Biology subjects

Truong, A.

Publications and source records attributed to Truong, A..

17 recordsLinked to original sources

bioRxiv2026

SpiraMed: A Stereotactic Helix-based Therapy Delivery system for the Human Brain

Stereotactic needle-based delivery remains the standard for local administration of Advanced Therapy Medicinal Products (ATMPs) to the human brain. ATMP administration typically involves multiple trajectories, presenting cumulative risks and prolonging surgery. Reflux-prone, patchy therapy coverage compromises clinical results. We demonstrate a novel approach, deploying a helical delivery catheter via a single access trajectory per target, referred to as SpiraMed. Helix retraction is synchronised with therapy delivery, enabling comprehensive target coverage in seconds. Helix pitch and diameter can be precisely tailored to patient-specific target volume and vascular anatomy, as part of the preoperative stereotactic surgical planning process. Testing in agarose phantoms, live sheep and cadaveric human brain confirms enhanced therapy delivery volume, delivery speed and target coverage, with reductions in reflux and predicted risk of bleeding complication. SpiraMed represents a new paradigm, promising to help deliver on the transformative potential of cell and gene therapies across the spectrum of human CNS disease.

bioengineering↗

bioRxiv2026

Legacy Effects of Early β-Adrenergic Stimulation Program Adipose Plasticity and Confer Metabolic Resilience in Obesity

Pathologic white adipose tissue (WAT) remodeling, characterized by fibrosis, inflammation, and adipocyte dysfunction, is a hallmark and driver of metabolic disease in obesity1. Here, we show that legacy effects of early physiological or pharmacological interventions driving adaptive adipose remodeling can mitigate maladaptive WAT remodeling and metabolic dysfunction when developing obesity later in life. Cold exposure or {beta}3-adrenergic receptor ({beta}3AR) agonism (CL316,243) induced thermogenic remodeling of WAT in male mice. After a prolonged recovery at room temperature, trained epididymal WAT reverted to an energy-storing state but retained a population of adipocytes resembling "metabolically flexible" visceral adipocytes found in human metabolically healthy obesity. The legacy of the antecedent treatment conferred lasting protection against glucose intolerance when later developing high fat diet (HFD)-induced obesity, with insulin sensitivity persisting for at least 20 weeks of overnutrition. This metabolic resilience was accompanied by healthy epididymal WAT expansion with reduced fibrosis and inflammation. Our findings demonstrate that short-term interventions, without genetic manipulation, can "train" adipose tissue, enhancing its long-term plasticity and conferring durable protection against future obesity-associated insulin resistance.

physiology↗

bioRxiv2026

Infection Tunes the Dynamics of Adenoviral E1A Disordered Regions

Intrinsically Disordered Proteins and protein regions (IDPs) are abundant in many viral proteomes and play diverse roles in the viral infectious cycles. The adenovirus Early Protein 1A (E1A) is one such viral IDP. E1A acts as a molecular hub that regulates viral infection by mediating interactions between viral and multiple host proteins. Like other IDPs, E1A exists in a flexible ensemble of conformations. Despite a demonstrated link between ensemble structure and function in E1A, no real-time measurement of its ensemble has been performed. Here, we use live cell FRET microscopy to measure the local ensemble structure of E1A in human cells, both in healthy cells and in cells infected with adenovirus. We found specific disordered regions undergo significant changes to their ensemble in infected cells. Furthermore, infection also alters the propensity of these regions to partition between the cytoplasm and nucleus, a hallmark of E1A function during infection. Our results showcase that the structural ensembles of viral IDPs are responsive to infection, and suggest that these may play a role in regulating infection progression. SignificanceIntegral to many viral proteomes are intrinsically disordered proteins and protein regions (IDPs), which target and rewire cellular pathways to ensure infection progression. During this process, the physical and chemical composition of the cell changes dramatically: metabolism is rewired, viral proteins are produced en masse, and as a result, the chemical composition of the host proteome is significantly altered. IDPs are known to be structurally sensitive to even mild changes in their environment, and their structural changes can result in a change to function. Here, using live cell FRET microscopy, we show that the structure and spatial localization of an adenovirus IDP, E1A, is altered in cells infected by the virus. Beyond a possible functional role for structural sensitivity in viral IDP function, our findings suggest that host IDPs may also be structurally altered by infection, with downstream functional consequences.

