Biology subjects

Bae, S.

Publications and source records attributed to Bae, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Characterization of Microbubble Cavitation in Theranostic Ultrasound-mediated Blood-Brain Barrier Opening and Gene Delivery

RationaleThe characterization of microbubble activity has proven critical in assessing the safety and efficacy of ultrasound-mediated blood-brain barrier (BBB) opening and drug and gene delivery. In this study, we build upon our previous work on theranostic ultrasound (ThUS)-mediated BBB opening (ThUS-BBBO) and conduct for the first time a comprehensive characterization of the role of microbubble cavitation in ThUS-BBBO, as well as its impact on gene delivery with adeno-associated viruses (AAV). MethodsA repurposed imaging phased array was used throughout the study to generate focused transmits and record microbubble activity through high-resolution power cavitation imaging (PCI). The cavitation of microbubbles under ThUS pulses was first characterized in flow phantom using pulse lengths ranging from 1.5 to 20 cycles and under varying microbubble flow rates using a separate single-element transducer a passive cavitation detector (PCD). A comprehensive in vivo study in mice was then conducted to characterize the in vivo microbubble activity under ThUS and correlate the resulting cavitation with AAV-mediated transgene delivery and expression. The transcranial microbubble activity was first detected in two mice using a PCD, to confirm the findings of the flow phantom study. Next, three mouse studies were conducted to evaluate the relationship between cavitation and AAV delivery; one with three different microbubble size distributions using polydisperse and size-isolated microbubbles, one with variable burst length and burst repetition frequency, and one with different AAV serotypes and injection doses. Electronic beam steering was used to induce bilateral BBB opening with 1.5 cycle on the left and 10 cycles on the right hemisphere. Cavitation dose was correlated with BBB opening volume, AAV transgene expression was evaluated with immunofluorescence staining and histological safety was assessed with T2* imaging and Hematoxylin and Eosin staining. ResultsFrequency domain analysis in the phantoms revealed a broadband-cavitation dominance at the shorter pulse lengths, while harmonic cavitation components are significantly increased for longer pulses. The PCD was better at detecting higher frequency harmonics, while the signal received by the theranostic array was more broadband dominated. Analysis of signals in the time domain showed that the longer pulses induce higher microbubble collapse compared to short pulses. In the transcranial in vivo experiments, the PCD was able to detect increased harmonic cavitation for 10-cycle pulses. The microbubble study showed that 3-5 m microbubbles resulted in the largest cavitation doses, BBBO volumes and AAV transgene expression compared to the smaller microbubble sizes. The burst sequence study revealed that the sequences with shorter bursts and faster burst repetition frequencies induce larger BBBO volumes and AAV transduction due to faster microbubble replenishment in the focal volume. Increased erythrocyte extravasation was observed on the hemisphere sonicated with 10-cycle pulses. Transgene expression was also increased with injection dose, without notable side effects during the three-week survival period. Finally, AAV9 was shown to be the serotype with the highest transduction efficiency compared to AAV2 and AAV5 at the same injected dose. ConclusionsThis is the first comprehensive study into the microbubble cavitation under theranostic ultrasound. The phantom and in vivo studies show that the mechanism of ThUS-BBBO is mainly transient cavitation dominant, as microbubble collapse increases with pulse length despite the increased harmonic frequency response. Increased cavitation dose resulted in larger BBBO volumes and transgene expression in vivo. While ThUS induced microhemorrhage for most of the studied conditions, it did not have an impact on the survival and behavior of the mice.

bioengineering↗

bioRxiv2024

Glycan analysis probes inspired by human lectins for investigating host-microbe crosstalk

Human lectins are critical carbohydrate-binding proteins that recognize diverse glycoconjugates from microorganisms and can play a key role in host-microbe interactions. Despite their importance in immune recognition and pathogen binding, the specific glycan ligands and functions of many human lectins remain poorly understood. Using previous proof-of-concept studies on selected lectins as the foundation for this work, we present ten additional glycan analysis probes (GAPs) from a diverse set of human soluble lectins, offering robust tools to investigate glycan-mediated interactions. We describe a protein engineering platform that enables scalable production of GAPs that maintain native-like conformations and oligomerization states, equipped with functional reporter tags for targeted glycan profiling. We demonstrate that the soluble GAP reagents can be used in various applications, including glycan array analysis, mucin- binding assays, tissue staining, and microbe binding in complex populations. These capabilities make GAPs valuable for dissecting interactions relevant to understanding host responses to microbes. The tools can be used to distinguish microbial from mammalian glycans, which is crucial for understanding the cross-target interactions of lectins in a physiological environment where both glycan types exist. GAPs have potential as diagnostic and prognostic tools for detecting glycan alterations in chronic diseases, microbial dysbiosis, and immune-related conditions.

