Biology subjects

Rojas, E.

Publications and source records attributed to Rojas, E..

14 recordsLinked to original sources

bioRxiv2026

ExPert infers causal perturbations from cell states across biological and clinical contexts

Pooled single-cell CRISPR screens have driven substantial effort toward models predicting the response to a perturbation. We posed the inverse question: which perturbation produced an observed cell state? Here we developed ExPert (Extracting Perturbations), which learns one prototype per perturbation in a latent space shared by 12 cellular contexts, trained on 8,964 perturbations and 19.5 million cells, and ranks 19,895 candidate genes per query, over half absent from training. ExPert more than doubled held-out-context recall over the best existing method with 1,000-fold less compute, and transferred without retraining to drugs, protein degradation and combinations. In alcohol-associated liver disease it implicated mRNA export, unchanged in expression, and we experimentally confirmed nuclear poly(A) retention and isoform switching. In primary mitochondrial disease it recovered the affected complex from blood, localized the signal to monocytes, and flagged a misdiagnosed control. Perturbation atlases can thus be queried in reverse, from state to likely driver.

systems biology↗

bioRxiv2026

Excitatory Dysfunction and Phenotypic Rescue in a Human Neuronal Model of SCN2A-Related Disorders

SCN2A-related disorders result from pathogenic variants in the gene encoding for the voltage-gated sodium channel Nav1.2. Collectively, these disorders result in variable age of onset epilepsy, autism spectrum disorder, and epileptic encephalopathies. While the mechanisms of haploinsufficiency resulting in autism spectrum disorder have been explored in detail, few studies report the impact of pathogenic missense variants in human neurons. In this work, we combined conventional electrophysiology and high-throughput all-optical electrophysiology assays to analyze the SCN2A p.M1879T pathogenic variant associated with early-onset epilepsy and developmental delay. In both platforms, iPSC-derived excitatory neurons expressing the disease variant showed greater firing at higher stimuli compared to the isogenic control neurons (corrected by CRISPR/Cas9), as well as changes to action potential shape (steeper slope and larger amplitude) with evoked firing. We used machine learning techniques on the optical physiology dataset to classify the two genotypes, finding that sodium channel blocking anti-seizure drugs could restore an isogenic phenotype. This work demonstrates proof of sodium channel blocker efficacy in a human neuronal model of SCN2A-related epilepsy and highlights the power of leveraging high-throughput all-optical electrophysiology for testing drug efficacy.

neuroscience↗

bioRxiv2026

A patient derived xenograft repository capturing clinical and molecular heterogeneity of large B-cell lymphoma

Large B-cell lymphomas (LBCLs) are a clinically and molecularly diverse group of malignancies with a rapidly evolving therapeutic landscape that has introduced new areas of clinical need such as post-CD19 chimeric antigen receptor T (CART19) progression. Patient derived xenograft (PDX) models are an important tool for mechanistic studies and preclinical evaluation of new therapies and can be generated from a variety of clinical contexts that capture tumor-intrinsic resistance mechanisms. We therefore undertook a comprehensive effort to generate PDX models that encompass the molecular landscape of LBCLs and include important clinical scenarios for new drug development. Here we describe the first 48 models within this publicly available repository, capturing the transcriptional and genetic subsets of LBCL. These models also include 23 generated from post-CART progression biopsies which reproduce patterns of progression driven by CD19 mutation or expression loss, as well as tumor cell-intrinsic CART19 resistance that we validate in vivo. STATEMENT OF SIGNIFICANCEYang et al. describe X-LYMPH (Xenografts of Lymphoma), a publicly available and molecularly annotated PDX repository that captures the heterogeneity of large B-cell lymphoma. X-LYMPH includes models of chimeric antigen receptor T cell resistance, providing a shared foundation for mechanistic research and therapeutic development for lymphomas.

