Biology subjects

Huang, Y. H.

Publications and source records attributed to Huang, Y. H..

13 recordsLinked to original sources

bioRxiv2026

RAD: A Read-structure Agnostic Demultiplexer for Single-Cell Long-Read Sequencing and Analysis

Single-cell long-read sequencing (LRS) techniques enable the analysis of full transcript sequences within a cell. However, the high error rate inherent to LRS introduces computational challenges for parsing information like cell barcode, and custom workflows are often required to handle complex read layouts, such as split combinatorial barcodes. We introduce an error-robust, read-structure agnostic demultiplexer (RAD). In RAD, users can easily specify read structure, such as adapter sequence and barcode relative position, and can rapidly extract these elements for each read. In addition to finding the barcode, RAD implements efficient barcode correction strategies for scenarios of knowing or not knowing the full barcode whitelist or having paired short-read single-cell sequencing data for a short whitelist. In synthetic and real-world benchmarks, RAD is faster and achieves significantly higher sensitivity than existing pipelines while having comparable precision. We show RAD can be applied to high-definition long-read spatial transcriptomic data and demonstrate single cell and spatial analysis of B cell isotype and secretion states.

bioinformatics↗

bioRxiv2025

Effects and mechanisms of monoclonal and polyclonal human antibodies in protection of humanized mice from HIV-1 challenge

Recent clinical trials of both active and passive immunization demonstrate the barriers to the successful development of efficacious preventative HIV-1 vaccines and prophylactic antibody treatments. More facile means to explore these interventions in preclinical models could fill key gaps in knowledge and contribute to identifying and optimizing the most promising interventions. Here we report simple adaptations to standard virus challenge strategies in the humanized mouse model that support evaluation of antibody-mediated protection from infection with fewer high stakes design choices and demonstrate evaluation of protection afforded by polyclonal human serum IgG antibodies (pAbs) and monoclonal antibodies (mAbs) with variable pharmacokinetic (PK) and functional profiles. Using these adaptations, we observed that both neutralization and Fc-mediated effector functions contribute to the in vivo antiviral activity of broadly-neutralizing antibody VRC01, that confounding of results due to differences in mAb PK can be overcome, and most promisingly, that polyclonal human serum IgG that exhibits potent neutralizing and Fc-effector function can protect from infection. Collectively, this work demonstrates insights into antibody-mediated protection and methods that hold promise in supporting testing the protection from HIV-1 afforded by human pAb responses induced by vaccination.

immunology↗

bioRxiv2025

Anti-CD206 CAR T Cell Treatment Restores Fibrosis-Induced Loss of Dermal White Adipose Tissue

Fibrosis drives pathology in the chronic autoimmune disease systemic sclerosis (SSc), which has the highest case fatality rate of any systemic autoimmune disease with no validated biomarkers or curative treatments. Our prior work has shown that CD206+ macrophages and dermal fibroblasts engage in cooperative mechanisms of inflammatory and fibrotic activation in SSc. Here, we designed a targeted immunotherapeutic approach to eliminate CD206+ macrophages using chimeric antigen receptor (CAR) T cells. We demonstrate that systemic delivery of a single dose of anti-CD206 CAR T cells restores dermal white adipose tissue (DWAT) in vivo. Notably, loss of subcutaneous fat is a well-recognized but poorly understood aspect of SSc pathogenesis that precedes the development of fibrosis and is driven by changes in lineage commitment of adipose-derived stem cells (ADSCs). Using snRNA-seq and a newly-developed in vitro co-culture model, we report that CD206high macrophages mediate ADSC shift from adipocytic to fibrotic activation in part through an IL-6-dependent mechanism. This report implicates a novel function for macrophages in the regulation of early SSc pathogenesis and is the first to establish the therapeutic efficacy of using CAR T cell immunotherapy to target macrophages in the treatment of SSc skin disease.

synthetic biology↗

bioRxiv2024

Temporal Dynamics of Nucleus Accumbens Neurons in Male Mice During Reward Seeking

The nucleus accumbens (NAc) regulates reward-motivated behavior, but the temporal dynamics of NAc neurons that enable "free-willed" animals to obtain rewards remain elusive. Here, we recorded Ca2+ activity from individual NAc neurons when mice performed self-paced lever-presses for sucrose. NAc neurons exhibited three temporally-sequenced clusters, defined by times at which they exhibited increased Ca2+ activity: approximately 0, -2.5 or -5 sec relative to the lever-pressing. Dopamine D1 receptor (D1)-expressing neurons and D2-neurons formed the majority of the -5-sec versus -2.5-sec clusters, respectively, while both neuronal subtypes were represented in the 0-sec cluster. We found that pre-press activity patterns of D1- or D2-neurons could predict subsequent lever-presses. Inhibiting D1-neurons at -5 sec or D2-neurons at -2.5 sec, but not at other timepoints, reduced sucrose-motivated lever-pressing. We propose that the time-specific activity of D1- and D2-neurons mediate key temporal features of the NAc through which reward motivation initiates reward-seeking behavior.