biochemistry↗

bioRxiv2024

Response of Serum-isolated Extracellular Vesicles to Focused Ultrasound Blood-Brain Barrier Opening

The blood-brain barrier (BBB) limits drug delivery to the brain and the movement of neurological biomarkers between the brain and blood. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) noninvasively opens the BBB, allowing increased molecular transport to and from the brain parenchyma. Despite being initially developed as a drug delivery method, FUS-BBBO has shown promise both as a neuroimmunotherapeutic modality, and as a way of improving neurological disease diagnosis via amplification of disease biomarker circulation. Recently, the role of extracellular vesicles (EVs) in modulating the neuroimmune system and in improving biomarker detection has sparked research interest. However, despite their potential role in modulating FUS-BBBO-induced neuroimmunotherapy and their ability to improve biomarker specificity after treatment, the EV response to FUS-BBBO had not been extensively characterized prior to this study. In this study, we investigated the effect of FUS-BBBO on EV concentration and content in the serum of mice and Alzheimers Disease (AD) patients. We observed a 164% increase in murine EV concentration one hour after treatment, as well as an increase in EV RNA associated with FUS-BBBO neuroimmunotherapy. Patient EV concentration also increased one hour after treatment and was dependent on the volume of BBB opening three days post-treatment. Furthermore, EV isolation was found to significantly enhance the amplification of AD biomarker detection by FUS-BBBO. Overall, we present the first evidence of altered murine and AD patient EV concentration and content in response to FUS-BBBO, providing evidence of EVs role within FUS-BBBO neuroimmunotherapy as well as their utility in improving FUS-BBBO biomarker amplification.

bioengineering↗

bioRxiv2024

PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation

Hearing loss is one of the most prevalent sensory disorders, but no commercial biological treatments are currently available. Here, we identified an East Asia-specific founder mutation, the homozygous c.220C>T mutation in MPZL2, that contributes to a significant proportion of hereditary deafness cases in our cohort study. We found that the disease-causing mutation could be targetable by adenine base editors (ABEs) that enable A{middle dot}T-to-G{middle dot}C base corrections without DNA double-strand breaks. To demonstrate this, we developed a humanized mouse model (hMPZL2Q74X/Q74X) that recapitulates human MPZL2 deafness and leads to progressive hearing loss. A PAM-flexible ABE variant with reduced bystander and off-target effects (ABE8eWQ-SpRY:sgRNA3) was packaged in dual adeno-associated viruses (AAVs) and injected into the inner ear of hMPZL2Q74X/Q74X mice and effectively corrected the mutation. This treatment significantly restored hearing function, improved inner ear structural integrity, and reversed altered gene expression. Base editing may hold therapeutic potential for hereditary deafness, including most cases of MPZL2 deafness.

genetics↗

bioRxiv2024

Hijacking and recording intracellular RNAs in human cells using eukaryotic reprogrammed tracrRNAs

Recording intracellular RNA expression events is critical for understanding cell states and biological processes, but no method without responsive promoters is available in mammalian cells. Here, we determined the sequence formula of eukaryotic reprogrammed tracrRNA, named euRptr, which can hijack intracellular RNAs to make a complex with CRISPR-associated tools. By leveraging the programmability of euRptr, we devised the system "CHEETAH" to directly capture and record intracellular RNAs in human cells. We validated that CHEETAH induces self-editing by hijacking endogenous mRNAs, that CHEETAH selectively records different viral RNA fragments on a DNA reporter using base editors with a shared-euRptr, and that CHEETAH enables multiplexed, time-resolved recording of viral RNAs on a DNA Tape using prime editors, providing a new avenue for investigating intracellular RNA in eukaryotes.