cancer biology↗

bioRxiv2026

Modular mRNA LNP design integrates RNA, lipid, and antigen engineering for protective vaccination

mRNA-lipid nanoparticle (LNP) vaccines are programmable, multi-component systems in which immune outcomes emerge from coupled control of nanoparticle chemistry, RNA regulatory architecture, and antigen design. Here we establish an integrated engineering framework that quantitatively maps how ionizable lipid identity, untranslated region (UTR) configuration, and 5' cap structure shape innate activation landscapes and thereby tune the magnitude, cellular distribution, and polarization of adaptive immunity. Benchmarking three ionizable lipids shows that lipid chemistry imprints distinct cytokine and chemokine milieus during dendritic cell-T cell priming that mirror downstream T cell activation phenotypes, identifying lipid structure as a determinant of pathway-selective activation; notably, our first-in-study lipid exhibits benchmark-comparable immunostimulatory profiles, supporting further translational evaluation. Using Crimean-Congo hemorrhagic fever virus (CCHFV) as a model high-consequence pathogen, we show that UTRs act as modular regulatory elements that redirect cytokine outputs, tuning effector versus proliferative programs and expanding helper polarization in an antigen-dependent manner. Cap structure functions primarily as a quantitative gain control, scaling cellular and humoral response magnitude without overriding antigen-defined polarization. Within this optimized platform, antigen architecture defines functional constraints on protection: structural modifications reshape immune hierarchies and antibody quality. Integrating these design axes yields an optimized mRNA-LNP vaccine encoding the CCHFV secreted glycoprotein complex (sGCs) that achieves 90% protection in an immunosuppressed murine lethal-challenge model with minimal clinical signs. Together, these data define generalizable design principles for rational, multi-parameter optimization of mRNA-LNP vaccines.

microbiology↗

bioRxiv2025

Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

The bacterial cell wall is a critical load-bearing structure, but is not thought to be an important permeability barrier since proteins freely diffuse through isolated cell wall sacculi and bacteria secrete proteins without the aid of any known channels or transporters in the wall. Using new genetically encoded probes to measure the permeability of the cell in situ at the single-cell level, we discovered that the size threshold determining whether proteins can pass through the Bacillus subtilis sacculus is smaller than was previously thought. We found that transport of small proteins (<10 kDa) through the sacculus was consistent with passive diffusion through discrete pores, while larger proteins (>15 kDa) required the generation of larger pores by inducing peptidoglycan hydrolysis unbalanced by synthesis. These data are consistent with physics-based models of diffusion through a random percolation network of finite thickness. Conversely, the ability of the innate immune factor phospholipase (15.2 kDa) to kill B. subtilis was inhibited by membrane de-polarization. The protective effect of de-polarization was dependent on latent peptidoglycan synthesis (decoupled from cell growth) by PBP1 - highlighting a new role for this enzyme - and on reduced peptidoglycan hydrolysis. These results demonstrate that the rapid peptidoglycan turnover that drives cell growth also promotes movement of phospholipase across the cell wall, identifying a quintessentially bacterial mechanism of active transport. Significance StatementGram-positive bacteria, which include many serious pathogens like Staphylococcus aureus, Listeria monocytogenes, and Clostridium difficile, are defined by their thick peptidoglycan cell wall. Here, we demonstrate that this structure is a critical permeability barrier that blocks antibacterial proteins like those used by the human innate immune system. Furthermore, this barrier function depends on the physiological state of the cell: the wall of non-growing cells is less permeable because peptidoglycan turnover during growth actively promotes transport of specific proteins through the cell wall. This prokaryotic paradigm for molecular transport has important implications for host-pathogen interactions since pathogenic bacteria often assume both non-growing and growing states during infection.

microbiology↗

bioRxiv2024

Loss of age-accumulated crh-1 circRNAs ameliorate amyloid β-induced toxicity in a C. elegans model for Alzheimers disease

