Biology subjects

Hong, G.

Publications and source records attributed to Hong, G..

16 recordsLinked to original sources

bioRxiv2026

Refractive index modulation by ultraviolet absorption of canonical amino acids for in vivo optical transparency

The inherent opacity of most mammalian tissues limits deep-tissue optical imaging and light delivery. In contrast, the natural transparency of certain species and ocular tissues has been hypothesized to involve proteins with unusually high refractive indices. Here, we systematically analyze the ultraviolet absorption and visible-range refractive index modulation of canonical amino acids to identify key contributors to high-refractive index proteins. We identify arginine as a leading candidate, combining strong ultraviolet absorption, efficient refractive index modulation, physiological pH, and biocompatibility. These properties are validated through successful achievement of optical transparency in both ex vivo and in vivo tissues. Our findings establish a foundation for using abundant endogenous biomolecules to achieve in vivo tissue transparency and suggest a strategy for engineering proteins enriched in high-performing amino acids to enable efficient, biocompatible tissue clearing.

biophysics↗

bioRxiv2026

Compact red-shifted near-infrared fluorescent proteins enable deep-tissue SWIR imaging with in vivo optical clearing.

Compact fluorescent proteins (FPs) with red-shifted emission are needed for deep-tissue short-wavelength infrared (SWIR) imaging. We engineered a GAF domain from the JSC1 cyanobacteriochrome of thermophilic Leptolyngbya sp. into three monomeric, biliverdin-binding FPs of 19.1 kDa: miRFP729nano, miRFP732nano and miRFP735nano, with excitation/emission maxima of 714/729, 716/732 and 719/735 nm, respectively. Their off-peak fluorescence beyond 1,000 nm was several-fold higher than that of miRFP718nano previously used for SWIR imaging. miRFP732nano functioned as a fusion tag, a component of target-stabilized nanobodies, and a reporter of NF-{kappa}B and AP-1 transcriptional activities. It enabled single-laser, dual-color three-photon imaging with EGFP to depths of [~]950 m in cortex and [~]300 m in spinal cord. In mice, miRFP732nano supported SWIR imaging of skeletal muscle, inflammatory signaling and intracellular targets. Combining SWIR detection with biocompatible 4-aminoantipyrine-based in vivo tissue clearing enhanced signal and image sharpness. These red-shifted NIR FPs expand the genetically encoded toolkit for deep-tissue imaging.

bioengineering↗

bioRxiv2026

Tartrazine clears live cells while preserving viability at high refractive indices and osmolality

Tissue-clearing techniques have transformed optical imaging of fixed specimens, yet their application to living systems remains limited by toxicity and removal of key tissue components. We recently demonstrated that absorbing molecules such as tartrazine can reversibly render live mouse skin transparent. Subsequently, it was reported that isotonic protein solutions can achieve ex vivo and in vivo cellular clearing. However, discrepancies remain regarding the optimal refractive index (RI) for live-cell clearing and the impact of elevated osmolality on cell viability. Here, using cultured mammalian cells, we systematically examine the dependence of optical contrast on medium RI and the effects of hyperosmolality. We find that, contrary to the recent report of an optimal RI of 1.36[~]1.37 for suspended cells, densely-packed adherent cells exhibit a monotonic decrease in phase contrast up to an RI of 1.41 with tartrazine. Moreover, even under highly hyperosmotic conditions ([~]1200 mOsm/kg), cultured cells exhibit minimal deformation and negligible loss of viability for up to 30 min in the clearing solution. These results demonstrate that tartrazine enables effective live-cell clearing at RI up to 1.41 while preserving viability under elevated osmolality, and motivate future studies to define optimal conditions for in vivo optical clearing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/717314v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ec9fbforg.highwire.dtl.DTLVardef@1ebe9e6org.highwire.dtl.DTLVardef@1492c3corg.highwire.dtl.DTLVardef@f75559_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Characterisation of novel Campylobacter jejuni Type VI secretion system (T6SS) effectors and exploration of the roles of the C. jejuni T6SS in bacterial antagonism and human host cell interaction

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

microbiology↗

bioRxiv2026

A Three-dimensional Analytical Framework for Retinal Microvasculature Reveals Layer-associated Vulnerability in Development and Neovascular Remodeling