genetics↗

bioRxiv2024

Bystander base editing interferes with visual function restoration in Leber congenital amaurosis

Base editors (BEs) have emerged as a powerful tool for gene correction with high activity. However, bystander base editing, a byproduct of BEs, presents challenges for precise editing. Here, we investigated the effects of bystander edits on phenotypic restoration in the context of Leber congenital amaurosis (LCA), a hereditary retinal disorder, as a therapeutic model. We observed that in rd12 of LCA model mice, the highest editing activity version of an adenine base editors (ABEs), ABE8e, generated substantial bystander editing, resulting in missense mutations despite RPE65 expression, preventing restoration of visual function. Through AlphaFold-based mutational scanning and molecular dynamics simulations, we identified that the ABE8e-driven L43P mutation disrupts RPE65 structure and function. Our findings underscore the need for more stringent requirements in developing precise BEs for future clinical applications.

genetics↗

bioRxiv2024

ADT-1004: A First-in-Class, Orally Bioavailable Selective pan-RAS Inhibitor for Pancreatic Ductal Adenocarcinoma

Here, we evaluated in vivo antitumor activity, target engagement, selectivity, and tumor specificity of ADT-1004, an orally bioavailable prodrug of ADT-007 having highly potent and selective pan-RAS inhibitory activity. ADT-1004 strongly blocked tumor growth and RAS activation in mouse PDAC models without discernable toxicity. As evidence of target engagement and tumor specificity, ADT-1004 inhibited activated RAS and ERK phosphorylation in PDAC tumors at dosages approximately 10-fold below the maximum tolerated dose and without discernable toxicity. ADT-1004 inhibited ERK phosphorylation in PDAC tumors. In addition, ADT-1004 blocked tumor growth and ERK phosphorylation in PDX PDAC models with KRASG12D, KRASG12V, KRASG12C, or KRASG13Q mutations. ADT-1004 treatment increased CD4+ and CD8+ T cells in the TME consistent with exhaustion and increased MHCII+ M1 macrophage and dendritic cells. ADT-1004 demonstrated superior efficacy over sotorasib and adagrasib in tumor models involving human PDAC cells resistant to these KRASG12C inhibitors. As evidence of selectivity for tumors from PDAC cells with mutant KRAS, ADT-1004 did not impact the growth of tumors from RASWT PDAC cells. Displaying broad antitumor activity in multiple mouse models of PDAC, along with target engagement and selectivity at dosages that were well tolerated, ADT-1004 warrants further development. SignificanceADT-1004 displayed robust antitumor activity in aggressive and clinically relevant PDAC models with unique tumor specificity to block RAS activation and MAPK signaling in RAS mutant cells. As a pan-RAS inhibitor, ADT-1004 has broad activity and potential efficacy advantages over allele-specific KRAS inhibitors by averting resistance. These findings support clinical trials of ADT-1004 for KRAS mutant PDAC.

cancer biology↗

bioRxiv2024

Intracortical injection of immune checkpoint inhibitor promotes monocyte/macrophage infiltration and restores microglial function and neuronal activity in an AD mouse model

Alzheimer's disease (AD) involves complex neuroimmune interactions. However, the role of immune checkpoint pathways in regulating the glial and neuronal functions of the AD brain remains unclear. This study aims to investigate how the brain-specific modulation of the PD-1/PD-L1 axis affects glial and neuronal function in an AD mouse model, which is characterized by the significant upregulation of microglial PD-1 and astrocytic PD-L1. A single intracortical administration of anti-PD-L1 antibody reshaped the glial microenvironment, which was accompanied by the restoration of impaired microglial process convergence. Astrocyte-specific Pd-l1 knockdown using the pSico system validated the essential role of astrocytic PD-L1 in enhancing microglial responses. Notably, the blockade of the PD-1/PD-L1 signaling pathway increased the microglial P2RY12 expression, a key marker of microglial homeostasis, which likely contributed to the restoration of neuronal hyperactivity. Collectively, these findings confirmed the potential of targeting the astrocyte-microglia PD-1/PD-L1 axis to mitigate AD pathology.

