Biology subjects

He, E.

Publications and source records attributed to He, E..

6 recordsLinked to original sources

bioRxiv2025

Focused Ultrasound Crosslinkable Granular Hydrogels

Minimally invasive delivery of biomaterials for tissue regeneration can be achieved using biomaterials delivered by injection that rapidly stabilize at the delivery site. Tissue regeneration has been shown to be dependent on material properties, with porosity strongly supporting revascularization and tissue regrowth. Here, a highly porous granular hydrogel system was designed that is compatible with the unique strengths of highly penetrating, minimal invasive focused ultrasound (FUS), to address this challenge. FUS offers well-defined spatial and temporal control over hydrogel crosslinking. We developed a composite granular hydrogel scaffold composed of two polyethylene glycol (PEG)-based components, microgels and fibers with FUS-responsive chemistry, and a pore-defining gelatin microgel component. Upon applying FUS, the bulk granular hydrogel stabilized through the formation of crosslinks between PEG components, with porosity designed by gelatin microgel melting. FUS-crosslinking parameters were determined that resulted in crosslinking both in vitro and a mouse cadaver model of minimally invasive delivery. The resulting granular hydrogels stability depended on the presence of fibers and exhibited viscoelastic properties comparable to granular hydrogels that were photocrosslinked. Hydrogels were highly porous and cytocompatible. This work defines a FUS-responsive granular system and extends the potential of FUS as a novel, noninvasive method for crosslinking regenerative hydrogel systems.

bioengineering↗

bioRxiv2024

I2SIM: Boosting High-Fidelity Isotropic Super-Resolution with Image Interference and Spatial-Spectrum Optimization

Spatial resolution is crucial for imaging subcellular structures. The advent of three-dimensional structured illumination microscopy (3D-SIM) greatly benefits the biology community, providing a powerful tool for imaging organelles with a two-fold resolution enhancement in all three dimensions. However, the axial resolution of 3D-SIM is limited to around 300 nm, which is inferior to its lateral resolution. Here, a novel method called image interference SIM (I2SIM) is reported, which utilizes two oppositely positioned objectives to detect fluorescence emission interference under three-beam excitation. By incorporating spectral modulation and spatial domain Frobenius-Hessian optimization, I2SIM achieves an axial resolution approximately twice that of 3D-SIM, reaching around 130 nm. Furthermore, the potential of I2SIM for imaging subcellular structures is demonstrated on various biological samples, including microtubules, actin filaments, and mitochondrial outer membranes. The enhanced optical sectioning capability can be utilized to resolve axial structures that are challenging to discern using ordinary 3D-SIM.

biophysics↗

bioRxiv2024

Sensitive detection of synthetic response to cancer immunotherapy driven by gene paralog pairs

Emerging immunotherapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell (CAR-T) therapy have revolutionized cancer treatment and have improved the survival of patients with multiple cancer types. Despite this success many patients are unresponsive to these treatments or relapse following treatment. CRISPR activation and knockout (KO) screens have been used to identify novel single gene targets that can enhance effector T cell function and promote immune cell targeting and eradication of tumors. However, cancer cells often employ multiple genes to promote an immunosuppressive pathway and thus modulating individual genes often has a limited effect. Paralogs are genes that originate from common ancestors and retain similar functions. They often have complex effects on a particular phenotype depending on factors like gene family similarity, each individual genes expression and the physiological or pathological context. Some paralogs exhibit synthetic lethal interactions in cancer cell survival; however, a thorough investigation of paralog pairs that could enhance the efficacy of cancer immunotherapy is lacking. Here we introduce a sensitive computational approach that uses sgRNA sets enrichment analysis to identify cancer-intrinsic paralog pairs which have the potential to synergistically enhance T cell-mediated tumor destruction. We have further developed an ensemble learning model that uses an XGBoost classifier and incorporates features such as gene characteristics, sequence and structural similarities, protein-protein interaction (PPI) networks, and gene coevolution data to predict paralog pairs that are likely to enhance immunotherapy efficacy. We experimentally validated the functional significance of these predicted paralog pairs using double knockout (DKO) of identified paralog gene pairs as compared to single gene knockouts (SKOs). These data and analyses collectively provide a sensitive approach to identify previously undetected paralog pairs that can enhance cancer immunotherapy even when individual genes within the pair has a limited effect.

bioinformatics↗

bioRxiv2024

The dorsal fan-shaped body is a neurochemically heterogeneous sleep-regulating center in Drosophila