neuroscience↗

bioRxiv2024

Cancer therapy via neoepitope-specific monoclonal antibody cocktails

BackgroundCellular heterogeneity presents a significant challenge to cancer treatment. Antibody therapies targeting individual tumor-associated antigens can be extremely effective but are not suited for all patients and often fail against tumors with heterogeneous expression as tumor cells with low or no antigen expression escape targeting and develop resistance. Simultaneously targeting multiple tumor-specific proteins with multiple antibodies has the potential to overcome this barrier and improve efficacy, but relatively few widely expressed cancer-specific antigens are known. In contrast, neoepitopes, which arise from mutations unique to tumor cells, are considerably more abundant. However, since neoepitopes are not commonly shared between individuals, a patient-customized approach is necessary and motivates efforts to develop an efficient means to identify suitable target mutations and isolate neoepitope-specific monoclonal antibodies. MethodsHere, focusing on the latter goal, we use directed evolution in yeast and phage display systems to engineer antibodies from non-immune, human antibody fragment libraries that are specific for neoepitopes previously reported in the B16F10 melanoma model. ResultsWe demonstrate proof-of-concept for a pipeline that supports rapid isolation and functional enhancement of multiple neoepitope peptide-targeted monoclonal antibodies and demonstrate their robust binding to B16F10 cells and potent effector functions in vitro. These antibodies were combined and evaluated in vivo for anti-cancer activity in tumor-bearing mice, where they suppressed B16F10 tumor growth and prolonged survival. ConclusionsThese findings emphasize the potential for clinical application of patient-customized antibody cocktails in the treatment of the many cancers poorly addressed by current therapies.

immunology↗

bioRxiv2024

Molecular and cellular rhythms in excitatory and inhibitory neurons in the mouse prefrontal cortex

Previous studies have shown that there are rhythms in gene expression in the mouse prefrontal cortex (PFC); however, the contribution of different cell types and potential variation by sex has not yet been determined. Of particular interest are excitatory pyramidal cells and inhibitory parvalbumin (PV) interneurons, as interactions between these cell types are essential for regulating the excitation/inhibition balance and controlling many of the cognitive functions regulated by the PFC. In this study, we identify cell-type specific rhythms in the translatome of PV and pyramidal cells in the mouse PFC and assess diurnal rhythms in PV cell electrophysiological properties. We find that while core molecular clock genes are conserved and synchronized between cell types, pyramidal cells have nearly twice as many rhythmic transcripts as PV cells (35% vs. 18%). Rhythmic transcripts in pyramidal cells also show a high degree of overlap between sexes, both in terms of which transcripts are rhythmic and in the biological processes associated with them. Conversely, in PV cells, rhythmic transcripts from males and females are largely distinct. Moreover, we find sex-specific effects of phase on action potential properties in PV cells that are eliminated by environmental circadian disruption. Together, this study demonstrates that rhythms in gene expression and electrophysiological properties in the mouse PFC vary by both cell type and sex. Moreover, the biological processes associated with these rhythmic transcripts may provide insight into the unique functions of rhythms in these cells, as well as their selective vulnerabilities to circadian disruption. Significance statementThis is the first study to examine translatomic rhythms in the mouse PFC with cell-type specificity. We find that the core molecular clock cycles in phase across cell types, indicating that previously described daily oscillations in the cortical excitation/inhibition balance are not the consequence of a phase offset between PV and pyramidal cells. Nevertheless, rhythmic transcripts and their associated biological processes differ by both sex and cell type, suggesting that molecular rhythms may play a unique role in different cell types and between sexes. Therefore, our results, such as the enrichment of transcripts associated with mitochondrial function in PV cells from males, point towards possible cell and sex-specific mechanisms that could contribute to psychiatric and cognitive diseases upon rhythm disruption.