neuroscience↗

bioRxiv2024

AI-generated small binder improves prime editing

The prime editing 2 (PE2) system comprises a nickase Cas9 fused to a reverse transcriptase utilizing a prime editing guide RNA (pegRNA) to introduce desired mutations at target genomic sites. However, the PE efficiency is limited by mismatch repair (MMR) that excises the DNA strand containing desired edits. Thus, inhibiting key components of MMR complex through transient expression of a dominant negative MLH1 (MLH1dn) exhibited approximately 7.7-fold increase in PE efficiency over PE2, generating PE4. Herein, by utilizing a generative artificial intelligence (AI) technologies, RFdiffusion and AlphaFold 3, we ultimately generated a de novo MLH1 small binder (named MLH1-SB), which bind to the dimeric interface of MLH1 and PMS2 to disrupt the formation of key MMR components. MLH1-SBs small size (82 amino acids) allowed it to be integrated into pre-existing PE architectures via the 2A system, creating a novel PE-SB platform. Resultantly, by incorporating MLH1-SB into PE7, we have developed an improved PE architecture called PE7-SB, which demonstrates the highest PE efficiency to date (29.4-fold over PE2 and 2.4-fold over PE7 in HeLa cells), providing an insight that generative AI technologies will boost up the improvement of genome editing tools.

bioengineering↗

bioRxiv2024

Short-term memory errors are strongly associated with a drift in neural activity in the posterior parietal cortex

Understanding the neural mechanisms behind short-term memory (STM) errors is crucial for unraveling cognitive processes and addressing deficits associated with neuropsychiatric disorders. This study examines whether STM errors arise from misrepresentation of sensory information or decay in these representations over time. Using 2-photon calcium imaging in the posterior parietal cortex (PPC) of mice performing a delayed match-to-sample task, we identified a subset of PPC neurons exhibiting both directional and temporal selectivity. Contrary to the hypothesis that STM errors primarily stem from mis-encoding during the sample phase, our findings reveal that these errors are more strongly associated with a drift in neural activity during the delay period. This drift leads to a gradual divergence away from the correct representation, ultimately leading to incorrect behavioral responses. These results emphasize the importance of maintaining stable neural representations in the PPC for accurate STM. Furthermore, they highlight the potential for therapeutic interventions aimed at stabilizing PPC activity during delay periods as a strategy for mitigating cognitive impairments in conditions like schizophrenia.

animal behavior and cognition↗

bioRxiv2024

Spatial transcriptomics unveils landscape of resistance to concurrent chemo-radiotherapy in hypopharyngeal squamous cell carcinoma: the role of SPP1+ macrophages

Hypopharyngeal squamous cell carcinoma (SCC) is a highly aggressive cancer with a poor prognosis, particularly in advanced stages where concurrent chemoradiotherapy (CCRT) is used for treatment. However, resistance to CCRT poses a significant challenge, often leading to treatment failure and disease progression. This study explores the tumor microenvironment (TME) of hypopharyngeal SCC to understand the molecular mechanisms underlying CCRT resistance. Using spatial transcriptomics (ST), we analyzed tissue samples from patients with locally advanced hypopharyngeal SCC, distinguishing between those who were CCRT-resistant and those who were CCRT-naive. The analysis revealed six distinct cellular clusters within the TME, including a prominent epithelio-immune cellular area in CCRT-resistant tissues. SPP1 was identified as a key gene with significantly higher expression in CCRT-resistant samples, specifically within macrophages. Further investigation showed that SPP1+ macrophages interacted with malignant epithelial cells through SPP1-CD44 and SPP1-ITGB1 ligand-receptor pairs. These interactions were primarily localized in the peri-tumoral and intra-tumoral regions, highlighting their potential role in driving CCRT resistance. Our findings suggest that SPP1+ macrophages contribute to the resistant phenotype in hypopharyngeal SCC by modulating the TME and interacting with cancer cells. Understanding these interactions offers valuable insights into the mechanisms of CCRT resistance and may inform the development of targeted therapies to improve patient outcomes.