The neurosensory retina is one of the most metabolically active tissues in the body and a uniquely accessible extension of the central nervous system, where neuronal and vascular structures can be visualized non-invasively. Its accessibility and highly organized laminar architecture make it a powerful model for studying vascular development and a window into systemic health. Although computational analyses of retinal images have enabled risk assessment for ocular and systemic diseases, most vascular studies rely on two-dimensional frameworks with limited resolution of capillary structure and layer-specific organization. Here, we present a high-resolution three-dimensional (3D) imaging and analysis pipeline enabling quantification of retinal microvasculature and extraction of structural and network metrics across vascular layers. We apply this approach to two mouse models of aberrant retinal vascular development: one with spontaneous postnatal chorioretinal neovascularization and another with disrupted neurovascular lattice formation and layered organization in early life. Across both pathologic contexts, 3D analysis enables detailed characterization of retinal vascular architecture and identifies early vulnerability within the intermediate plexus, the vascular network that bridges the superficial and deep retinal layers, as a sensitive indicator of abnormal remodeling and neovascularization. This framework enables precise characterization of retinal vasculature and establishes a foundation for identifying new retinal biomarkers with potential relevance to neurovascular and systemic disease.

bioengineering↗

bioRxiv2025

Color-neutral and reversible tissue transparency enables longitudinal deep-tissue imaging in live mice

Light scattering in biological tissue presents a significant challenge for deep in vivo imaging. Our previous work demonstrated the ability to achieve optical transparency in live mice using intensely absorbing dye molecules, which created transparency in the red spectrum while blocking shorter-wavelength photons. In this paper, we extend this capability to achieve optical transparency across the entire visible spectrum by employing molecules with strong absorption in the ultraviolet spectrum and sharp absorption edges that rapidly decline upon entering the visible spectrum. This new color-neutral and reversible tissue transparency method enables optical transparency for imaging commonly used fluorophores in the green and yellow spectra. Notably, this approach facilitates tissue transparency for structural and functional imaging of the live mouse brain labeled with yellow fluorescent protein and GCaMP through the scalp and skull. We show that this method enables longitudinal imaging of the same brain regions in awake mice over multiple days during development. Histological analyses of the skin and systemic toxicology studies indicate minimal acute or chronic damage to the skin or body using this approach. This color-neutral and reversible tissue transparency technique opens new opportunities for noninvasive deep-tissue optical imaging, enabling long-term visualization of cellular structures and dynamic activity with high spatiotemporal resolution and chronic tracking capabilities. Significance StatementTissue scattering represents a major barrier to deep tissue imaging in vivo. We recently showed that tissue can be rendered transparent in the red spectrum using intensely absorbing dye molecules. Here, we introduce a new, color-neutral and reversible tissue transparency approach. We demonstrate longitudinal structural and functional imaging in the deep tissue of awake mice.

bioengineering↗

bioRxiv2025

A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

The DNA damage response (DDR) is a central regulator of cancer cell fate, coordinating both pro-death and pro-survival pathways in response to genotoxic stress. Here, we reveal an unexpected role for the DDR at the cell surface, in mediating immune evasion from complement-dependent cytotoxicity (CDC), an innate immune mechanism exploited by therapeutic monoclonal antibodies (mAbs). In the context of diffuse large B-cell lymphoma (DLBCL), where the anti-CD20 mAb rituximab utilizes CDC, we show that genotoxic chemotherapy induces expression of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59, thereby reducing CDC sensitivity and compromising rituximab activity. In this setting, CD59 emerged as the dominant DDR-induced inhibitor of complement-mediated killing. A high-throughput kinase inhibitor screen identified checkpoint kinase 1 (Chk1) as a critical mediator of this response. Mechanistically, DNA damage activates Chk1, enhancing CD59 transcription via an Sp1-bound promoter. Co-immunoprecipitation mass spectrometry revealed a Chk1 dependent remodelling of Sp1-associated complexes to a transcriptionally active state with recruitment of the histone acetyltransferase KAT2A. These findings expand the role of the DDR in immune resistance at the tumor cell surface, and highlight a negative interaction between chemotherapy and monoclonal antibodies that may require sequential administration or targeting of the Chk1- Sp1-CD59 axis. SignificanceThe DNA Damage Response upregulates complement-protective proteins, extending its role in modulating immune evasion at the cell surface, with direct implications for combinations of chemotherapy and monoclonal antibodies widely used in cancer.

