Biology subjects

Yao, P.

Publications and source records attributed to Yao, P..

At least 19 recordsLinked to original sources

bioRxiv2026

A hypoxia-sensitive medullary nucleus modulates cerebral blood flow via a disynaptic pathway to cortex

The RVLM (rostral ventral lateral medulla) region of the brainstem is implicated as a controller of both systemic and cerebral blood flow (CBF). Past studies leave open the question of how the RVLM stabilizes CBF in awake animals. Here, we focus on CBF regulation by the adenergic subpopulation of RVLM neurons (RVLMD{beta}h). Hypoxic challenge increases the variability of the activity of RVLMD{beta}h neurons along with increased CBF. Experimental photoactivation of RVLMD{beta}h neurons leads to rapid vasodilation of pial arterioles across the cortical mantle and increased CBF, which is only then followed by an increase in cortical activity. No significant changes in systemic physiology are observed. Virus tracing establishes disynaptic pathways from the RVLM to neocortex with predominant relays involving the lateral hypothalamus and the zona incerta subthalamic nuclei. Chemogenetic inhibition of those nuclei led to a 70 % reduction in the ability of photoactivated RVLMD{beta}h neurons to induce cortical vasodilation and increase CBF. In toto, these findings reveal a major RVLM subcortical pathway to drive transcortical increases in CBF and represent an adaptive mechanism to inform the cerebral vasculature about environmental shifts in pO2.

neuroscience↗

bioRxiv2025

Aptamer-Enabled Discovery and Clinical Analysis of Exosomal Surface Biomarkers in Hepatocellular Carcinoma

Exosomal membrane proteins hold promise as cancer biomarkers but face detection challenges due to low abundance and conformational sensitivity. We developed AptEx-ID, a microbead-displayed aptamer platform enabling high- throughput screening of hepatocellular carcinoma (HCC)-specific exosomal surface proteins. Aptamers, selected via competitive binding to tumor-derived versus healthy exosomes, demonstrated high affinity and specificity. Clinical validation using 50 HCC and 30 control plasma samples identified a 7-aptamer panel via machine learning, achieving 93.75% accuracy and 0.95 ROC-AUC. These aptamers further revealed HCC-associated biomarkers (e.g., IMPDH1, ASCC3, GTSE1), validated by siRNA knockdown and biophysical assays. Post-treatment monitoring showed aptamer signal reduction in responsive patients, highlighting their prognostic utility. This platform integrates biomarker discovery, diagnostics, and therapeutic monitoring, offering a robust approach for HCC management through exosomal surface proteome profiling.

molecular biology↗

bioRxiv2025

Upstream open reading frame inactivation augments GATA4 translation and cardiomyocyte hypertrophy in mice

Upstream open reading frames (uORFs) are short peptide-encoding sequences located in the 5 untranslated region (5 UTR) of mRNAs, enabling translational repression of main (m)ORFs. While uORFs are found in [~]50% of mRNAs in humans, our understanding of their biological function remains limited. This study aims to elucidate the role of the uORF in the 5 UTR of the Gata4 (GATA binding protein 4) gene in cardiac biology by inactivating its start codon ({Delta}uORF) in the mouse genome. Our investigation reveals that mice with Gata4 uORF inactivation manifest spontaneous cardiac hypertrophy without apparent fibrosis as they age. Utilizing single-nucleus RNA sequencing (snRNA-seq), we uncovered significant transcriptional variations between wild-type (WT) and {Delta}ORF mice. Notably, mRNAs associated with sarcomeres and contractile functions show heightened expression levels, reflecting the hypertrophic phenotype. Notably, at least nine upregulated genes are GATA4-bound targets in mouse ventricles. Functional assessments of isolated primary adult cardiomyocytes confirmed enhanced hypertrophy and contractility in {Delta}ORF mice. Additionally, we employed single-nucleus transposase-accessible chromatin (snATAC)-seq to investigate changes in chromatin accessibility. Our results indicated increased accessibility within specific transcription-regulatory elements linked to elevated gene transcription. These putative cis-regulatory elements (pCREs) are significantly enriched in MEF2 (myocyte enhancer factor 2) binding motifs. In vitro luciferase reporter assays further supported the regulatory potential of three of these pCREs, highlighting their role in the transcriptional enhancement of three GATA4 target genes bound by MEF2 and GATA4. These findings illuminate the role of uORF in negatively regulating GATA4 protein expression and cardiomyocyte hypertrophy at the organismal level and provide a novel therapeutic target for cardiac pathogenesis.

biochemistry↗

bioRxiv2025

Glutamate indicators with increased sensitivity and tailored deactivation rates

Identifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neurons thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide range of naturalistic synaptic transmission, including in the vibrissal cortex layer 4 and in hippocampal CA1 dendrites. iGluSnFR4 increases the sensitivity and scale (4s) or speed (4f) of tracking information flow in neural networks in vivo.