cancer biology↗

bioRxiv2024

Cellular dsRNA interactome reveals the regulatory map of exogenous RNA sensing

RNA-binding proteins (RBPs) provide a critical post-transcriptional regulatory layer in determining RNA fate. Currently, UV crosslinking followed by oligo-dT pull-down is the golden standard in identifying the RBP repertoire, but such method is ineffective in capturing RBPs that recognize double-stranded RNAs (dsRNAs). Here, we utilize anti-dsRNA antibody immunoprecipitation followed by quantitative mass spectrometry to comprehensively identify RBPs bound to cellular dsRNAs without any external stimulus. Notably, our dsRNA interactome contains proteins involved in sensing N6-methyladenosine RNAs and stress granule components. We further perform targeted CRISPR-Cas9 knockout functional screening and discover proteins that can regulate the interferon (IFN) response during exogenous RNA sensing. Interestingly, depleting dsRBPs mostly attenuate the IFN response, and they act as antiviral factors during human {beta}-coronavirus HCoV-OC43 infection. Our dsRNA interactome capture provides an unbiased and comprehensive characterization of putative dsRBPs and will facilitate our understanding of dsRNA sensing in both physiological and pathological contexts.

molecular biology↗

bioRxiv2024

CELLama: Foundation Model for Single Cell and Spatial Transcriptomics by Cell Embedding Leveraging Language Model Abilities

Large-scale single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) have transformed biomedical research into a data-driven field, enabling the creation of comprehensive data atlases. These methodologies facilitate detailed understanding of biology and pathophysiology, aiding in the discovery of new therapeutic targets. However, the complexity and sheer volume of data from these technologies present analytical challenges, particularly in robust cell typing, integration and understanding complex spatial relationships of cells. To address these challenges, we developed CELLama (Cell Embedding Leverage Language Model Abilities), a framework that leverage language model to transform cell data into sentences that encapsulate gene expressions and metadata, enabling universal cellular data embedding for various analysis. CELLama, serving as a foundation model, supports flexible applications ranging from cell typing to the analysis of spatial contexts, independently of manual reference data selection or intricate dataset-specific analytical workflows. Our results demonstrate that CELLama has significant potential to transform cellular analysis in various contexts, from determining cell types across multi-tissue atlases and their interactions to unraveling intricate tissue dynamics.

bioinformatics↗

bioRxiv2024

Parent attention-orienting behavior is associated with neural entropy in infancy

Parents play a significant role in directing infants attention to environmental stimuli via joint attention. We hypothesized that infants whose parents provide more bids for joint attention will display a more complex neural response when viewing social scenes. Sixty-one 8-month-old infants underwent electroencephalography (EEG) while viewing videos of joint-and parallel-play and participated in a parent-infant free play interaction. EEG data was analyzed using multiscale entropy, which quantifies moment-to-moment neural variability. Free play interactions were coded for parent alternating gaze, a behavioral mechanism for directing attention to environmental cues. We found a significant positive association between parent alternating gaze and neural entropy in frontal and central brain regions. These results suggest a relationship between parent behavior and infant neural mechanisms that regulate social attention, underlying the importance of parent cues in the formation of neural networks in infancy.

neuroscience↗

bioRxiv2024

Transcranial Functional Ultrasound Imaging Detects Focused Ultrasound Neuromodulation Induced Hemodynamic Changes in Mouse and Nonhuman Primate Brains In Vivo

BackgroundFocused ultrasound (FUS) is an emerging non-invasive technique for neuromodulation in the central nervous system (CNS). Functional ultrasound imaging (fUSI) leverages ultrafast Power Doppler Imaging (PDI) to detect changes in cerebral blood volume (CBV), which correlate well with neuronal activity and thus hold promise to monitor brain responses to FUS. ObjectiveInvestigate the immediate and short-term effects of transcranial FUS neuromodulation in the brain with fUSI by characterizing hemodynamic responses. MethodsWe designed a setup that aligns a FUS transducer with a linear array to allow immediate subsequent monitoring of the hemodynamic response with fUSI during and after FUS neuromodulation (FUS-fUSI) in lightly anesthetized mice. We investigated the effects of varying pressures and transducer positions on the hemodynamic responses. ResultsWe found that higher FUS pressures increase the size of the activated brain area, as well as the magnitude of change in CBV and could show that sham sonications did not produce hemodynamic responses. Unilateral sonications resulted in bilateral hemodynamic changes with a significantly stronger response on the ipsilateral side. FUS neuromodulation in mice with a cranial window showed distinct activation patterns that were frequency-dependent and different from the activation patterns observed in the transcranial model. ConclusionfUSI is hereby shown capable of transcranially monitoring online and short-term hemodynamic effects in the brain during and following FUS neuromodulation.

