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Gomez, M.

Publications and source records attributed to Gomez, M..

At least 19 recordsLinked to original sources
bioRxiv2026

Combining interstrand crosslinking agents with histone deacetylase inhibitors against high grade IDH mutant gliomas

Mismatch repair (MMR) deficiency contributes to temozolomide (TMZ) resistance in a subset of isocitrate dehydrogenase 1/2 mutant (IDHmut) gliomas, creating a need for therapies that retain activity despite MMR loss. We evaluated the novel interstrand crosslinking agent KL-50, alone and in combination with the histone deacetylase inhibitor belinostat, in patient-derived IDHmut glioma models. MSH6 knockout or constitutive expression established four matched MMR-proficient and MMR-deficient culture models. Drug responses were assessed using live cell count and cytotoxicity assays, and KL-50 monotherapy was tested in intracranial BT142 orthotopic xenografts. LOEWE synergy analysis, RNA sequencing, and a genome-wide CRISPR knockout screen assessed combination activity and potential mechanisms. MMR deficiency increased TMZ GI values 2.2- to 30.4-fold, whereas KL-50 retained antiproliferative activity and showed enhanced cytotoxicity in selected MMR-deficient models. In TMZ-naive, MMR-proficient BT142 xenografts, KL-50 increased median survival from 107.5 to 194 days. Two of eight treated mice had no histologically detectable residual tumor after 220 days post-engraftment. KL-50 and belinostat exhibited synergistic cytotoxicity in BT142, 905, and TS603 IDHmut glioma cultures. Transcriptomic gene ontology analyses identified downregulation of DNA repair pathways following histone deacetylase inhibition, including terms for homologous recombination and double-strand break repair. The CRISPR screen identified depletion of PKMYT1-targeting gRNAs during KL-50 treatment, implicating this mitotic checkpoint kinase as a candidate determinant of drug sensitivity. PKMYT1 expression was also reduced by histone deacetylase inhibition. These findings support further preclinical development of KL-50, alone and in combination with belinostat, for IDHmut gliomas including tumors with MMR-associated TMZ resistance.

cancer biology↗
bioRxiv2026

Endothelial β3-Adrenergic Receptor activation prevents pulmonary hypertension

BackgroundPulmonary hypertension (PH) is a progressive vascular disease characterized by endothelial dysfunction, vascular remodeling and increased pulmonary vascular resistance. The {beta}3-adrenergic receptor ({beta}3-AR) has been implicated in cardiovascular regulation and cardioprotective mechanisms; however, its role in pulmonary vascular disease remains poorly understood. We investigated whether activation of {beta}3-AR protects pulmonary endothelial function and prevents the development of pre-capillary PH. Methods{beta}3-AR expression was evaluated in pulmonary endothelium from patients with Chronic Obstructive Pulmonary Disease (COPD) and in murine models of hypoxia-induced PH. Genetic mouse models including {beta}3-AR knockout (KO) and conditional {beta}3-AR overexpression in endothelial cells (EC) or in smooth muscle cells (SMC), were used to determine cell-specific roles. Pharmacological activation of {beta}3-AR was achieved using the selective {beta}3-agonist mirabegron in hypoxia-induced PH mice and monocrotaline-induced PH rats. Pulmonary vascular reactivity and vasodilatory responses to {beta}3-AR stimulation were evaluated by wire myography in isolated pulmonary arteries. Mechanistic studies were performed in human pulmonary artery endothelial cells (HPAEC) under hypoxic conditions, in human pulmonary arterial smooth muscle cells (HPASMC) and in endothelial nitric oxide synthase (NOS3) KO mice. Results{beta}3-AR was upregulated in pulmonary endothelium of COPD patients and mice exposed to chronic hypoxia. Genetic deletion of {beta}3-AR aggravated PH, whereas endothelial-specific overexpression attenuated the disease phenotype, reducing right ventricular systolic pressure (RVSP), vascular remodeling and right ventricular (RV) hypertrophy. Activation of {beta}3-AR with mirabegron improved pulmonary hemodynamics, reduced vascular remodeling and preserved RV function. {beta}3-AR activation promoted endothelial nitric oxide synthase (eNOS)-dependent NO production, indirectly inhibiting SMC proliferation. Additionally, {beta}3-AR activation improved mitochondrial fitness in endothelial cells by increasing uncoupling protein 2 (UCP2) expression, reducing reactive oxygen species (ROS) generation and preventing mitochondrial fragmentation. ConclusionsThese findings identify endothelial {beta}3-AR as a previously unrecognized regulator of pulmonary vascular homeostasis and provide a strong translational rationale for targeting the {beta}3-adrenergic pathway in PH. Given that mirabegron is already approved for clinical use, our results support its repurposing as a therapeutic strategy for pre-capillary forms of PH.