neuroscience↗

bioRxiv2025

Peroxisomal Dysfunction Drives Loss of Dynamic Nutrient Responses to Initiate Metabolic Inflexibility During Aging

Aging results in a loss of metabolic flexibility during fasting, characterized by the inability to acutely switch between metabolic substrates and reduced lipid mobilization. However, the drivers of these effects intracellularly remain unclear. Here, in Caenorhabditis elegans, we show that loss of coordinated inter-organelle dynamics causally initiates metabolic inflexibility with age. In young animals, peroxisomes emerge as the priming orchestrators of the fasting response, simultaneously governing lipid droplets (LDs) utilization and mitochondrial bioenergetics. With age, peroxisomal priming is lost, leading to mitochondrial fragmentation and impaired dynamic nutrient responses during fasting. Notably, dietary restriction (DR) exerts a rejuvenating effect on peroxisomal function, thereby preserving mitochondrial integrity and promoting longevity. Our study uncovers the expansive network of organelles enabling lipid mobilization during youth, providing critical context to the poorly understood role of peroxisomes in actively maintaining organelle homeodynamics and metabolic flexibility throughout the aging process.

cell biology↗

bioRxiv2025

Hetero-multicellular stromal cells incorporate into scaffold-free 3D cultures of epithelial cancer cells to drive invasion

Breast cancer (BC) is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids (PDOs) and patient-derived xenograft organoids (PDXOs) are pathophysiologically relevant, they are costly to propagate, difficult to manipulate and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. Advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these hetero-multicellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells(THP-1) and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent of the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, hetero-multicellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in hetero-multicellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression. SUMMARYThere is a critical need for 3D cancer models that capture hetero-cellular crosstalk to study cancer metastasis. Our study presents the generation of hetero-multicellular stromal-epithelial in a scaffold and scaffold free environment that can be used to study invasion and cellular spatial distributions.

cancer biology↗

bioRxiv2024

Beta burst characteristics and coupling within the sensorimotor cortical-subthalamic nucleus circuit in Parkinson's disease

BackgroundBursts of exaggerated subthalamic nucleus (STN) beta activity contribute to clinical impairments in Parkinsons disease (PD). Few studies have explored the characteristics and coupling of bursts across the sensorimotor cortical-STN circuit. ObjectiveWe sought to (1) establish the characteristics of sensorimotor cortical and STN bursts during naturalistic behaviours, and (2) determine the predictability of STN bursts from motor cortical recordings. MethodsWe analysed 1,478 hours of wirelessly streamed bilateral sensorimotor cortical and STN recordings from 5 PD patients. ResultsSTN bursts were longer than cortical bursts and had shorter inter-burst intervals. Long bursts (>200ms) in both structures displayed temporal overlap (>30%), with an estimated cortico-STN conduction delay of 8ms. Furthermore, approximately 27% of all STN bursts were preceded by a cortical burst. ConclusionCortical beta bursts tend to precede STN beta bursts, with short delays. However, subcortical mechanisms are also likely to contribute to STN burst initiation and propagation.

neuroscience↗

bioRxiv2024

Novel Imaging Tools to Study Mitochondrial Dynamics in Caenorhabditis elegans

Mitochondria exhibit a close interplay between their structure and function. Understanding this intricate relationship requires advanced imaging techniques that can capture the dynamic nature of mitochondria and their impact on cellular processes. However, much of the work on mitochondrial dynamics has been done in single celled organisms or in vitro cell culture. Here, we introduce novel genetic tools for live imaging of mitochondrial networks in the nematode Caenorhabditis elegans, addressing a pressing need for advanced techniques in studying organelle dynamics within live intact multicellular organisms. Through a comprehensive analysis, we directly compare our tools with existing methods, demonstrating their advantages for visualizing mitochondrial morphology and contrasting their impact on organismal physiology. We reveal limitations of conventional techniques, while showcasing the utility and versatility of our approaches, including endogenous CRISPR tags and ectopic labeling. By providing a guide for selecting the most suitable tools based on experimental goals, our work advances mitochondrial research in C. elegans and enhances the strategic integration of diverse imaging modalities for a holistic understanding of organelle dynamics in living organisms.

cell biology↗

bioRxiv2024

FAK and p130Cas modulate stiffness-mediated early transcription and cellular metabolism

