Biology subjects

Chu, E.

Publications and source records attributed to Chu, E..

12 recordsLinked to original sources

bioRxiv2026

Expression-linked promoter selection (ELiPS) engineers short, strong ubiquitous promoters for gene therapy applications

Adeno-associated virus (AAV)-based gene therapy has made steady progress towards efficient delivery to numerous target cell populations, yet the viruss 5 kb packaging limit remains a challenge for effective and in some cases cell-selective cargo expression. Here, we introduce Expression-Linked Promoter Selection (ELiPS), a high-throughput platform for generating and functionally screening >106 engineered, short promoter variants using an AAV expression platform. ELiPS relies on a Golden Gate cloning method to build random oligomers of selected transcription factor binding sites (TFBSs) upstream of a minimal promoter, GFP, and a unique 3 barcode. As a proof of concept, to engineer short ([~]250 bp), synthetic, ubiquitous promoters, we applied ELiPS to build two libraries composed of TFBSs for ubiquitously expressed transcription factors (TFs) and screened them via AAV-mediated transduction in vitro. This strategy identified promoters with expression surpassing human cytomegalovirus (CMV) and CAG in vitro, and one variant was capable of driving therapeutic expression of B-domain-deleted Factor VIII (BDDFVIII) in vivo at levels comparable to a liver-specific promoter benchmark. ELiPS thus establishes a scalable framework for promoter discovery, enabling the design of compact, ubiquitous or cell-selective expression cassettes that enable further precision and efficacy in AAV-based gene therapies.

bioengineering↗

bioRxiv2026

Metabolic signals regulate resuscitation speed of antibiotic persister bacteria during infection

All living organisms adjust their metabolism in response to environmental changes. Under unfavorable conditions, organisms enter a state of dormancy by halting metabolism, enabling survival. Dormant bacteria become highly tolerant to antibiotics-a phenomenon called persistence. Here, we demonstrate that selective metabolic reprogramming controls the resuscitation speed of persister after antibiotic exposure. Using multi-omics and in silico modeling, we found that dormant bacteria reprogram metabolic pathways to modulate persister awakening. Accumulation of L-serine and reduction of arginine drive rapid resuscitation. L-serine promotes cysteine biosynthesis and motility while reducing energy metabolism to facilitate rapid resuscitation. In contrast, arginine slows regrowth from dormancy by enhancing ethanol-aldehyde and energy metabolism. L-serine and arginine can, respectively, promote or inhibit the regrowth of antibiotic persister cells in macrophages and mouse models, and regulate the awakening speed of Salmonella, E. coli, and methicillin-resistant Staphylococcus aureus (MRSA). These findings suggest new strategies to target chronic bacterial infections. TeaserL-serine speeds and arginine slows the awakening of antibiotic persisters, revealing targets for chronic infection.

microbiology↗

bioRxiv2026

Frontal Brain Injury Reduces Sensitivity to Reward-Predictive Cues and Remodels the Nucleus Accumbens

Traumatic brain injuries (TBIs) are more than mere lesions and generate a persistent secondary pathology. This, combined with functional reorganization of circuits post-injury, may explain the increased risk for psychiatric disorders in patients with TBI. In the current studies, we demonstrate that frontal TBI changed the Pavlovian behavioral response to reinforcer-predicting cues and reduced the motivational value of cues. TBI also chronically impaired decision-making on a gambling-like task with reinforcer-paired cues. To investigate how these changes occur, we evaluated the nucleus accumbens (NAc) core. At a subacute time point (14 days), we confirmed reduced input to the NAc with optogenetics and evaluated electrophysiological and transcriptional changes. TBI increased neuronal excitability and the single nucleus RNA sequencing profile indicated a substantial stress and inflammatory response, but also high indicators of plasticity, particularly in D1- and D2-positive medium spiny neurons. To evaluate how these subacute changes transitioned to chronic NAc dysfunction, we measured immunohistochemical surrogates of activity post-mortem and recorded calcium activity from the NAc after TBI during Pavlovian conditioning. TBI reduced histological markers of activity and reduced cue-evoked calcium activity. Overall, these data indicate that substantial reorganization of the NAc occurs following frontal brain injury. A primary effect of this is to reduce the salience of environmental cues linked to outcomes. The inability to properly process outcomes could contribute to broader psychiatric symptoms after TBI, including impairments in decision-making, behavioral flexibility, and impulsivity but also presents a potential treatment target.