pharmacology and toxicology↗
bioRxiv2026

MicroRNA regulation of stress-survival signalling and protein quality control in human heatstroke

Heatstroke is a life-threatening condition in which heat-shock and unfolded-protein responses are strongly activated but fail to prevent proteostasis disruption and severe cellular injury. Whether post-transcriptional regulation contributes to this mismatch remains unknown. We integrated small RNA sequencing with mRNA profiling in peripheral blood mononuclear cells from patients with classical heatstroke and matched heat-exposed controls recruited during the Hajj pilgrimage. mRNA profiling was performed in 19 cases and 19 controls, and miRNA sequencing in 17 cases and 16 controls from the same cohort. Differentially expressed miRNAs were integrated with 4,462 differentially expressed mRNAs using high-confidence inverse-expression miRNA-mRNA pairs. Twenty-six miRNAs mapped to 376 mRNA targets, forming 414 regulatory pairs and two opposing programmes. Programme A, comprising 16 downregulated miRNAs, was associated with activation of PI3K-mTOR, NRF2 oxidative stress and HIF-1 signalling, consistent with stress-survival signalling. Programme B, comprising 10 upregulated miRNAs, was associated with suppression of stress-granule components and fatty-acid {beta}-oxidation genes, consistent with impaired protein quality control and reduced metabolic flexibility. miR-92a-3p emerged as a central regulatory node, and its target PIK3R3 connected 9 of the 10 enriched pathways. These findings suggest a post-transcriptional regulatory layer that could contribute to the limited protection afforded by activated stress defences in human heatstroke. FundingKing Abdullah International Medical Research Center (RC18/373/R). Key pointsO_LIHeatstroke can occur even when major cellular stress defences are activated, raising the question of why heat-shock and unfolded-protein responses are insufficient to prevent injury under extreme heat. C_LIO_LITo understand this mismatch, we extend our previous human heatstroke transcriptomic study by analysing post-transcriptional regulation by microRNAs (miRNAs). C_LIO_LIThe miRNA-regulated network showed activation of stress-survival signalling pathways, including PI3K-mTOR, NRF2 and HIF-1, while stress-granule and fatty-acid {beta}-oxidation pathways were attenuated. C_LIO_LITranslation arrest was active but stress-granule signalling was suppressed. Normally, translation arrest generates pools of non-translating mRNAs that stress granules sort, store or return to translation when conditions improve; their uncoupling therefore suggests impaired coordination of the cellular stress response. C_LIO_LIThese findings suggest a post-transcriptional trade-off in human heatstroke, in which short-term stress-survival signalling is favoured at the cost of protein quality control, metabolic flexibility and recovery capacity under extreme heat. C_LI

physiology↗
bioRxiv2026

Sexual selection does not predict long-term population trends in birds

Sexual selection is a major evolutionary force, yet its demographic consequences remain unclear. While experimental studies often report positive effects of sexual selection on traits linked to population performance, comparative studies often find null or negative associations with population persistence. One explanation for this discrepancy is that the demographic consequences of sexual selection depend on ecological context, particularly variation in mortality and fecundity. Here, we used six decades of abundance data and test whether sexual selection predicts population trends across 738 bird species from Europe and North America. We quantify sexual selection using complementary proxies capturing different components of sexual selection: mating system, sexual dichromatism, sexual size dimorphism and relative testes mass. We further assess whether the effect of sexual selection in population trends is mediated by mortality and fecundity. Across all proxies, we found no evidence that sexual selection is associated with population trends. This result is consistent across continents and robust to variation in mortality and fecundity. Our findings suggest that, despite its central role in shaping phenotypic evolution, sexual selection does not translate into consistent effects on long-term population trends at macroecological scales. More broadly, these results highlight a potential disconnect between evolutionary processes and population dynamics.

evolutionary biology↗
bioRxiv2026

EpiFlow: multidimensional single-cell epigenetic profiling by spectral flow cytometry