neuroscience↗

bioRxiv2024

An EEG Signature of MCH Neuron Activities Predicts Cocaine Seeking

BackgroundIdentifying biomarkers that predict substance use disorder (SUD) propensity may better strategize anti-addiction treatment. The melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH) critically mediates interactions between sleep and substance use; however, their activities are largely obscured in surface electroencephalogram (EEG) measures, hindering the development of biomarkers. MethodsSurface EEG signals and real-time Ca2+ activities of LH MCH neurons (Ca2+MCH) were simultaneously recorded in male and female adult rats. Mathematical modeling and machine learning were then applied to predict Ca2+MCH using EEG derivatives. The robustness of the predictions was tested across sex and treatment conditions. Finally, features extracted from the EEG-predicted Ca2+MCH either before or after cocaine experience were used to predict future drug-seeking behaviors. ResultsAn EEG waveform derivative - a modified theta-to-delta ratio (EEG Ratio) - accurately tracks real-time Ca2+MCH in rats. The prediction was robust during rapid eye movement sleep (REMS), persisted through REMS manipulations, wakefulness, circadian phases, and was consistent across sex. Moreover, cocaine self-administration and long-term withdrawal altered EEG Ratio suggesting shortening and circadian redistribution of synchronous MCH neuron activities. In addition, features of EEG Ratio indicative of prolonged synchronous MCH neuron activities predicted lower subsequent cocaine seeking. EEG Ratio also exhibited advantages over conventional REMS measures for the predictions. ConclusionsThe identified EEG Ratio may serve as a non-invasive measure for assessing MCH neuron activities in vivo and evaluating REMS; it may also serve as a potential biomarker predicting drug use propensity.

neuroscience↗

bioRxiv2024

Hierarchal single-cell lineage tracing reveals differential fate commitment of CD8 T-cell clones in response to acute infection.

Generating balanced populations of CD8 effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of CD8 differentiation remains unclear. We used CARLIN, a processive lineage recording mouse model with single-cell RNA-seq and TCR-seq to track endogenous antigen-specific CD8 T cells during acute viral infection. We identified a diverse repertoire of expanded T-cell clones represented by seven transcriptional states. TCR enrichment analysis revealed differential memory- or effector-fate biases within clonal populations. Shared Vb segments and amino acid motifs were found within biased categories despite high TCR diversity. Using single-cell CARLIN barcode-seq we tracked multi-generational clones and found that unlike unbiased or memory-biased clones, which stably retain their fate profiles, effector-biased clones could adopt memory- or effector-bias within subclones. Collectively, our study demonstrates that a heterogenous T-cell repertoire specific for a shared antigen is composed of clones with distinct TCR-intrinsic fate-biases.

immunology↗

bioRxiv2023

Anesthetic oxygen use and sex are critical factors in the FLASH sparing effect

IntroductionUltra-high dose-rate (UHDR) radiation has been reported to spare normal tissue compared to conventional dose-rate (CDR) radiation. However, reproducibility of the FLASH effect remains challenging due to varying dose ranges, radiation beam structure, and in-vivo endpoints. A better understanding of these inconsistencies may shed light on the mechanism of FLASH sparing. Here, we evaluate whether sex and/or use of 100% oxygen as carrier gas during irradiation contribute to the variability of the FLASH effect. MethodsC57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm2 diameter area of the right leg skin using the Mobetron linear accelerator. The primary post-radiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female) skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions. ResultsIn the UHDR group, time to ulceration was significantly shorter in mice that received 100% oxygen compared to room air, and amongst them female mice ulcerated sooner compared to males. However, no significant difference was observed between male and female UHDR mice that received room air. Oxygen measurements showed significantly higher tissue oxygenation using 100% oxygen as the anesthesia carrier gas compared to room air, and female mice showed higher levels of tissue oxygenation compared to males under 100% oxygen. ConclusionThe FLASH sparing effect is significantly reduced using oxygen during anesthesia compared to room air. The FLASH sparing was significantly lower in female mice compared to males. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

cancer biology↗

bioRxiv2023

Resident memory T cell precursors in tumor draining lymph nodes require type-1 IFN for optimal differentiation

Resident memory (Trm) cells play an essential role in anti-tumor immunity. However, little is known about the precursors that differentiate into protective Trm populations against cancer. Here we employed an established model of B16 melanoma neoadjuvant anti-CD4 therapy, to track tumor antigen-specific CD8+ T cells through tissues and across time; from their priming as effectors to their differentiation into Trm. We show that tumor-draining lymph nodes (TDLNs) contain Teff cells that begin to express canonical Trm markers CD103 and CD69. These tumor-specific Teff cells seeded skin and tumor during the effector phase of the response, although egress from these tissues was not required Trm development in LNs. Paired scRNAseq/scTCRseq was used to identify Teff clonotypes in TDLNs and trace their differentiation, in real-time, into Trm populations. We found that expanded clonotypes favored the Trm fate and were unlikely to co-differentiate into other lineages. Precursors of Trm (pre-Trm) clonotypes that subsequently seeded populations throughout tumors, LNs, and skin, were characterized by early expression of tissue residency, stemness, and type-1 IFN sensing genes. These multipotent pre-Trm cells sensed plasmacytoid dendritic cell-derived type-1 interferons in TDLNs, and their expression of interferon alpha receptor was required for their formation of Trm populations in LNs but not in skin. These findings reveal the defining features of pre-Trm cells in response to tumor antigens, and reveal a previously unappreciated role for type-1 IFNs in programming regional Trm immunity to cancer. One Sentence SummaryAnti-tumor effector CD8 T cells adopt early characteristics of tissue residency and stemness, and rely on the sensing of type-1 interferons for their local differentiation into resident memory T cells.

