Biology subjects

Wong, K.

Publications and source records attributed to Wong, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Anxiety and risk-taking behavior maps onto opioid and alcohol polysubstance consumption patterns in male and female mice

Polysubstance use is prevalent in the population but remains understudied in preclinical models. Alcohol and opioid polysubstance use is associated with negative outcomes, worse treatment prognosis, and higher overdose risk; but underlying mechanisms are still being uncovered. Examining factors that motivate use of one substance over another in different contexts in preclinical models will better our understanding of polysubstance use and improve translational value. Here we assessed baseline anxiety-like and locomotive behavior and then measured voluntary consumption of multiple doses of alcohol and fentanyl in group housed male and female mice using our novel Socially Integrated Polysubstance (SIP) system. Fifty-six male (n=32) and female (n=24) adult mice were housed in groups of 4 for one week with continuous access to food, water, two doses of ethanol (5% and 10%) and two doses of fentanyl (5 ug/ml and 20 ug/ml). Our analyses revealed sex differences across multiple domains - female mice consumed more liquid in the dark cycle, had higher activity, a higher preference for both ethanol and fentanyl over water, and their fentanyl preference increased over the seven days. Furthermore, both male and female mice displayed polysubstance consumption patterns, with female mice displaying more prolonged polysubstance use across days in the SIP chambers. We then used machine-learning techniques to reveal underlying relationships between baseline behavioral phenotypes and subsequent polysubstance consumption patterns, where anxiety- and risk-taking-like behavioral phenotypes mapped onto discrete patterns of polysubstance use, preference, and escalation. By simulating more translationally relevant substance use and improving our understanding of the motivations for different patterns of consumption, this study contributes to the developing preclinical literature on polysubstance use with the goal of facilitating better treatment outcomes and novel therapeutic strategies.

neuroscience↗

bioRxiv2024

An ingestible, battery-free, tissue-adhering robotic interface for non-invasive and chronic electrostimulation of the gut

Ingestible electronics have the capacity to transform our ability to effectively diagnose and potentially treat a broad set of conditions. Current applications could be significantly enhanced by addressing poor electrode-tissue contact, lack of navigation, short dwell time, and limited battery life. Here we report the development of an ingestible, battery-free, and tissue-adhering robotic interface (IngRI) for non-invasive and chronic electrostimulation of the gut, which addresses challenges associated with contact, navigation, retention, and powering (C-N-R-P) faced by existing ingestibles. We show that near-field inductive coupling operating near 13.56 MHz was sufficient to power and modulate the IngRI to deliver therapeutically relevant electrostimulation, which can be further enhanced by a bio-inspired, hydrogel-enabled adhesive interface. In swine models, we demonstrated the electrical interaction of IngRI with the gastric mucosa by recording conductive signaling from the subcutaneous space. We further observed changes in plasma ghrelin levels, the "hunger hormone," while IngRI was activated in vivo, demonstrating its clinical potential in regulating appetite and treating other endocrine conditions. The results of this study suggest that concepts inspired by soft and wireless skin-interfacing electronic devices can be applied to ingestible electronics with potential clinical applications for evaluating and treating gastrointestinal conditions.

bioengineering↗

bioRxiv2023

Coral species-specific loss and physiological legacy effects are elicited by extended marine heatwave

Marine heatwaves are increasing in frequency and intensity, with potentially catastrophic consequences for marine ecosystems such as coral reefs. An extended heatwave and recovery time-series that incorporates multiple stressors and is environmentally realistic can provide enhanced predictive capacity for performance under climate change conditions. We exposed common reef-building corals in Hawai i, Montipora capitata and Pocillopora acuta, to a 2-month period of high temperature and high pCO2 conditions or ambient conditions in a factorial design, followed by 2 months of ambient conditions. High temperature, rather than high pCO2, drove multivariate physiology shifts through time in both species, including decreases in respiration rates and endosymbiont densities. Pocillopora acuta exhibited more significantly negatively altered physiology, and substantially higher bleaching and mortality than M. capitata. The sensitivity of P. acuta appears to be driven by higher baseline rates of photosynthesis paired with lower host antioxidant capacity, creating an increased sensitivity to oxidative stress. Thermal tolerance of M. capitata may be partly due to harboring a mixture of Cladocopium and Durusdinium spp., whereas P. acuta was dominated by other distinct Cladocopium spp. Only M. capitata survived the experiment, but physiological state in heatwave exposed M. capitata remained significantly diverged at the end of recovery relative to individuals that experienced ambient conditions. In future climate scenarios, particularly marine heatwaves, our results indicate a species-specific loss of corals that is driven by baseline host and symbiont physiological differences as well as Symbiodiniaceae community compositions, with the surviving species experiencing physiological legacies that are likely to influence future stress responses.