The epigenetic landscape of individual cells determines their identity and function, yet current methods for profiling chromatin modifications at single-cell resolution remain low-throughput, costly, or limited in parametric depth. Here we present EpiFlow, a spectral flow cytometry-based platform that enables the simultaneous quantification of 16 epigenetic markers, including histone post-translational modifications, DNA methylation, and hydroxymethylation, at the single-cell level. We demonstrate that EpiFlow is robust across species from yeast to mammals and resolves biologically meaningful epigenetic transitions during the cell cycle, stem cell differentiation, germinal centre B cell maturation, diabetic liver remodelling, and seizure-induced chromatin reprogramming. High-dimensional integration of EpiFlow data enables cell-type classification based solely on epigenetic profiles in liver, brain, blood, and cancer. Furthermore, EpiFlow detects on-target and off-target/indirect effects of epigenetic drugs in a high-throughput-compatible format. Collectively, these results establish EpiFlow as a broadly applicable platform for single-cell epigenetic analysis in basic, pharmaceutical, and translational research.

cell biology↗
bioRxiv2025

Stable Inheritance of Transgene and Yellow Fluorescent Protein Gene Expression in Progeny of Transgenic Cacao (Theobroma cacao) Plants

Genetic engineering tools have the potential to rapidly and precisely improve the genome of slow-to-breed cacao. We previously developed an efficient protocol for transforming cacao using cotyledonary explants derived from secondary somatic embryos via Agrobacterium tumefaciens. In this study, we demonstrate that our transformation protocol is successful in elite cultivars, INIAPG-038 and Matina 1-6, producing fertile seeds with stable visual marker inheritance regardless of whether the transgenic plants were used as the pollen or ovule donor. Three vectors were used in the transformations, each containing genes for enhanced yellow fluorescent protein (eyfp) and neomycin phosphotransferase II (nptII). Three transgenic INIAPG-038 events and one transgenic Matina 1-6 event were used to evaluate seed fertility and the stability of transgene inheritance in cacao seeds and plants. The T1 progeny of these four transgenic events were analyzed for YFP expression and transgene presence. YFP expression segregated at a 1:1 ratio in all events when the transgenic plants were crossed with non-transgenic plants, while a 3:1 segregation was observed when transgenic events were crossed with each other. The transgenic plants exhibited a normal phenotype compared to non-transgenic control plants, producing seeds with a 97% germination rate.

plant biology↗
bioRxiv2025

Morphological plasticity in a reef-building coral is context-dependent and trades off with resistance to thermal stress

In sessile, long-lived organisms, such as reef-building corals, phenotypic plasticity may play a key role in mitigating the impacts of climate change. Plasticity is often context-dependent and has costs and limits, ultimately resulting in adaptive, neutral, or maladaptive effects. However, our understanding of the mechanisms that maintain variation in, and therefore drive the evolution of, plasticity is still limited. We use a field-based transplant experiment to show context-dependent tradeoffs to morphological plasticity in Acropora cervicornis under real-world conditions driven by climate change. We find that while morphological plasticity is neutral under some conditions, it trades off with thermal tolerance during moderate temperature stress during a marine heatwave. Additionally, we find that plasticity changes over time, underlining the importance of its temporal dynamics in long-lived organisms. Our results demonstrate a key role for environmental change in the eco-evolutionary dynamics of plasticity by demonstrating context-dependent costs under real-world thermal stress.

evolutionary biology↗
bioRxiv2025

Slow RNAPII elongation enhances naive-pluripotency rewiring while preserving replication fork speed

DNA replication and transcription must be intricately coordinated, as both machineries navigate the same chromatin landscape to ensure genome stability and proper cell function. Here, we uncover that a global imbalance between their elongation rates-- specifically, slowed transcriptional elongation alongside rapid replication fork progression--does not elicit replicative stress. Instead, this uncoupling accelerates the acquisition of naive pluripotency during in vitro de-differentiation, revealing an unexpected link between transcription kinetics and cell plasticity. Mechanistically, we show that the transition to naive pluripotency is accompanied by a distinctive alternative splicing program indicative of reduced RNAPII elongation, both in vitro and in vivo. These findings redefine the functional relationship between replication and transcription dynamics and uncover transcriptional velocity as a tunable layer of control over cellular identity transitions. HighlightsO_LIReplication and transcription elongation rates can be uncoupled genome-wide. C_LIO_LISlow transcription elongation accelerates the acquisition of naive pluripotency during in vitro de-differentiation. C_LIO_LIHigh replication fork speed is maintained in slow-transcribing cells during cell state transitions. C_LIO_LIAlternative splicing is distinctly regulated at the naive and primed pluripotency states, both in vitro and in vivo. C_LI

molecular biology↗
bioRxiv2025

Lack of consistent effect of dietary fiber on immune checkpoint blockade efficacy across diverse murine tumor models