neuroscience↗

bioRxiv2025

Cyclin-dependent kinase 9 inhibitors as oncogene signaling modulators in combination with targeted therapy for the treatment of colorectal cancer

BackgroundColorectal cancer (CRC) is the second deadliest cancer worldwide and new treatment options are urgently needed. Cyclin dependent kinase 9 (CDK9) promotes aberrant RNA transcription in cancer and is a promising target for cancer therapies. MethodsUsing CRC cell lines as well as newly established patient-derived organoid models of CRC, we studied the clinically promising CDK9 inhibitors (AZD4573, BAY1125152/VIP152/enitociclib, and NVP2) to determine their therapeutic potential. We investigated the efficacy and mechanisms of action through cell growth and cytotoxicity assays, RNAseq, immunoblotting, and IHC. ResultsWe found CDK9 inhibitors to be highly potent against CRC, through suppression of proliferation and induction of apoptosis. Our results demonstrated CDK9 inhibitors to be broadly active against a set of CRC models derived from a diverse patient population. Our mechanistic studies showed significant suppression of the Mitogen-active protein kinase (MAPK) signaling pathway due to CDK9 inhibitor treatment, suggesting that CDK9 inhibitor efficacy could be enhanced when combined with MAPK pathway inhibitors. As proof-of-concept, we found that CDK9 inhibitors and MEK inhibitors could be combined to synergistically suppress CRC growth and survival. ConclusionsCDK9 inhibitors show promising activity against patient-derived models of CRC. MAPK signaling is particularly suppressed by CDK9 inhibitors. Combining CDK9 inhibitors and targeted therapy against MAPK signaling pathway may be a viable strategy worthy of further investigation preclinically and clinically. State of translational relevanceNovel therapies for chemotherapy-refractory CRC remain urgently needed. Rapid validation of efficacy in appropriate translational models can provide the necessary rationale to test new therapies in clinical trials. Using newly established patient-derived organoids, we validate the preclinical efficacy of transcriptional CDK9 inhibitors against CRC. This is a therapeutic class that has been scarcely evaluated for efficacy against CRC. We found that CDK9 inhibitors potently inhibit patient-derived organoid models of CRC. Our mechanistic work also reveals that clinically achievable concentrations of CDK9 inhibitor treatment results in suppression of MAPK signaling. As proof-of-concept of the potential actionability of our findings, we demonstrate that CDK9 inhibitors can be combined with MEK inhibitors to synergistically suppress CRC growth. Our study provides new patient-derived CRC models as a resource available to others for therapeutic testing, as well as a roadmap for novel hypotheses about synergistic combination treatments using CDK9 inhibitors.

cancer biology↗

bioRxiv2025

High Throughput Screening Identifies Small Molecules that Synergize with MRTX1133 Against Acquired Resistant KRAS G12D Mutated CRC

Colorectal cancer (CRC) remains a significant clinical challenge, with a 5-year survival rate of 10%. Over half of all CRCs harbor mutations in the KRAS gene, leading to poor response to standard therapy. This underscores the crucial need for novel therapeutics targeting KRAS and overcoming the growing barrier of resistance. To address these critical challenges, we conducted a high-throughput screen to identify small molecules that synergize with KRAS G12D inhibitor MRTX1133 against CRC. Through screening a 2,652 kinase inhibitor library, we discovered that Osimertinib and its analogs strongly synergize with MRTX1133 against both parental and MRTX1133-resistant cells. The top compound from the screen, NT-1, is a chemical analog of Osimertinib. NT-1 strongly synergized with MRTX1133 to suppress EGFR/MAPK signaling and induce apoptosis in an MRTX1133-resistant patient-derived organoid model of CRC. We present novel small molecule combinations with the potential to overcome the limitations of MRTX1133 with direct clinical translational applications. One Sentence SummaryHigh throughput screening and validation in colon cancer PDOs identifies novel KRAS inhibitor combinations with potential for clinical translation.

cancer biology↗

bioRxiv2024

Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and Comorbid Klebsiella pneumoniae Lung Infection in Mice