Circular RNAs (circRNAs) are non-coding RNAs mostly derived from exons of protein-coding genes via a back-splicing process. The expression of hundreds of circRNAs accumulates during healthy aging and is associated with Alzheimers disease (AD), characterized by the accumulation of amyloid-beta (A{beta}) proteins. In C. elegans, many circRNAs were previously found to accumulate during aging, with loss of age-accumulated circRNAs derived from the CREB gene (circ-crh-1) to increase mean lifespan. Here, we used C. elegans to study the effects of age-accumulated circRNAs on the age-related onset of A{beta}-toxicity. We found that circ-crh-1 mutations delayed A{beta}-induced muscle paralysis and lifespan phenotypes in a transgenic C. elegans strain expressing a full-length human A{beta}-peptide (A{beta}1-42) selectively in muscle cells (GMC101). The delayed A{beta} phenotypic defects were associated with inhibiting the deposition of A{beta} aggregates, and thus, genetic removal of circ-crh-1 provides protection against A{beta}-induced toxicity. Consistent with a detrimental role for age-accumulated circRNAs in AD, circ-crh-1 expression level is elevated after induction of A{beta} during aging, whereas linear crh-1 mRNA expression remains unchanged. Finally, we show that a circ-crh-1 upregulated collagen gene, col-49, promotes A{beta}-induced paralysis. Taken together, our results show that the loss of an age-accumulated circRNA exerts a protective role on A{beta}-induced toxicity, demonstrating the utility of C. elegans for studying circRNAs in AD and its relationship to aging.

neuroscience↗

bioRxiv2023

CREBBP lysine acetyltransferase domain mutations create zombie enzymes that alter chromatin loading dynamics and prevent EP300 redundancy

Epigenetic regulation of gene expression is fundamental to cell state transitions. A prominent example of this is the epigenetically-regulated generation of transitory cells states within the germinal center (GC) reaction that are required for humoral immunity. The deregulation of these processes through somatic mutations can give rise to B-cell lymphoma, thus studying these mutations can provide insight into fundamental mechanisms underlying epigenetic regulation of gene expression as well as GC B-cell and lymphoma biology. Here we show that classes of mutations in the CREBBP lysine acetyltransferase gene result in different structure, catalytic activity and function. We discover that CREBBP and its paralog EP300 are dynamically and reciprocally loaded onto chromatin during a signal-responsive cell state transitions within the GC. Mutation of CREBBP in GC-derived lymphoma interrupt signal-responsive CREBBP loading onto chromatin, while also inhibiting EP300 redundancy. These observations lead to a model in which CREBBP or EP300 loss of function through acetyltransferase domain mutations, which occur frequently in both hematopoietic and solid tumors, create a zombie enzyme that retards the function of the active paralog by binding limiting concentrations of transcription factor substrate and preventing signal-responsive enhancer activation.

cancer biology↗

bioRxiv2023

CD8 T-cell dysfunction is linked with CAR T-cell failure and can be mitigated by a non-alpha IL-2 agonist, pegenzileukin

Chimeric antigen receptor (CAR) T-cell therapy has been a breakthrough for relapsed or refractory large B-cell lymphoma (rrLBCL). However, suboptimal CAR T-cell activity can lead to therapeutic failure and dismal outcome. Using single cell RNA-sequencing of rrLBCL tumors, we identify a prominent population of clonally expanded dysfunctional CAR+ CD8 T-cells indicative of ongoing tumor cell engagement, proliferation, and dysfunction at the time of progression from CAR T-cell therapy. Furthermore, we show that rrLBCL patient-derived CAR T-cells are more prone to dysfunction and loss of cytotoxicity compared to healthy donor-derived CAR T-cells. Using both antigen-driven and CAR-driven models of T-cell dysfunction, we show that pegenzileukin, a non-alpha IL2 agonist, can prevent T-cell dysfunction. In both in vitro and in vivo CAR T-cell models, pegenzileukin improved T-cell expansion and tumor control. This provides pre-clinical rational for use of pegenzileukin in combatting T-cell dysfunction, a central mechanism of CAR T-cell failure. HIGHLIGHTSO_LITumor-infiltrating CD8 CAR T-cells show clonal expansion and dysfunction at the time of progression. C_LIO_LIrrLBCL patient-derived CAR T-cells are more prone to dysfunction compared to healthy-donor-derived CAR T-cells. C_LIO_LIPegenzileukin, a non-alpha IL2 agonist, rescues antigen- and CAR-driven CD8 T-cell dysfunction and improves CAR T-cell responses in vivo. C_LI