Immune checkpoint blockade (ICB) has transformed cancer treatment, but success rates remain low in most cancers. Recent research suggest that dietary fiber enhances ICB response in melanoma patients and murine preclinical models through microbiome-dependent mechanisms. Yet, the robustness of this effect across cancer types and dietary contexts remains unclear. Specifically, prior literature compared grain-based chow (high fiber) to low-fiber purified diet, but these diets differ also on other dimensions including phytochemicals. Here we investigated, in mice fed grain-based chow or purified diets with differing quantities of isolated fibers (cellulose and inulin), metabolite levels and ICB activity in multiple tumor models. The blood and fecal metabolome were relatively similar between mice fed high- and low-fiber purified diets, but differed massively between mice fed purified diets or chow, identifying the factor as diet type, independent of fiber. Tumor growth studies in three implantable and two spontaneous genetically engineered tumor models revealed that fiber has a weaker impact on ICB (anti-PD-1) efficacy than previously reported. In some models, dietary modulation impacted ICB activity, but not in a consistent direction across models. In none of the models did we observe the pattern expected if fiber controlled ICB efficacy: strong efficacy in both chow and high-fiber purified diet but low efficacy in low-fiber purified diet. Thus, dietary fiber appears to have limited or inconsistent effect on ICB efficacy in mouse models, and other dietary factors that correlate with fiber intake may underlie the clinical correlations between fiber consumption and immunotherapy outcomes.

cancer biology↗
bioRxiv2025

Fluoxetine Delivery for Wound Treatment Through an Integrated Bioelectronic Device - Pharmacokinetic Parameters and Safety Profile in Swine

Wound infections are a significant medical challenge, often leading to chronicity or systemic infection. Selective serotonin reuptake inhibitors (SSRIs) have emerged as potential non-antibiotic candidates with demonstrated ability to limit growth and biofilm formation in Gram-negative bacteria, in addition to their pro-healing activity. Here, we compared direct delivery of the SSRI fluoxetine by topical bolus dosing to delivery from an iontophoresis bandage device with an actuator for temporally controlled drug delivery, in a porcine excisional wound model. Device delivery of fluoxetine resulted in a maximum concentration of 12.25 ng fluoxetine per mg tissue, compared to 2.926 ng/mg following bolus dosing, and tissue fluoxetine levels were higher after application using the device than after bolus dosing across the range of doses tested (p=0.0041). The half-life of fluoxetine in the wound tissue was 0.988 {+/-} 0.256 days. Fluoxetine was not detected in the pig plasma, and plasma serotonin levels were not affected by the topical application. Fluoxetine delivery using the device, but not bolus delivery, produced tissue concentrations above the minimum inhibitory concentration (MIC) for some clinically important species of bacteria. The experimental device can effectively deliver topical fluoxetine to the wound, producing higher tissue concentrations of fluoxetine at lower cumulative doses compared to bolus dosing, and with minimal risk of off-target effects. The device may simplify wound treatment by reducing the burden for daily drug application, possibly increasing adherence to a prescribed treatment regimen.

bioengineering↗
bioRxiv2024

Design and Validation of a Wearable Imaging System for Automated Wound Monitoring in Porcine Model

Effective wound monitoring has the potential to guide treatment regiments and improve healing outcomes, yet current clinical assessment methods remain largely subjective and labor-intensive. To address this challenge, we present a high-resolution wearable imaging system designed for continuous wound monitoring. The system integrates a 64 MP camera with a plano-convex lens housed in an enclosure measuring 73 mm in diameter and 36.1 mm in height, and features a custom printed circuit board (PCB) for programmable LED illumination. The 3D-printed device enclosure is designed to accommodate a silicone bioelectronic device and can be securely attached using a commercially available ostomy skin barrier. In porcine model validation studies, the system successfully captured daily wound progression over periods up to 7 days. The captured images were wirelessly transmitted to a processing unit where DeepMapper, a machine learning algorithm, processed z-stacked images and performed multi-level feature extraction to predict wound healing stages and indicate potential complications such as infection. This imaging system enables automated analysis of wound progression and supports the development of smart wound care platforms for personalized treatment strategies. The integrated design approach demonstrates the feasibility of creating compact, high-resolution imaging systems suitable for clinical wound monitoring applications.