Traumatic brain injury (TBI) is a leading cause of long-term disability, and infections such as pneumonia represent a common and serious complication for TBI patients in the acute and subacute post-injury period. While the acute effects of infections have been documented, their long-term consequences on neurological and behavioral recovery as well as the potential precipitation of seizures after TBI remain unclear. This study aimed to investigate the chronic effects of Klebsiella pneumoniae infection following TBI, focusing on post-traumatic seizure development and neurobehavioral changes. Using a mouse model, we assessed the long-term effects of TBI and K. pneumoniae infection both in isolation and in combination. We found that, while infection with K. pneumoniae resulted in loss of body weight and increased mortality compared to vehicle-inoculated mice, there was no additional mortality in TBI animals. Further, although TBI alone induced chronic hyperactivity and reduced anxiety-like behaviors, K. pneumoniae lung infection had no lasting effect on these long-term outcomes. Thirdly, while TBI resulted in both spontaneous and evoked seizures long-term post-injury, early post-injury K. pneumoniae infection did not affect late onset seizure susceptibility. Together with recent findings on acute outcomes in this combined insult model of TBI and K. pneumoniae infection, this study suggests that K. pneumoniae does not significantly alter long-term neurobehavioral outcomes or the development of post-traumatic epilepsy. This research highlights the need to further explore the interplay between additional immune insults such as infection that may influence long-term recovery.

neuroscience↗

bioRxiv2024

A post-injury immune challenge with lipopolysaccharide following adult traumatic brain injury alters neuroinflammation and the gut microbiome acutely, but has little effect on chronic outcomes

Patients with a traumatic brain injury (TBI) are susceptible to hospital-acquired infections, presenting a significant challenge to an already-compromised immune system. The consequences and mechanisms by which this dual insult worsens outcomes are poorly understood. This study aimed to explore how a systemic immune stimulus (lipopolysaccharide, LPS) influences outcomes following experimental TBI in young adult mice. Male and female C57Bl/6J mice underwent controlled cortical impact or sham surgery, followed by 1 mg/kg i.p. LPS or saline-vehicle at 4 days post-TBI, before behavioral assessment and tissue collection at 6 h, 24 h, 7 days or 6 months. LPS induced acute sickness behaviors including weight loss, transient hypoactivity, and increased anxiety-like behavior. Early systemic immune activation by LPS was confirmed by increased spleen weight and serum cytokines. In brain tissue, gene expression analysis revealed a time course of inflammatory immune activation in TBI or LPS-treated mice (e.g., IL-1{beta}, IL-6, CCL2, TNF), which was exacerbated in TBI+LPS mice. This group also presented with fecal microbiome dysbiosis at 24 h post-LPS, with reduced bacterial diversity and changes in the relative abundance of key bacterial genera associated with sub-acute neurobehavioral and immune changes. Chronically, TBI induced hyperactivity and cognitive deficits, brain atrophy, and increased seizure susceptibility, similarly in vehicle and LPS-treated groups. Together, findings suggest that an immune challenge with LPS early after TBI, akin to a hospital-acquired infection, alters the acute neuroinflammatory response to injury, but has no lasting effects. Future studies could consider more clinically-relevant models of infection to build upon these findings.

neuroscience↗

bioRxiv2022

Single-Cell Multi-Omic Roadmap of Human Fetal Pancreatic Development

The critical cellular transitions that govern human pancreas development are largely unknown. We performed large-scale single-cell RNA-sequencing (scRNA-Seq) to interrogate human fetal pancreas development from 8-20 weeks post conception. We identified 103 distinct cell types, including four novel endocrine progenitor subtypes displaying unique transcriptional features and differentiation potency. Integration with single-nucleus Assay for Transposase Accessible Chromatin Sequencing (snATAC-Seq) identified candidate regulators of human endocrine cell fate and revealed development-specific regulatory annotation at diabetes risk loci. Comparison of in vitro stem cell-derived and endogenous endocrine cells predicted aberrant genetic programs leading to the generation of off-target cells. Finally, knock-out studies revealed that the gene FEV regulates human endocrine differentiation. This work establishes a roadmap of human pancreatic development, highlights previously unappreciated cellular diversity and lineage dynamics, and provides a blueprint for understanding pancreatic disease and physiology, as well as generating human stem cell-derived islet cells in vitro for regenerative medicine purposes.

developmental biology↗

bioRxiv2018

Combinations of low-level and high-level neural processes can account for distinct patterns of context-dependent choice