cancer biology↗

bioRxiv2025

Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. How this pathogen interacts with the host defence machinery of human intestinal epithelial cells (IECs) and is involved in pathogenesis remains elusive. Bacterial pathogens utilise strategies to gain access to the eukaryotic cell machinery that can involve subversion of biological processes in host. Unfolded protein response (UPR) is a highly conserved host cell stress response to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and is a conserved evolutionary response against invading pathogens. Several bacterial pathogens can induce the UPR for their own survival and thus design a dual scenario where UPR can both protect and facilitate pathogen evasion. Herein, we investigated whether UPR represents a virulence mechanism exploited by C. jejuni during bacterial invasion in human IECs. Our data show that following C. jejuni infection, we observe upregulation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) pathways of the UPR, albeit differentially in human T84 and Caco-2 cells. Chemical induction of UPR by thapsigargin in host cells reduced intracellular survival of C. jejuni while conversely pretreatment with UPR inhibitors increased intracellular survival of C. jejuni and attenuated IL-8 release. Finally, we show that C. jejuni mutant C. jejuni capsular polysaccharide and flagella contribute to UPR activation in IECs. Collectively, these findings provide mechanistic insights into how C. jejuni infection leads to UPR activation and inflammation, potentially contributing to downstream C. jejuni-mediated damage. Author SummaryCampylobacter jejuni is one of the most prevalent bacterial causes of foodborne human gastroenteritis worldwide. The common symptoms of campylobacteriosis include diarrhoea, abdominal pain and fever. C. jejuni-induced diarrhoea is associated with disruption of tight junctions and adherens junctions, downregulation of epithelial sodium channels and disruption of bile reabsorption. In addition, C. jejuni-induced diarrhoea in part result from intestinal inflammation caused by increased C. jejuni colonisation and invasion due to dysfunctional intestinal barrier. C. jejuni was reported to cause an inflammatory signalling cascade via ligand recognition by Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerisation domain (NOD) in IECs. However, other potential mechanisms of C. jejuni-induced inflammation, with respect to the IEC responses remains unknown. In this study, we demonstrate UPR activation following C. jejuni infection. The UPR is a conserved eukaryotic regulatory response which is initiated to relieve ER stress and recover ER homeostasis, however several bacteria have evolved to exploit UPR pathways for their own survival and replication, creating a dual scenario where UPR can both protect and facilitate pathogen evasion. This study shows that the activation of the PERK pathway of host UPR is activated in the presence of live C. jejuni or its structural components, flagella and a capsular polysaccharide. Furthermore, we propose that although activation of the UPR is a conserved evolutionary response against invading pathogens, UPR induction induces inflammation which potentially contributes to C. jejuni-mediated damage.

microbiology↗

bioRxiv2024

Chronic recording of brain activity in awake toads

BackgroundAmphibians represent an important evolutionary transition from aquatic to terrestrial environments and they display a large variety of complex behaviors despite a relatively simple brain. However, their brain activity is not as well characterized as that of many other vertebrates, partially due to physiological traits that have made electrophysiology recordings difficult to perform in awake and moving animals. New methodWe implanted flexible mesh electronics in the cane toad (Rhinella marina) and performed extracellular recordings in the telencephalon of anesthetized toads and partially restrained, awake toads over multiple days. ResultsWe recorded brain activity over five consecutive days in awake toads and over a 15 week period in a toad that was anesthetized during recordings. We were able to perform spike sorting and identified single- and multi-unit activity in all toads. Comparison with existing methodsTo our knowledge, this is the first report of a modern method to perform electrophysiology in non-paralyzed toads over multiple days, though there are historical references to short term recordings in the past. ConclusionsImplementing flexible mesh electronics in amphibian species will allow for advanced studies of the neural basis of amphibian behaviors.

neuroscience↗

bioRxiv2023

Direct-print three-dimensional electrodes for large-scale, high-density, and customizable neural interfaces

Silicon-based planar microelectronics is a powerful tool for scalably recording and modulating neural activity at high spatiotemporal resolution, but it remains challenging to target neural structures in three dimensions (3D). We present a method for directly fabricating 3D arrays of tissue-penetrating microelectrodes onto silicon microelectronics. Leveraging a high-resolution 3D printing technology based on 2-photon polymerization and scalable microfabrication processes, we fabricated arrays of 6,600 microelectrodes 10-130 {micro}m tall and at 35-m pitch onto a planar silicon-based microelectrode array. The process enables customizable electrode shape, height and positioning for precise targeting of neuron populations distributed in 3D. As a proof of concept, we addressed the challenge of specifically targeting retinal ganglion cell (RGC) somas when interfacing with the retina. The array was customized for insertion into the retina and recording from somas while avoiding the axon layer. We verified locations of the microelectrodes with confocal microscopy and recorded high-resolution spontaneous RGC activity at cellular resolution. This revealed strong somatic and dendritic components with little axon contribution, unlike recordings with planar microelectrode arrays. The technology could be a versatile solution for interfacing silicon microelectronics with neural structures and modulating neural activity at large scale with single-cell resolution.