physiology↗

bioRxiv2023

Rational design of a SOCS1-edited tumor infiltrating lymphocyte therapy for solid tumors using CRISPR/Cas9 screens

Cell therapies such as Tumor Infiltrating Lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the anti-tumor activity of T cell therapies, large-scale in vitro and in vivo CRISPR/Cas9 screens were performed, with the suppressor of cytokine signaling 1 (SOCS1) gene identified as a top T cell-enhancing target. In murine CD8 T cell therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of T central memory cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cell (Texprog) cells in tumors. A comprehensive CRISPR tiling screen of the SOCS1 coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with a sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo anti-tumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.

immunology↗

bioRxiv2023

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

cell biology↗

bioRxiv2023

Identification of small molecule inhibitors of G3BP-driven stress granule formation

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

cell biology↗

bioRxiv2022

Socially Integrated Polysubstance (SIP) system: an open-source solution for continuous monitoring of polysubstance fluid intake in group housed mice

Despite impressive results from neuroscience research using rodent models, there is a paucity of successful translation from preclinical findings to effective pharmacological interventions for treatment of substance use disorder (SUD) in humans. One potential reason for lack of translation from animal models is difficulty in accurately replicating the lived experience of people who use drugs. Aspects of substance use in humans that are often not modeled in animal research include but are not limited to 1) voluntary timing and frequency of substance intake, 2) social environment during substance use, and 3) access to multiple substances and multiple concentrations of each substance. Critically, existing commercial equipment that allows for social housing and voluntary polysubstance use (e.g., home cage monitoring system) is prohibitively expensive and no open-source solutions exist. With these goals in mind, here we detail development of the Socially Integrated Polysubstance (SIP) system, an open-source and lower cost solution that allows for group housed rodents to self-administer multiple substances with continuous monitoring and measurement. In our current setup, each SIP cage contains four drinking stations, and each station is equipped with a RFID sensor and sipper tube connected to a unique fluid reservoir. Using this system, we can track which animal (implanted with unique RFID transponder) visits which drinking location and the amount they drink during each visit (in 20 ul increments). Using four flavors of Kool-Aid, here we demonstrate that the SIP system is reliable and accurate with high temporal resolution for long term monitoring of substance intake and behavior tracking in a social environment. The SIP cage system is a first step towards designing an accessible and flexible rodent model of substance use that more closely resembles the experience of people who use drugs.

neuroscience↗

bioRxiv2022

Positive allosteric modulation of α5-GABA A Receptor in the 5XFAD mouse model has cognitive and neurotrophic benefits

INTRODUCTIONReduced somatostatin (SST) and SST-expressing GABAergic neurons are well-replicated findings in Alzheimers disease (AD) and are associated with cognitive deficits. SST cells inhibit pyramidal cell dendrites through 5-GABA-A receptors (5-GABAA-R). 5-GABAAR positive allosteric modulation (5-PAM) has procognitive and neurotrophic effects in stress and aging models. METHODSWe tested whether 5-PAM (GL-II-73) could reverse cognitive deficits and neuronal spine loss in early and late stages of {beta}-amyloid deposition in the 5xFAD model (N=48/study; 50% female). RESULTSAcute or chronic administration of GL-II-73 reversed spatial working memory in 5xFAD mice at 2 and 5 months of age. Chronic GL-II-73 treatment reversed 5xFAD-induced loss of spine density, spine count and dendritic length at both time points, despite {beta}-amyloid accumulation. DISCUSSIONThese results demonstrate procognitive and neurotrophic effects of GL-II-73 in early and late stages of Alzheimer-related {beta}-amyloid deposition. This suggests 5-PAM as a novel {beta}-amyloid-independent symptomatic therapeutic approach.

pharmacology and toxicology↗

bioRxiv2022

Multisensory inputs control the regulation of time investment for mating by sexual experience in male Drosophila melanogaster

Males have finite resources to spend on reproduction. Thus, males rely on a time investment strategy to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. Here we report a novel form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; we refer to this plasticity as shorter-mating-duration (SMD). SMD is a plastic behavior and requires sexually dimorphic taste neurons. We identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Using a cost-benefit model and behavioral experiments, we further show that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, our study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation. ONE SENTENCE SUMMARYMale flies use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating.

neuroscience↗

bioRxiv2022

Self-assembly of cellular neighborhoods converts stochastic signaling into sustained olfactory neurogenesis