bioengineering↗

bioRxiv2024

Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors

CRISPR-Cas9 nucleases are versatile tools for genetic engineering cells and function by producing targeted double-strand breaks (DSBs) in the DNA sequence. However, the unintended production of large deletions (>100 bp) represents a challenge to the effective application of this genome-editing system. We optimized a long-range amplicon sequencing system and developed a k-mer sequence-alignment algorithm to simultaneously detect small DNA alteration events and large DNA deletions. With this workflow, we determined that CRISPR-Cas9 induced large deletions at varying frequencies in cancer cell lines, stem cells, and primary T cells. With CRISPR interference screening, we determined that end resection and the subsequent TMEJ [DNA polymerase theta-mediated end joining] repair process produce most large deletions. Furthermore, base editors and prime editors also generated large deletions despite employing mutated Cas9 "nickases" that produce single-strand breaks. Our findings reveal an important limitation of current genome-editing tools and identify strategies for mitigating unwanted large deletion events.

genetics↗

bioRxiv2023

Connectivity at home: A data-driven connectivity modeling framework for home range movements in heterogeneous landscapes

The landscape connectivity of cities is increasingly recognized as crucial for biodiversity conservation and ecosystem services. Yet, modelling ecological connectivity in cities remains challenging because landscape resistance is often based on expert judgment rather than empirical evidence, leading to varying modelling results and limited use for planning. We developed and tested a data-driven framework for empirically parametrizing resistance and movement-distance parameters in functional connectivity models from movement-proxy data - information on the presence/absence of animal movement from direct observation or camera traps. At each step, we ensured that the connectivity model reflected the behavioural and spatial properties of the observations. We applied the framework for the common blackbird (Turdus merula) in Munich, Germany. We used observations of flying blackbirds as movement-proxy data in a logistic regression framework, testing alternative combinations of resistance and movement distances. Model selection identified the parameter sets best supported by the data. The resulting parameters were validated using repeated out-of-sample validation and compared against an expert-based connectivity model. Connectivity derived from empirically estimated parameters increased the probability of observing flying blackbirds. Across repeated validations, the empirical model achieved a mean AUC of 0.76 and R2 of 0.17. It performed moderately better than the expert-based model. Depending on their height, buildings exhibited varying resistance to flying blackbirds. Results indicate that expert assessments may oversimplify urban barriers. The approach provides a transparent, reproducible framework for using movement-proxy data to derive maps of landscape resistance. It offers a step toward more data-driven urban connectivity modelling.

ecology↗

bioRxiv2023

Detection of Blood Volume Reduction and Vasoconstriction Following Focused Ultrasound Blood-Brain Barrier Opening Using Ultrasound Flow Imaging

Microbubble-mediated focused ultrasound (FUS) offers a non-invasive treatment for transient and localized blood-brain barrier (BBB) opening for drug delivery or immunostimulation. It is known that FUS-induced BBB opening is accompanied by blood flow changes, vasoconstriction, and vasodilation, as validated by optical microscopy through a cranial window. In this study, we introduce a novel method for quantifying vascular changes after FUS-induced BBB opening by employing ultrasound flow imaging in mice. We acquired pre-FUS and post-FUS ultrasound flow images with the same microbubble concentration in the brain. Contrast-enhanced power Doppler (CEPD) images and ultrasound localization microscopy images were obtained to evaluate changes in cerebral blood volume and vessel diameter at the sonicated region of the brain. Our findings demonstrate that FUS leads to a reduction in blood volume at the treated region, with vasoconstriction being more dominant than vasodilation. Furthermore, we show that transcranial CEPD can detect local blood reduction following FUS, which spatially coincides with the edema region identified in T2-weighted MRI. Our findings suggest that ultrasound flow imaging has the potential to serve as a cost-effective and immediate monitoring tool for evaluating the safety and efficacy of FUS-induced BBB opening.

bioengineering↗