immunology↗

bioRxiv2023

Cue- versus reward-encoding basolateral amygdala projections to nucleus accumbens

In substance use disorders, drug use as unconditioned stimulus (US) reinforces drug taking. Meanwhile, drug-associated cues (conditioned stimulus, CS) also gain incentive salience to promote drug seeking. The basolateral amygdala (BLA) is implicated in both US- and CS-mediated responses. Here, we show that two genetically distinct BLA neuronal types, expressing Rspo2 versus Ppp1r1b, respectively, project to the nucleus accumbens (NAc) and form monosynaptic connections with both dopamine D1 and D2 receptor-expressing neurons. While intra-NAc stimulation of Rspo2 or Ppp1r1b presynaptic terminals establishes intracranial self-stimulation (ICSS), only Ppp1r1b-stimulated mice exhibit cue-induced ICSS seeking. Furthermore, increasing versus decreasing the Ppp1r1b-to-NAc, but not Rspo2-to-NAc, subprojection increases versus decreases cue-induced cocaine seeking after cocaine withdrawal. Thus, while both BLA- to-NAc subprojections contribute to US-mediated responses, the Ppp1r1b subprojection selectively encodes CS-mediated reward and drug reinforcement. Such differential circuit representations may provide insights into precise understanding and manipulation of drug-versus cue-induced drug seeking and relapse. TeaserTwo subprojections from the amygdala to nucleus accumbens differentially encode cue-vs reward-mediated reinforcement.

neuroscience↗

bioRxiv2020

AMPK activation by metformin promotes survival of dormant ER+ breast cancer cells

PurposeDespite adjuvant anti-estrogen therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental DesignMimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant anti-estrogen therapy. ResultsDormant breast cancer cells exhibited upregulated 5 adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the anti-diabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. ConclusionsAMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer. Statement of Translational RelevanceDormant cancer cells that survive adjuvant therapy can ultimately give rise to recurrent/advanced tumors that frequently develop resistance to all approved therapies. Patients with early-stage estrogen receptor alpha (ER)-positive breast cancer are typically treated with surgical resection followed by [≥]5 years of adjuvant anti-estrogen therapy that neutralizes ER and suppresses, but often does not eliminate, tumor-initiating cells. Estrogen withdrawal, which mimics aromatase inhibitor therapy, induced activation of the metabolic sensor 5 adenosine monophosphate-activated protein kinase (AMPK) and upregulated fatty acid oxidation (FAO) in preclinical models. Treatment with the anti-diabetes AMPK-activating drug metformin or high dietary fat intake promoted survival of dormant ER+ breast cancer cells, while anti-anginal drugs that inhibit FAO induced clearance of dormant tumor cells. These findings caution against using AMPK modulators with anti-estrogens in patients with ER+ breast cancer, and warrant testing of FAO inhibitors as anti-cancer agents in combination with anti-estrogens.

cancer biology↗

bioRxiv2020

Cocaine triggers glial-mediated synaptogenesis

Synaptogenesis is essential in forming new neurocircuits during development, and this is mediated in part by astrocyte-released thrombospondins (TSPs) and activation of their neuronal receptor, 2{delta}-1. Here, we show that this developmental synaptogenic mechanism is utilized during cocaine experience to induce spinogenesis and the generation of AMPA receptor-silent glutamatergic synapses in the adult nucleus accumbens (NAc). Specifically, cocaine administration activates NAc astrocytes, and preventing this activation blocks cocaine-induced generation of silent synapses. Furthermore, knockout of TSP2, or pharmacological inhibition or viral-mediated knockdown of 2{delta}-1, prevents cocaine-induced generation of silent synapses. Moreover, disrupting TSP2-2{delta}-1-mediated spinogenesis and silent synapse generation in the NAc occludes cue-induced cocaine seeking after withdrawal from cocaine self-administration and cue-induced reinstatement of cocaine seeking after drug extinction. These results establish that silent synapses are generated by an astrocyte-mediated synaptogenic mechanism in response to cocaine experience and embed critical cue-associated memory traces that promote cocaine relapse.

neuroscience↗