immunology↗

bioRxiv2022

Chronic noise unexpectedly increases fitness of a freshwater zooplankton

Although there is an increasing interest in the effects of anthropogenic noise on underwater wildlife, most studies focus on marine mammals and fishes while many other taxa of substantial ecological importance are still overlooked. This is the case for zooplankton species which ensure the coupling between primary producers and fishes in pelagic food webs. Here, we measured lifespan, reproduction, and mobility of laboratory-raised water fleas Daphnia magna, a widespread freshwater zooplankton species, in response to continuous broadband noise. Surprisingly, we found a significant increase in survival and fecundity, leading to a higher individual fitness when considering total offspring production and a slight increase in the population growth rate according to the Euler-Lotka equation. Exposed water fleas were found to be slower than control individuals, and we discussed potential links between mobility and fitness. Our results can have implications in aquaculture and for in-lab studies (e.g., in ecotoxicology) where the acoustic environment receives little attention. Chronic broadband noise can be associated with certain human activities, but the consequences for natural Daphnia populations might differ as reduced velocity could have negative outcomes when considering competition and predation. Our work is one of the few showing an effect of noise on individual fitness and suggests that noise should be better accounted for in laboratory studies.

ecology↗

bioRxiv2022

A freshwater zooplankton in the face to boat noise pollution

AO_SCPLOWBSTRACTC_SCPLOWAmong the numerous questions about human impacts on ecosystems, there is a growing interest for acoustic pollution. First studies on underwater acoustic pollution focused, and showed effects, on vertebrates behaviours. Knowledge on the effects on invertebrates is more limited and there is a huge lack concerning zooplankton species, although widely used as bioindicators in chemical pollution. Consequently, it is critical to assess the impact of noise on zooplanktons fitness (survival and fecundity). Here, isolated water fleas, Daphnia magna, were reared from birth to death in the presence or absence of motorboat noises. Effects on lifespan and clonal offspring production (e.g., clutch size, number of offspring produced along life) were assessed and chronic exposure to boat noise did not affect Daphnias fitness. The spectral and temporal features of the sounds could explain the results. This study highlights the importance of integrating noise pollution into ecotoxicological research to understand, and prevent, human impacts on communities.

ecology↗

bioRxiv2022

From behaviour to complex communities: Resilience to anthropogenic noise in a fish-induced trophic cascade

Sound emissions from human activities represent a pervasive environmental stressor. Individual responses in terms of behaviour, physiology or anatomy are well documented but whether they propagate through nested ecological interactions to alter complex communities needs to be better understood. This is even more relevant for freshwater ecosystems that harbour a disproportionate fraction of biodiversity but receive less attention than marine and terrestrial systems. We conducted a mesocosm investigation to study the effect of chronic exposure to motorboat noise on the dynamics of a freshwater community including phytoplankton, zooplankton, and roach as a planktivorous fish. As expected under the trophic cascade hypothesis, roach predation induced structural changes in the planktonic communities. Surprisingly, although roach changed their feeding behaviour in response to noise, the dynamics of the roach-dominated planktonic communities did not differ between noisy and noiseless mesocosms. This suggests that the top-down structuring influence of roach on planktonic communities might be resilient to noise and reveals the difficulties on extrapolating impacts form individual responses to complex communities.

ecology↗

bioRxiv2022

Feedback linking cell envelope stiffness, curvature, and synthesis enables robust rod-shaped bacterial growth