neuroscience↗

bioRxiv2022

Campylobacter jejuni modulates reactive oxygen species production and NADPH oxidase 1 expression in human intestinal epithelial cells

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (Nox1), a key ROS-generating enzyme in IECs and antioxidant defence genes cat and sod1. Furthermore, inhibition of Nox1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery.

microbiology↗

bioRxiv2021

Differential heating of metal nanostructures by radio frequencies: a theoretical study

Nanoparticles with strong absorption of incident radio frequency (RF) or microwave irradiation are desirable for remote hyperthermia treatments. While controversy has surrounded the absorption properties of spherical metallic nanoparticles, other geometries such as prolate and oblate spheroids have not received sufficient attention for application in hyperthermia therapies. Here, we use the electrostatic approximation to calculate the relative absorption ratio of metallic nanoparticles in various biological tissues. We consider a broad parameter space, sweeping across frequencies from 1 MHz to 10 GHz, while also tuning the nanoparticle dimensions from spheres to high-aspect-ratio spheroids approximating nanowires and nanodiscs. We find that while spherical metallic nanoparticles do not offer differential heating in tissue, large absorption cross sections can be obtained from long prolate spheroids, while thin oblate spheroids offer minor potential for absorption. Our results suggest that metallic nanowires should be considered for RF- and microwave-based wireless hyperthermia treatments in many tissues going forward.

biophysics↗

bioRxiv2020

Through-scalp deep-brain stimulation in tether-free, naturally-behaving mice with widefield NIR-II illumination

Neural modulation techniques with electricity, light and other forms of energy have enabled the deconstruction of neural circuitry. One major challenge of existing neural modulation techniques is the invasive brain implants and the permanent skull attachment of an optical fiber for modulating neural activity in the deep brain. Here we report an implant-free and tether-free optical neuromodulation technique in deep-brain regions through the intact scalp with brain-penetrant second near-infrared (NIR-II) illumination. Macromolecular infrared nanotransducers for deep-brain stimulation (MINDS) demonstrate exceptional photothermal conversion efficiency of 71% at 1064 nm, the wavelength that minimizes light attenuation by the brain in the entire 400-1700 nm spectrum. Upon widefield 1064-nm illumination >50 cm above the mouse head at a low incident power density of 10 mW/mm2, deep-brain neurons are activated by MINDS-sensitized TRPV1 channels with minimal thermal damage. Our approach could open opportunities for simultaneous neuromodulation of multiple socially interacting animals by remotely irradiating NIR-II light to stimulate each subject individually.

neuroscience↗

bioRxiv2019

Single-cell profiles of retinal neurons differing in resilience to injury reveal neuroprotective genes

Neuronal types in the central nervous system differ dramatically in their resilience to injury or insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ~80% of RGCs within 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC, characterized transcriptomic, physiological, and morphological changes that preceded degeneration, and identified genes selectively expressed by each type. Finally, using loss- and gain-of-function assays in vivo, we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury, and demonstrates that differential gene expression can be used to reveal molecular targets for intervention.

neuroscience↗

bioRxiv2018

Optical Sectioning of Live Mammal with Near-Infrared Light Sheet

Deep-tissue three-dimensional optical imaging of live mammals in vivo with high spatiotemporal resolution in non-invasive manners has been challenging due to light scattering. Here, we developed near-infrared (NIR) light sheet microscopy (LSM) with optical excitation and emission wavelengths up to ~ 1320 nm and ~ 1700 nm respectively, far into the NIR-II (1000-1700 nm) region for 3D optical sectioning through live tissues. Suppressed scattering of both excitation and emission photons allowed one-photon optical sectioning at ~ 2 mm depth in highly scattering brain tissues. NIR-II LSM enabled non-invasive in vivo imaging of live mice, revealing never-before-seen dynamic processes such as highly abnormal tumor microcirculation, and 3D molecular imaging of an important immune checkpoint protein, programmed-death ligand 1 (PD-L1) receptors at the single cell scale in tumors. In vivo two-color near-infrared light sheet sectioning enabled simultaneous volumetric imaging of tumor vasculatures and PD-L1 proteins in live mammals.

bioengineering↗

bioRxiv2018

Structure of the Human Volume Regulated Anion Channel

SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.

biochemistry↗