bioengineering↗
bioRxiv2024

Accelerating Wound Healing Through Deep Reinforcement Learning: A Data-Driven Approach to Optimal Treatment

Advancements in bioelectronic sensors and actuators have paved the way for real-time monitoring and control of wound healing progression. Real-time monitoring allows for precise adjustment in treatment strategies that align with an individuals unique biological response. However, due to the complexities of human-drug interactions and a lack of predictive models it is challenging to determine just how one should adjust drug dosage to achieve the desired biological response. This work proposes an adaptive closed-loop control framework that integrates deep learning, optimal control, and reinforcement learning to update treatment strategies in real-time with the goal of accelerating wound closure. The proposed approach eliminates the need for mathematical modeling of complex nonlinear wound healing dynamics. We demonstrate the convergence of the controller via an in silico experimental setup, where the proposed approach successfully accelerates the wound healing process by 17.71%. Finally, we share the experimental setup and results of an in vivo implementation to highlight the translational potential of our work. Our data-driven model estimates a 40% acceleration in wound closure.

systems biology↗
bioRxiv2024

DeepMapper: Attention-Based AutoEncoder for System Identification in Wound Healing and Stage Prediction

Advancements in bioelectronic sensors and actuators have paved the way for real-time monitoring and control of wound healing progression. Real-time monitoring allows for precise adjustment in treatment strategies that align with an individuals unique biological response. However, due to the complexities of human-drug interactions and a lack of predictive models it is challenging to determine just how one should adjust drug dosage to achieve the desired biological response. This work proposes an adaptive closed-loop control framework that integrates deep learning, optimal control, and reinforcement learning to update treatment strategies in real-time with the goal of accelerating wound closure. The proposed approach eliminates the need for mathematical modeling of complex nonlinear wound healing dynamics. We demonstrate the convergence of the controller via an in silico experimental setup, where the proposed approach successfully accelerates the wound healing process by 17.71%. Finally, we share the experimental setup and results of an in vivo implementation to highlight the translational potential of our work. Our data-driven model estimates a 40% acceleration in wound closure.

systems biology↗
bioRxiv2024

Wireless bioelectronic device for wound healing

Wireless bioelectronic actuators have been developed to deliver targeted treatments over multiple days while continuously monitoring delivery, thereby improving wound healing. Specifically, these devices can deliver charged biomolecules such as fluoxetine cations (Flx+) and electric field (EF) in freely moving pigs. Treatments can be controlled and monitored in real time via WiFi, with options for both user-specified delivery rates and durations, as well as automated closed-loop (CL) control. The devices are engineered to handle various failure scenarios that may arise in dynamic, real-world experiments--such as communication or power interruptions--ensuring that valuable experimental data is collected with minimal disruption. The ion pump features eight drug reservoirs and channels arranged around a 20 mm diameter-wound, with a central ground electrode (0 V). When voltages above 0 V are applied to the outer channels, currents flow from the reservoirs and channels into the wound, delivering Flx+ and/or EF depending on the reservoir solution. The device records applied voltages and currents locally to a microSD card at a high sampling rate, while simultaneously transmitting real-time measurements via a local WiFi network to a wound healing algorithm running on a nearby laptop. CL control of current/delivery rate is performed by an onboard microcontroller unit (MCU) and current-source microchips, based on instructions received from the wound healing algorithm. A graphical user interface (GUI) provides intuitive user control and real-time data visualization, with support for multiple devices. In vivo studies over seven days showed that Flx+-treated wounds had a 20% lower M1/M2 macrophage ratio and 41.67% greater re-epithelialization compared to controls (standard-of-care), demonstrating the actuators potential to enhance wound healing.

biochemistry↗
bioRxiv2024

Predicting neuronal firing from calcium imaging using a control theoretic approach

