Maturation of resting membrane potential in Dup15q-derived neurons was similar to neurons from unaffected control subjects, but Dup15q neurons had delayed action potential maturation and increased synaptic event frequency and amplitude. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared to control neurons, in part due to disruption of KCNQ2 channels. Together these data point to multiple mechanisms underlying hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

neuroscience↗

bioRxiv2017

Systemic Activation Coordinates The Heat Shock Response Of The Insulin/IGF-1 Pathway In Caenorhabditis elegans

Exposure to high temperatures has an adverse effect on cellular processes and results in activation of the cellular heat shock response (HSR), a highly conserved program of inducible genes to maintain protein homeostasis1. The insulin/IGF-1 signaling (IIS) pathway, which has diverse roles from metabolism to stress response and longevity, is activated as part of the HSR2-4. Recent evidence suggest that the IIS pathway is able to affect proteostasis non-autonomously5,6, yet it is not known if it is activated autonomously in stressed cells or systemically as part of an organismic program. In Caenorhabditis elegans, the single forkhead box O (FOXO) homologue DAF-16 functions as the major target of the IIS pathway7 and, together with the heat-shock factor HSF-1, induce the expression of small heat shock proteins in response to heat shock8-10,3. Here we use a novel microfluidic device that allows precise control of the spatiotemporal temperature profile to show that cellular activation of DAF-16 integrates local temperature sensation with systemic signals. We demonstrate that DAF-16 activation in head sensory neurons is essential for DAF-16 activation in other tissues, but show that no known thermosensory neuron is individually required. Our findings demonstrate that systemic and cell-autonomous aspects of stress response act together to facilitate a coordinated cellular response at the organismic level.

systems biology↗