Sleep is a behavior that is conserved throughout the animal kingdom. Yet, despite extensive studies in humans and animal models, the exact function or functions of sleep remain(s) unknown. A complicating factor in trying to elucidate the function of sleep is the complexity and multiplicity of neuronal circuits that are involved in sleep regulation. It is conceivable that distinct sleep-regulating circuits are only involved in specific aspects of sleep and may underlie different sleep functions. Thus, it would be beneficial to assess the contribution of individual circuits in sleeps putative functions. The intricacy of the mammalian brain makes this task extremely difficult. However, the fruit fly Drosophila melanogaster, with its simpler brain organization, available connectomics, and unparalleled genetics offers the opportunity to interrogate individual sleep-regulating centers. In Drosophila, neurons projecting to the dorsal Fan-Shaped Body (dFB) have been proposed to be key regulators of sleep, particularly sleep homeostasis. We recently demonstrated that the most widely used genetic tool to manipulate dFB neurons, the 23E10-GAL4 driver, expresses in two sleep-regulating neurons (VNC-SP neurons) located in the Ventral Nerve Cord (VNC), the fly analog of the vertebrate spinal cord. Since most data supporting a role for the dFB in sleep regulation have been obtained using 23E10-GAL4, it is unclear whether the sleep phenotypes reported in these studies are caused by dFB neurons or VNC-SP cells. A recent publication replicated our finding that 23E10-GAL4 contains sleep-promoting neurons in the VNC. However, it also proposed that the dFB is not involved in sleep regulation at all, but this suggestion was made using genetic tools that are not dFB-specific and a very mild sleep deprivation protocol. In this study, using a newly created dFB-specific genetic driver line, we demonstrate that the majority of 23E10-GAL4 dFB neurons can promote sleep when activated and that these neurons are involved in sleep homeostasis. We also show that dFB neurons require stronger stimulation than VNC-SP cells to promote sleep. In addition, we demonstrate that dFB-induced sleep can consolidate Short-Term Memory (STM) into Long-Term Memory (LTM), suggesting that the benefit of sleep on memory is not circuit-specific. Finally, we show that dFB neurons are neurochemically heterogeneous and can be divided in 3 populations. Most dFB neurons express both glutamate and acetylcholine, while a minority of cells express only one of these two neurotransmitters. Importantly, dFB neurons do not express GABA, as previously suggested. Using neurotransmitter-specific dFB tools, our data also points at cholinergic dFB neurons as particularly potent at regulating sleep and sleep homeostasis.

neuroscience↗

bioRxiv2023

Rational design of immune gene therapy combinations via in vivo CRISPR activation screen of tumor microenvironment modulators

The hostile tumor microenvironment (TME) is major challenge for cancer immunotherapies. Here, we design and perform TME-targeted in vivo CRISPR activation (CRISPRa) screens to uncover factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Through adeno-associated virus (AAV) delivery, multiplexed activation of pooled immunoregulatory genes encoding antigen presentation, cytokine, and co-stimulation molecules (APCM) leads to enhanced anti-tumor immunity. APCM screen in metastatic tumors identifies Cd80, Tnfsf14, Cxcl10, Tnfsf18, Tnfsf9, and Ifng as the top immunostimulatory candidates. AAV-mediated delivery of these factors individually or in combination shows anti-tumor efficacy across different cancer models. Further optimization pinpoints Ifng+Tnfsf9+Il12b(Il12/Il23) as a potent therapeutic combination, leading to increased IFN-{gamma}+CD8+ and tissue-resident memory T cells. APCM therapy synergizes with CAR-T cell therapy against human solid tumors in vivo. APCM-based CRISPRa screen and gene activation systems can thus be leveraged for the rapid generation of off-the-shelf immune gene therapies against solid tumors.

immunology↗

bioRxiv2023

Multiplexed repression of immunosuppressive genes as combinatorial cancer immunotherapy

Checkpoint blockade immunotherapy is a potent class of cancer treatment, however, the complex immunosuppressive tumor microenvironment (TME) often requires multi-agent combinations to be effective. Current cancer immunotherapy combination approaches are cumbersome, usually involving one-drug-at-a-time scheme. Here, we devise Multiplex Universal Combinatorial Immunotherapy via Gene-silencing (MUCIG), as a versatile approach for combinatorial cancer immunotherapy. We harness CRISPR-Cas13d to efficiently target multiple endogenous immunosuppressive genes on demand, allowing us to silence various combinations of multiple immunosuppressive factors in the TME. Intratumoral AAV-mediated administration of MUCIG (AAV-MUCIG) elicits significant anti-tumor activity with several Cas13d gRNA compositions. TME target expression analysis driven optimization led to a simplified off-the-shelf MUCIG targeting a four gene combination (PGGC: Pdl1, Galectin9, Galectin3 and Cd47). AAV-PGGC shows significant in vivo efficacy in syngeneic tumor models. Single cell and flow profiling revealed that AAV-PGGC remodeled the TME by increasing CD8+ T cell infiltration and reducing myeloid-derived immunosuppressive cells (MDSCs). MUCIG thus serves as a universal method to silence multiple immune genes in vivo, and can be delivered via AAV as a therapeutic approach.

immunology↗