Olfactory neurogenesis occurs continuously throughout the lives of vertebrates, including in humans, and relies on the rapid, unceasing differentiation and integration of neurons into a complex multicellular network. The system-wide regulation of this intricate choreography is poorly understood; in particular, it is unclear how progenitor cells convert stochastic fluctuations in cell-cell signaling, over both space and time, into streamlined fate decisions. Here, we track single-cell level multicellular dynamics in the developing zebrafish olfactory epithelium, perturb signaling pathways with temporal specificity, and find that the continuous generation of neurons is driven by the spatially-restricted self-assembly of transient groups of progenitor cells, i.e. cellular neighborhoods. Stochastic modeling and validation of the underlying genetic circuit reveals that neighborhood self-assembly is driven by a tightly regulated bistable toggle switch between Notch signaling and the transcription factor Insulinoma-associated 1a that is responsive to inter-organ retinoic acid signaling. Newly differentiating neurons emerge from neighborhoods and, in response to brain-derived neurotrophic factor signaling, migrate across the olfactory epithelium to take up residence as apically-located, mature sensory neurons. After developmental olfactory neurogenesis is complete, inducing injury results in a robust expansion of neighborhoods, followed by neuroregeneration. Taken together, these findings provide new insights into how stochastic signaling networks spatially pattern and regulate a delicate balance between progenitors and their neuronal derivatives to drive sustained neurogenesis during both development and regeneration.

developmental biology↗

bioRxiv2022

Full-length optic nerve regeneration in the absence of genetic manipulations

The inability of mature retinal ganglion cells (RGCs) to regenerate axons after optic nerve injury can be partially reversed by manipulating cell-autonomous and/or -non-autonomous factors, among which are neuroimmune interactions. We report here that preconditioning resulting from a mild lens injury (conditioning LI, cLI) prior to optic nerve damage induces far greater axon regeneration than LI or the pro-inflammatory agent zymosan after nerve injury or preconditioning with Zymosan. Unlike other instances of immune-supported regeneration, cLI is unaltered by depleting mature neutrophils, T cells or blocking receptors for identified inflammation-associated growth factors (Oncomodulin, SDF1, CCL5), and is only partially diminished by suppressing peripheral monocyte recruitment. Repeated LI leads to full-length optic nerve regeneration, and pharmacological removal of local resident macrophages with the colony stimulating factor 1 receptor (CSF-1R) inhibitor PLX5622 enables some axons to re-innervate the brain in just 6 weeks. Thus, cell non-autonomous interventions not involving genetic manipulations can induce high levels of optic nerve regeneration, paving the way to uncover potent, translatable therapeutic targets for CNS repair.

neuroscience↗

bioRxiv2022

The mechanosensitive ion channel Piezo1 modulates the migration and immune response of microglia

Microglia are the brains resident immune cells, performing surveillance to promote homeostasis and healthy functioning. While chemical signaling in microglia is well-studied, the mechanical cues regulating their function are less well-understood. Here, we investigate the role of the mechanosensitive ion channel Piezo1 in microglia migration, pro-inflammatory cytokine production and stiffness sensing. We demonstrated the functional expression of Piezo1 in microglia and identified genes whose expression was affected by conditional Piezo1 knockout in transgenic mice. Functional assays revealed that Piezo1-deficiency in microglia enhanced migration towards amyloid {beta}-protein, and decreased levels of pro-inflammatory cytokines produced upon stimulation by lipopolysaccharide, both in vitro and in vivo. The phenomenon could be mimicked or reversed using a Piezo1-specific agonist or antagonist. Finally, we also showed that Piezo1 mediated the effect of substrate stiffness-induced migration and cytokine expression. Altogether, we show that Piezo1 is an important molecular mediator for microglia, its activation modulating their migration and immune responses.

neuroscience↗

bioRxiv2022

HIRA loss transforms FH-deficient cells

Fumarate Hydratase (FH) is a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle. Germline mutations of FH lead to HLRCC, a cancer syndrome characterised by a highly aggressive form of renal cancer(1). Although HLRCC tumours metastasise rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas (2). How Fh1-deficient cells overcome these tumour suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR/Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of HIRA enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss enables the activation of MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

cancer biology↗

bioRxiv2022

Metagenomic analysis of coprolites from three Late Pleistocene megaherbivores from the Southwestern United States.