biophysics↗

bioRxiv2026

Direct Binding of miR-155 to FLT3 Regulates Key Cellular Functions in Acute Myeloid Leukemia

Mutation in FLT3 protein is one of the most common mutations in acute myeloid leukemia (AML). Most patients with FLT3-ITD mutation detected at diagnosis or acquired during treatment display poor prognosis and resistance to tyrosine kinase inhibitors or chemotherapy. Existing clinical and pre-clinical data implicate miR-155 in the carcinogenesis of hematological cancers, including FLT3-assocaited AML. However, the role of miR-155 in regulating FLT3-ITD mutation remains elusive. In this study, we have applied loss-of-function studies using wild-type and mutated leukemic cell line models to validate the functional effect of miR-155 inhibition in leukemic cells. Our bioinformatics analysis indicates that FLT3 has a binding site for miR-155 which makes it a direct target of miR-155. Specific targeting of miR-155 by miR-155 inhibitor induced cell apoptosis and reduced FLT3-ITD-mediated cell proliferation and survival. Our data suggests that miR-155 could be a potential therapeutic target for FLT3-associated AML.

cancer biology↗

bioRxiv2025

A Co-Evolutionary Multi-Objective Framework for Joint Topology and Parameter Discovery in Gene Regulatory Networks

Inferring gene regulatory networks (GRN) poses a complex discrete-continuous, multi-objective optimization problem that requires the simultaneous discovery of both network topology and kinetic parameters. Current methods are often limited because they address these two challenges separately. We propose a novel co-evolutionary multi-objective framework that evolves topology and parameter populations in tandem to optimize three competing objectives: dynamic fidelity, network simplicity, and robustness. This algorithm utilizes biologically-inspired operators and is grounded in theoretical results on Pareto optimality for mixed discrete-continuous spaces. In computational experiments on synthetic benchmarks, our approach showed superior performance, attaining the lowest mean squared error in three of four test cases against single-objective and topology-fixed methods. The framework successfully discovers multiple functionally equivalent network configurations, a finding that highlights the degeneracy inherent in biological systems and offers practical insights for synthetic biology, where function is often prioritized over precise structural accuracy.

systems biology↗

bioRxiv2025

A Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP) for Extracellular Electron Transfer

Electroactive microbes can be used as components in electrical devices to leverage their unique behavior for biotechnology, but they remain challenging to engineer because the bioelectrochemical systems (BES) used for characterization are low-throughput. To overcome this challenge, we describe the development of the Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP), which allows for samples to be arrayed and characterized in individually addressable microwells. This device reliably reports on the current generated by electroactive bacteria on the minute time scale, decreasing the time for data acquisition by several orders of magnitude compared to conventional BES. Also, this device increased the throughput of screening engineered biological components in cells, quickly identifying mutants of the membrane protein wire MtrA in Shewanella oneidensis that retain the ability to support extracellular electron transfer (EET). BiCASP is expected to enable the design of new components for bioelectronics by supporting directed evolution of electroactive proteins. The bigger pictureDevices that interface microbes and materials, known as bioelectronics, can be used to sense environmental chemicals in real time, generate energy from sugars, and synthesize chemicals. While these devices leverage the unique capabilities of living systems as components in devices, such as their ability to convert chemical information in the environment into electrical information at the cell surface, it remains challenging to engineer these cellular components and their biomolecules for new applications, largely because commercially available bioelectrochemical systems for monitoring current generated by electroactive microbes are costly and require large culture volumes, needs continuous monitoring for days to obtain stable signals, and multichannel potentiostats to monitor multiple microbes in parallel. To overcome these challenges, we created the Bioelectrochemical Crossbar Architecture Screening Platform or BiCASP that is easy to fabricate, enables parallel analysis of microbial samples in flexible arrayed formats, and yields a stable signal on the minute time scale. This device is expected to enable the application of combinatorial protein engineering methods, such as directed evolution, to proteins that control microbial current production, by allowing for fast screening of cells expressing protein mutant libraries. As a proof-of-concept, we demonstrate that this device can screen for cells that express mutants of decaheme cytochromes that retain the ability to electrically connect cells to electrodes. This device will simplify the engineering of cells and proteins that function as electrical switches as well as the diversification of bioelectronic devices for real-time sensing of chemicals in the environment. Furthermore, BiCASP is promising as a high-throughput screening (HTS) platform, enabling rapid, parallel analysis of cellular and molecular interactions of diverse biological systems through label-free electrochemical methods. Such capabilities could transform drug discovery, personalized medicine, and functional genomics, supporting systematic genetic and chemical screens even at single-cell resolution. HighlightsO_LIA high-throughput screening platform with individual addressability C_LIO_LIA device with a flexible crossbar architecture that simplifies current analysis C_LIO_LIReproducible detection of real-time cellular current on the minute time scale C_LIO_LIThe device can be used to screen a library for cells with functional protein wires C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/663982v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@c451d1org.highwire.dtl.DTLVardef@d7a50corg.highwire.dtl.DTLVardef@194e986org.highwire.dtl.DTLVardef@16f7af9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Sensitizing Immune-Refractory Ovarian Tumors via p53 Mutation-Tailored Immunotherapy