Context effects have been explained by either high-level cognitive processes or low-level neural adjustments but not their combination. It is currently unclear how these processes interact to shape individuals responses to context. Here, we used a large cohort of human subjects in experiments involving choice between two or three gambles in order to study the dependence of context effects on neural adaptation and individuals risk attitudes. We found no evidence that neural adaptation on long timescales (~100 trials) contributes to context effects. However, we identified two groups of subjects with distinct patterns of responses to decoys, both of which depended on individuals risk aversion. Subjects in the first group exhibited strong, consistent decoy effects and became more risk averse due to decoy presentation. In contrast, subjects in the second group did not show consistent decoy effects and became more risk seeking. The degree of change in risk aversion due to decoy presentation was positively correlated with the initial degrees of risk aversion. To explain these results and reveal underlying neural mechanisms, we developed a new model that incorporates both low- and high-level processes to fit individuals choice behavior. We found that observed decoy effects can be explained by a combination of adjustments in neural representations and competitive weighting of reward attributes, both of which depend on risk aversion but in opposite directions. Altogether, our results demonstrate how a combination of low- and high-level processes shapes multi-attribute choice, modulates overall risk preference, and explains distinct behavioral phenotypes. Significance statementA large body of experimental work has illustrated that the introduction of a new, and often irrelevant, option can influence preference among the existing options, a phenomenon referred to as context or decoy effects. Although context effects have been explained by high-level cognitive processes--such as comparisons and competitions between attributes--or low-level adjustments of neural representations, it is unclear how these processes interact to shape individuals responses to context. Here, we show that both high-level cognitive processes and low-level neural adjustments shift risk preference during choice between multiple options but in opposite directions. Moreover, we demonstrate that a combination of these processes can account for distinct patterns of context effects in human subjects.

neuroscience↗

bioRxiv2018

Influence of expected reward on perceptual decision making

Perceptual decision making is influenced by reward expected from alternative options or actions, but the underlying neural mechanisms are currently unknown. More specifically, it is debated whether reward effects are mediated through changes in sensory processing and/or later stages of decision making. To address this question, we conducted two experiments in which human subjects made saccades to what they perceived to be the first or second of two visually identical but asynchronously presented targets, while we manipulated expected reward from correct and incorrect responses on each trial. We found that unequal reward caused similar shifts in target selection (reward bias) between the two experiments. Moreover, observed reward biases were independent of the individuals sensitivity to sensory signals. These findings suggest that the observed reward effects were determined heuristically via modulation of decision-making processes instead of sensory processing and thus, are more compatible with response bias rather than perceptual bias. To further explain our findings and uncover plausible neural mechanisms, we simulated our experiments with a cortical network model and tested alternative mechanisms for how reward could exert its influence. We found that our observations are more compatible with reward-dependent input to the output layer of the decision circuit. Together, our results suggest that during a temporal judgment task, the influence of reward information on perceptual choice is more compatible with changing later stages of decision making rather than early sensory processing.

neuroscience↗

bioRxiv2018

Salience-driven value construction for adaptive choice under risk

Decisions we face in real life are inherently risky and can result in one of many possible outcomes. However, most of what we know about choice under risk is based on studies that use options with only two possible outcomes (simple gambles), so it remains unclear how the brain constructs reward values for more complex risky options faced in real life. To address this question, we combined experimental and modeling approaches to examine choice between pairs of simple gambles and pairs of three-outcome gambles in male and female human subjects. We found that subjects evaluated individual outcomes of three-outcome gambles by multiplying functions of reward magnitude and probability. To construct the overall value of each gamble, however, most subjects differentially weighted possible outcomes based on either reward magnitude or probability. These results reveal a novel dissociation between how reward information is processed when evaluating complex gambles: valuation of each outcome is based on an integrated value whereas combination of possible outcomes relies on a single piece of reward information. We show that differential weighting of possible outcomes enabled subjects to make decisions more easily and quickly. Together, these findings reveal a plausible mechanism for how salience, in terms of possible reward magnitude or probability, can influence the construction of subjective values for complex gambles. They also point to separable neural mechanisms for how reward value controls choice and attention in order to allow for more adaptive decision making.

neuroscience↗

bioRxiv2017

Functional Compensation Between Hematopoietic Stem Cells In Vivo

In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about whether and how individual stem cells compensate for the functional deficiencies of other stem cells. Functional compensation between stem cells is critically important during disease progression and treatment. Here, we show how individual hematopoietic stem cells (HSCs) in a mouse heterogeneously compensate for the deficiencies of other HSCs during lymphopoiesis by increasing their clonal expansion at specific differentiation stages. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We have identified the molecular regulators and signaling pathways in HSCs that are involved in this process. Our data demonstrate how stem cells interact with each other to constitute a coordinated network that is robust enough to withstand minor functional disruptions. Exploiting the innate compensation capacity of stem cell networks may improve the prognosis and treatment of many diseases.

systems biology↗