Bacterial growth is remarkably robust to environmental fluctuations, yet the mechanisms of growth-rate homeostasis are poorly understood. Here, we combine theory and experiment to infer mechanisms by which Escherichia coli adapts its growth rate in response to changes in osmolarity, a fundamental physicochemical property of the environment. The central tenet of our theoretical model is that cell-envelope expansion is only sensitive to local information such as enzyme concentrations, cell-envelope curvature, and mechanical strain in the envelope. We constrained this model with quantitative measurements of the dynamics of E. coli elongation rate and cell width after hyperosmotic shock. Our analysis demonstrated that adaptive cell-envelope softening is a key process underlying growth-rate homeostasis. Furthermore, our model correctly predicted that softening does not occur above a critical hyperosmotic shock magnitude and precisely recapitulated the elongation-rate dynamics in response to shocks with magnitude larger than this threshold. Finally, we found that to coordinately achieve growth-rate and cell-width homeostasis, cells employ direct feedback between cell-envelope curvature and envelope expansion. In sum, our analysis points to new cellular mechanisms of bacterial growth-rate homeostasis and provides a practical theoretical framework for understanding this process. Significance StatementThe bacterial cell envelope is the critical structure that defines cell size and shape, and its expansion therefore defines cell growth. Although size, shape, and growth rate are important cellular variables that are robust to environmental fluctuations, the feedback mechanisms by which these variables influence cell-envelope expansion are unknown. Here, we explore how E. coli cells achieve growth-rate and cell-width homeostasis during fluctuations in osmolarity, a key environmental property. A biophysical model in which the cell envelope softens after an osmotic shock and envelope expansion depends directly on local curvature quantitatively recapitulated all experimental observations. Our study elucidates new mechanisms of bacterial cell morphogenesis and highlights the deep interplay between global cellular variables and the mechanisms of cell-envelope expansion.

microbiology↗

bioRxiv2020

Bacterial evolution in high osmolarity environments

Bacteria must maintain a cytosolic osmolarity higher than that of their environment in order to take up water. High osmolarity environments therefore present a formidable stress to bacteria. To explore the evolutionary mechanisms by which bacteria adapt to high osmolarity environments, we selected Escherichia coli in media with a variety of osmolytes and concentrations for 250 generations. Adaptation was osmolyte-dependent, with sorbitol stress generally resulting in increased fitness in conditions with higher osmolarity, while selection in high concentrations of proline resulted in increased fitness specifically on proline. Consistent with these phenotypes, sequencing of the evolved populations showed that passaging in proline resulted in specific mutations in an associated metabolic pathway that increases the ability to utilize proline for growth, while evolution in sorbitol resulted in mutations in many different genes that generally improve growth in high osmolarity conditions at the expense of growth at low osmolarity. High osmolarity decreased growth rate but increased mean cell volume compared with growth on proline as the sole carbon source, demonstrating that osmolarity-induced changes in growth rate and cell size follow an orthogonal relationship from the classical Growth Law relating cell size and nutrient quality. Isolates from a sorbitol-evolved population that capture the likely temporal sequence of mutations revealed by metagenomic sequencing demonstrate a tradeoff between growth at high and low osmolarity. Our study highlights the utility of experimental evolution for dissecting complex cellular networks and environmental interactions, particularly in the case of behaviors that can involve both specific and general metabolic stressors. ImportanceFor bacteria, maintaining higher internal solute concentrations than the environment allows cells to take up water. As a result, survival is challenging in high osmolarity environments. To investigate how bacteria adapt to high osmolarity environments, we evolved Escherichia coli in a variety of high osmolarity solutions for hundreds of generations. We found that evolved populations adopted different strategies to improve their growth depending on the osmotic passaging condition, either generally adapting to high osmolarity conditions or better metabolizing the osmolyte as carbon source. Single-cell imaging demonstrated that enhanced fitness was coupled to faster growth, and metagenomic sequencing revealed mutations that reflect growth tradeoffs across osmolarities. Our study demonstrates the utility of long-term evolution experiments for probing adaptation during environmental stress.

microbiology↗

bioRxiv2020

A mechanical cusp catastrophe imposes a universal developmental constraint on the shapes of tip-growing cells

Understanding the mechanistic basis for cell morphology is a central problem in biology. Evolution has converged on tip growth many times, yielding filamentous cells, yet tip-growing cells display a range of apical morphologies. To understand this variability, we measured the spatial profiles of cell-wall expansion for three species that spanned the phylogeny and morphology of tip-growth. Profiles were consistent with a mechanical model whereby the wall was stratified and stretched by turgor pressure during cell growth. We calculated the spatial profiles of wall mechanical properties, which could be accurately fit with an empirical two-parameter function. Combined with the mechanical model, this function yielded a "morphospace" that accounted for the shapes of diverse tip-growing species. However, natural shapes were bounded by a cusp bifurcation in the morphospace that separated thin, fast-growing cells from (nonexistent) wide, slow-growing cells. This constraint has important implications for our understanding of the evolution of tip-growing cells.

cell biology↗