1.BackgroundDetermining the life-history traits of extinct species is often difficult from skeletal remains alone, limiting the accuracy of studies modeling past ecosystems. However, the analysis of the degraded endogenous bacterial DNA present in paleontological fecal matter (coprolites) may enable the characterization of specific traits such as the hosts digestive physiology and diet. An issue when evaluating the microbial composition of coprolites is the degree to which the microbiome is representative of the hosts original gut community versus the changes that occur in the weeks following deposition due to desiccation. Analyses of paleontological microorganisms are also relevant in the light of recent studies linking the Late Pleistocene and Early Holocene extinctions with modern-day zoonotic pathogen outbreaks. MethodsShotgun sequencing was performed on ancient DNA (aDNA) extracted from coprolites of the Columbian mammoth (Mammuthus Columbi), Shasta ground sloth (Nothrotheriops shastensis) and paleontological bison (Bison sp.) collected from caves on the Colorado Plateau, Southwestern USA. The novel metagenomic classifier MTSv, parameterized for studies of aDNA, was used to assign bacterial taxa to sequencing reads. The resulting bacterial community of coprolites was then compared to those from modern fecal specimens of the African savannah elephant (Loxodonta africana), the brown-throated sloth (Bradypus variegatus) and the modern bison (Bison bison). Both paleontological and modern bison fecal bacterial communities were also compared to those of progressively dried cattle feces to determine whether endogenous DNA from coprolites had a microbiome signal skewed towards aerobic microorganisms typical of desiccated fecal matter. ResultsThe diversity of phyla identified from coprolites was lower than modern specimens. The relative abundance of Actinobacteria was increased in coprolites compared to modern specimens, with fewer Bacteroidetes and Euryarchaeota. Firmicutes had a reduced relative abundance in the mammoth and bison coprolites, compared to the African savanna elephants and modern bison. There was a significant separation of samples in NMDS plots based on their classification as either paleontological or modern, and to a lesser extent, based on the host species. Increasingly dried cattle feces formed a continuum between the modern and paleontological bison samples. ConclusionOur results reveal that any coprolite metagenomes should always be compared to desiccated modern fecal samples from closely related hosts fed a comparable diet to determine the degree to which the coprolite metagenome is a result of desiccation versus true dissimilarities between the modern and paleontological hosts. Also, a large-scale desiccation study including a variety of modern species may shed light on life-history traits of extinct species without close extant relatives, by establishing the proximity of coprolite metagenomes with those from dried modern samples.

paleontology↗

bioRxiv2022

Ingestible capsule for detecting labile inflammatory biomarkers in situ

Transient molecules in the gastrointestinal (GI) tract, such as nitric oxide and hydrogen sulfide, are key signals and mediators of inflammatory bowel disease (IBD). Because these molecules are extremely short-lived in the body, they are difficult to detect. To track these reactive molecules in the GI tract, we have developed a miniaturized device that integrates genetically-engineered probiotic biosensors with a custom-designed photodetector and readout chip. Leveraging the molecular specificity of living sensors, we genetically encoded bacteria to respond to IBD-associated molecules by luminescing. Low-power electronic readout circuits (nanowatt power) integrated into the device convert the light from just 1 L of bacterial culture into a wireless signal. We demonstrate biosensor monitoring in the GI tract of small and large animal models and integration of all components into a sub-1.4 cm3 ingestible form factor capable of supporting wireless communication. The wireless detection of short-lived, disease-associated molecules could support earlier diagnosis of disease than is currently possible, more accurate tracking of disease progression, and more timely communication between patient and their care team supporting remote personalized care.

synthetic biology↗

bioRxiv2021

WESTPA 2.0: High-performance upgrades for weighted ensemble simulations and analysis of longer-timescale applications

The weighted ensemble (WE) family of methods is one of several statistical-mechanics based path sampling strategies that can provide estimates of key observables (rate constants, pathways) using a fraction of the time required by direct simulation methods such as molecular dynamics or discrete-state stochastic algorithms. WE methods oversee numerous parallel trajectories using intermittent overhead operations at fixed time intervals, enabling facile interoperability with any dynamics engine. Here, we report on major upgrades to the WESTPA software package, an open-source, high-performance framework that implements both basic and recently developed WE methods. These upgrades offer substantial improvements over traditional WE. Key features of the new WESTPA 2.0 software enhance efficiency and ease of use: an adaptive binning scheme for more efficient surmounting of large free energy barriers, streamlined handling of large simulation datasets, exponentially improved analysis of kinetics, and developer-friendly tools for creating new WE methods, including a Python API and resampler module for implementing both binned and "binless" WE strategies. Table of Contents/Abstract ImageFor the manuscript "WESTPA 2.0: High-performance upgrades for weighted ensemble simulations and analysis of longer-timescale applications" by Russo et al. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/471280v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@575710org.highwire.dtl.DTLVardef@151c3dorg.highwire.dtl.DTLVardef@1f0f525org.highwire.dtl.DTLVardef@6e5956_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control

Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two timepoints, 1 day and 4 days, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

systems biology↗

bioRxiv2021

Genomically Mined Acoustic Reporter Genes Enable On-Demand In Vivo Monitoring of Tumor-Homing Bacteria

A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics, identified through a phylogenetic screen of candidate gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue, and stable long-term expression. We demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.

bioengineering↗