Two-photon calcium imaging has become a powerful tool to explore the functions of neurons and the connectivity of their circuitry. Frequently, fluorescent calcium indicators are taken as a direct measure of neuronal activity. These indicators, however, are slow relative to behavior, obscuring functional relationships between an animals movements and the true neuronal activity. As a consequence, the firing rate of a neuron is a more meaningful metric. Converting calcium imaging data to the firing of a neuron is nontrivial. State of the art methods depend largely on neural networks or non-mechanistic processes, which may yield acceptable correlations between calcium dynamics and the frequency at which a neuron fires, but do not illuminate the underlying chemical exchanges within the neuron or require significant data to be trained on. Leveraging modeling frameworks from chemical reaction networks (CRN) coupled with modern control architectures, a new algorithm is presented based off of fully deterministic ordinary differential equations (ODEs). This framework utilizes model predictive control to challenge state of the art correlation scores while retaining the interpretability that comes inherent with a model. Moreover, these computations can be done in real time, thus enabling online experimentation informed by neuronal firing rates. To demonstrate the use cases of this architecture, it is tested on a ground truth dataset courtesy of the spikefinder challenge. Finally, the predictive power of the methodology put forward here is underscored by demonstrating its ability to aid in the creation of novel indicators utilized in two-photon imaging. Author summaryWe put forward a novel approach to infer when neurons fire as a function of calcium concentration. These calcium recordings are useful for imaging whole populations of neurons, such as those found in the brain, but act only as a proxy for the true underlying spiking occurrences. Moreover, these calcium traces are unfortunately noisy. To uncover the actual firing times we apply modern control architectures to a framework built from chemical reaction equations. The result is accurate, fast enough to provide information in real time, and highly interpretable. More broadly this analysis process aids understanding in contexts where methods of measurement obfuscate the desired ground truth information. Not just capable of inference, prediction is possible thanks to the fully mechanistic formulation provided here. Specifically, we may quantify how indicators, which allow the tracking of calcium, behave in the presence of differing conditions. In more general terms, this framework also enables understanding of the tools used to measure the desired underlying signals.

neuroscience↗
bioRxiv2024

Gene editing of the E3 ligase PIRE1 fine-tunes ROS production for enhanced bacterial disease resistance in tomato

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of Solanum lycopersicum (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using CRISPR-cas9, we generated mutants in two S. lycopersicum PIRE homologs (SlPIRE). SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens Pseudomonas syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and PIRE is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning PIRE as a promising target for improving overall plant health.

plant biology↗
bioRxiv2024

The topography of DNA replication origins in Eukarya: GGN clusters, landmark nucleosomes, CDC6 and G4 structures

BackgroundWe recently identified tandem repeats of GGN triplets as a sequence motif strongly enriched in the DNA replication origins (ORIs) of the Arabidopsis thaliana genome, where it correlates with the ORI strength quantified through short nascent strands sequencing. ResultsHere we show that clusters of four or more GGN repeats separated by short tracts (<7 nucleotides) are present in more than 65% ORIs of six model eukaryotic organisms: Leishmania major, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens. The measured percentages vary with the experimental technique adopted for ORI determination. For all studied techniques and organisms, ORIs are significantly prone to be located within or near strong GGN clusters, although these are neither necessary nor sufficient for ORI activation. Interestingly, for same experimental technique, the association between ORIs and GGN clusters was stronger al later developmental stages for all organisms for which we could perform this comparison. In accordance with a biophysical model of nucleosome positioning, the GGN clusters strongly favor nucleosome occupancy. At the same time, both GGN clusters and ORIs occur frequently within 1kb from nucleosome-depleted regions (NDR). We propose a structural model based on chromatin secondary structure in which the NDR and the well-positioned nucleosome at the GGN are close in space, which may favor functional interactions. We hypothesize that the presence of GGN at ORIs arose in part for promoting the above nucleosome organization Moreover, at least in Arabidopsis, the replication protein CDC6 is very strongly associated with GGN clusters. NMR experiments showed that clusters of at least four GGN can form G-quadruplex (G4) in vitro. Our data support the view that GGN clusters are formed through the interplay of mutational processes (GC skew at ORIs plus triplet expansion), and that similar mutational processes, i.e. AT skew at transcription start sites (TSS) and ORIs, might facilitate the formation of the NDR, thus favoring the evolvability of chromatin. ConclusionsGGN clusters are easily evolvable sequence motifs enriched at ORIs of eukaryotic genomes. They favor G4 secondary structure and a nucleosome organization that may unify the apparent discrepancy between ORIs of higher eukaryotes and yeast.

genomics↗
bioRxiv2023

Development of a robotic cluster for automated and scalable cell therapy manufacturing

The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. Here, we present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic clusters expansion module was demonstrated by activating and expanding human CD8+ T cells. The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies.

bioengineering↗