High-grade serous ovarian cancer demonstrates limited responsiveness to immune checkpoint inhibitors, owing in part to immunosuppressive environments shaped by nearly universal p53 aberrations. Utilizing an immunocompetent mouse model and individual p53 mutations, we identified a dependence of the p53-R270H mutation (equivalent of human R273H) on regulatory T cells (Tregs) and the PD-1/PD-L1 axis. Analysis of patient datasets associated R273H with elevated levels of two p53 targets, PD-L1 and amphiregulin (AREG), a Tregs growth factor. In contrast to p53-R172H tumors, where there was limited activity, dual antibody therapy targeting AREG and PD-L1 selectively and effectively inhibited R270H tumors. This involved polarization toward M1 macrophages, infiltration of CD8+ T cells, diminished Ly6G+ neutrophils and downregulation of interleukin-4. In patient-derived R273C organoids, the combination treatment reduced the CD4/CD8 ratio. This study is the first to establish a mutation-tailored therapeutic approach that leverages the capacity of p53 to modulate immunosuppressive mechanisms.

cancer biology↗

bioRxiv2024

Towards optimizing diversifying base editors for high-throughput studies of single-nucleotide variants

Determining the phenotypic effects of single nucleotide variants is critical for understanding the genome and interpreting clinical sequencing results. Base editors, including diversifying base editors that create C>N mutations, are potent tools for installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across [~]200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, [~]4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA to create G>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. The DivA-BE editors efficiently diversified their target sites, an ideal characteristic for discovering functional variants. These and other findings provide a comprehensive analysis of how design features influence the activity of several popular base editors.

genomics↗

bioRxiv2024

Regulating ferredoxin electron transfer using nanobody and antigen interactions

Fission and fusion can be used to generate new regulatory functions in proteins. This approach has been used to create ferredoxins (Fd) whose cellular electron transfer is dependent upon small molecule binding. To investigate whether Fd fragments can be used to monitor macromolecular binding reactions, we investigated the effects of fusing fragments of Mastigocladus laminosus Fd to nanobodies and their protein antigens. When Fd fragments arising from fission were fused to green fluorescent protein (GFP) and three different anti-GFP nanobodies, split proteins were identified that supported Fd-mediated electron transfer from Fd-NADP reductase (FNR) to sulfite reductase (SIR) in Escherichia coli. However, the order of nanobody and antigen fusion to the Fd fragments affected cellular electron transfer. Insertion of these anti-GFP nanobodies within Fd had differing effects on electron transfer. One domain-insertion variant was unable to support cellular electron transfer unless it was coexpressed with GFP, while others supported electron transfer in the absence of GFP. These findings show how Fds can be engineered so that their electron transfer is regulated by macromolecules, and they reveal the importance of exploring different nanobody homologs and fusion strategies when engineering biomolecular switches.

synthetic biology↗

bioRxiv2024

Protein surface chemistry encodes an adaptive resistance to desiccation

Cellular desiccation - the loss of nearly all water from the cell - is a recurring stress in an increasing number of ecosystems that can drive protein unfolding and aggregation. For cells to survive, at least some of the proteome must resume function upon rehydration. Which proteins tolerate desiccation, and the molecular determinants that underlie this tolerance, are largely unknown. Here, we apply quantitative and structural proteomic mass spectrometry to show that certain proteins possess an innate capacity to tolerate rehydration following extreme water loss. Structural analysis points to protein surface chemistry as a key determinant for desiccation tolerance, which we test by showing that rational surface mutants can convert a desiccation sensitive protein into a tolerant one. Desiccation tolerance also has strong overlap with cellular function, with highly tolerant proteins responsible for production of small molecule building blocks, and intolerant proteins involved in energy-consuming processes such as ribosome biogenesis. As a result, the rehydrated proteome is preferentially enriched with metabolite and small molecule producers and depleted of some of the cells heaviest consumers. We propose this functional bias enables cells to kickstart their metabolism and promote cell survival following desiccation and rehydration. TeaserProteins can resist extreme dryness by tuning the amino acids on their surfaces.

biophysics↗

bioRxiv2023

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

neuroscience↗

bioRxiv2023

Laboratory evolution identifies elongated flavodoxins that support electron transfer to sulfite reductases

Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. While dozens of Fld-partner oxidoreductases have been discovered, these only represent a subset of the oxidoreductases that couple with ferredoxin (Fd) protein electron carriers. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a Fd, we evaluated the ability of Flds to transfer electrons from a Fd-NADP reductase (FNR) to a Fd-dependent SIR using growth complementation of a microbe with a sulfur metabolism defect. We show that Flds from cyanobacteria complement the growth of this microbe when coexpressed with an FNR and an SIR that evolved to couple with a plant Fd. To better understand the interaction of Fld with these partner oxidoreductases, we evaluated the effect of peptide insertion on Fld-mediated electron transfer. We observe a high insertion sensitivity within regions predicted to be proximal to the cofactor and partner binding sites and a high insertion tolerance within the loop that is used to differentiate short- and long-chain flavodoxins. These results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of peptide-insertion tolerance is influenced by interactions with oxidoreductase partners in electron transfer pathways.

biochemistry↗

bioRxiv2021

Functional analysis of a common BAG3 allele associated with protection from heart failure

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism for such protection remains elusive. One of the variants in this allele block is coding, changing cysteine to arginine at position 151 of BAG3 (rs2234962-BAG3C151R). Here, we use induced pluripotent stem cells (iPSC) to test if the BAG3C151R variant alters protein and cellular function in human cardiac myocytes. Quantitative protein interaction network analysis identified specific changes in BAG3C151R protein interaction partners in cardiomyocytes but not in iPSCs or an immortalized cell line. Knockdown of BAG3 interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151R variant have improved response to proteotoxic stress in an allele dose-dependent manner. This study suggests that the BAG3C151R variant increases cardiomyocyte protection from stress by enhancing the recruitment of factors critical to the maintenance of myofibril integrity, hinting that this variant could be responsible for the cardioprotective effect of the haplotype block. By revealing specific changes in preferential binding partners of the BAG3C151R protein variant, we also identify potential targets for the development of novel cardioprotective therapies.

genetics↗

bioRxiv2021

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein aggregates within processing bodies

RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.

cell biology↗

bioRxiv2020

SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in SMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1s role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.

cancer biology↗

bioRxiv2019

Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

Genetically-engineered immune cell therapies have been in development for decades1-3 and recently have proven effective to treat some types of cancer4. CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies5,6. Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo. Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

immunology↗

bioRxiv2019

Identification and mitigation of pervasive off-target activity in CRISPR-Cas9 screens for essential non-coding elements

Pooled CRISPR-Cas9 screens have recently emerged as a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we conducted a genome-scale screen for essential CTCF loop anchors in the K562 leukemia cell line. Surprisingly, the primary drivers of signal in this screen were single guide RNAs (sgRNAs) with low specificity scores. After removing these guides, we found that there were no CTCF loop anchors critical for cell growth. We also observed this effect in an independent screen fine-mapping the core motifs in enhancers of the GATA1 gene. We then conducted screens in parallel with CRISPRi and CRISPRa, which do not induce DNA damage, and found that an unexpected and distinct set of off-targets also caused strong confounding growth effects with these epigenome-editing platforms. Promisingly, strict filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and allowed for the identification of essential enhancers, which we validated extensively. Together, our results show off-target activity can severely limit identification of essential functional motifs by active Cas9, while strictly filtered CRISPRi screens can be reliably used for assaying larger regulatory elements.

genomics↗