Cellular metabolism is influenced by the stiffness of the extracellular matrix. Focal adhesion kinase (FAK) and its binding partner, p130Cas, transmit biomechanical signals about substrate stiffness to the cell to regulate a variety of cellular responses, but their roles in early transcriptional and metabolic responses remain largely unexplored. We cultured mouse embryonic fibroblasts with or without siRNA-mediated FAK or p130Cas knockdown and assessed the early transcriptional responses of these cells to placement on soft and stiff substrates by RNA sequencing and bioinformatics analyses. Exposure to the stiff ECM altered the expression of genes important for metabolic and biosynthetic processes, and these responses were influenced by knockdown of FAK and p130Cas. Our findings reveal that FAK-p130Cas signaling mechanotransduces ECM stiffness to early transcriptional changes that alter cellular metabolism and biosynthesis.

genomics↗

bioRxiv2023

Cardiomyocyte-specific Loss of Glutamyl-prolyl-tRNA Synthetase Leads to Disturbed Protein Homeostasis and Dilated Cardiomyopathy

Glutamyl-prolyl-tRNA synthetase (EPRS1), an aminoacyl-tRNA synthetase (ARS) ligating glutamic acid and proline to their corresponding tRNAs, plays an essential role in decoding proline codons during translation elongation. The physiological function of EPRS1 in cardiomyocytes (CMs) and the potential effects of CM-specific loss of EPRS1 remain unknown. Here, we found that heterozygous Eprs1 knockout in CMs does not cause any significant changes in CM hypertrophy induced by pressure overload, while homozygous knockout leads to dilated cardiomyopathy, heart failure, and lethality at around 1 month after Eprs1 deletion. Transcriptomic profiling of early-stage Eprs1 knockout hearts suggests a significantly decreased expression of multiple ion channel genes and an increased gene expression in proapoptotic pathways and integrated stress response. Proteomic analysis shows decreased protein expression of multi-aminoacyl-tRNA synthetase complex components, fatty acid, and branched-chain amino acid metabolic enzymes, as well as a compensatory increase in cytosolic translation machine-related proteins. Immunoblot analysis indicated that multiple proline-rich proteins were reduced at the early stage, which might contribute to cardiac dysfunction of Eprs1 knockout mice. Taken together, this study demonstrates the physiological and molecular outcome of loss-of-function of EPRS1 in vivo and provides valuable insights into the potential side effects on CMs resulting from the EPRS1-targeting therapeutic approach.

pathology↗

bioRxiv2023

Spatiotemporal development of growth and death zones in expanding bacterial colonies driven by emergent nutrient dynamics

Bacterial colony growth on hard agar is commonplace in microbiology; yet, what occurs inside a growing colony is complex even in the simplest cases. Robust colony expansion kinetics featuring a linear radial growth and a saturating vertical growth indicates a common developmental program which is elucidated here for Escherichia coli cells using a combination of modeling and experiments. Radial colony expansion is found to be limited by mechanical factors rather than nutrients as commonly assumed. In contrast, vertical expansion is limited by glucose depletion inside the colony, an effect compounded by reduced growth yield due to anaerobiosis. Carbon starvation in the colony interior results in substantial cell death within 1-2 days, with a distinct death zone that expands with the growing colony. Overall, the development of simple colonies lacking EPS production and differentiation is dictated by an interplay of mechanical constraints and emergent nutrient gradients arising from obligatory metabolic processes.

microbiology↗

bioRxiv2023

Secondary structures that regulate mRNA translation provide insights for ASO-mediated modulation of cardiac hypertrophy

Translation of upstream open reading frames (uORFs) typically abrogates translation of main (m)ORFs. The molecular mechanism of uORF regulation in cells is not well understood. Here, we identified a double-stranded RNA (dsRNA) structure residing within the GATA4 uORF that augments uORF translation and inhibits mORF translation. Antisense oligonucleotides (ASOs) that disrupt this dsRNA structure promote mORF translation, while ASOs that base-pair immediately downstream (i.e., forming a bimolecular double-stranded region) of either the uORF or mORF start codon enhance uORF or mORF translation, respectively. Human cardiomyocytes and mice treated with a uORF-enhancing ASO showed reduced cardiac GATA4 protein levels and increased resistance to cardiomyocyte hypertrophy. We further show the general utility of uORF-dsRNA- or mORF- targeting ASO to regulate mORF translation for other mRNAs. Our work demonstrates a regulatory paradigm that controls translational efficiency and a useful strategy to alter protein expression and cellular phenotypes by targeting or generating dsRNA downstream of a uORF or mORF start codon. Bullet points for discoveriesO_LIdsRNA within GATA4 uORF activates uORF translation and inhibits mORF translation. C_LIO_LIASOs that target the dsRNA can either inhibit or enhance GATA4 mORF translation. C_LIO_LIASOs can be used to impede hypertrophy in human cardiomyocytes and mouse hearts. C_LIO_LIuORF- and mORF-targeting ASOs can be used to control translation of multiple mRNAs. C_LI

molecular biology↗

bioRxiv2023

Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His10 fusion in Escherichia coli. The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.

biochemistry↗

bioRxiv2023

FAM210A Regulates Mitochondrial Translation and Maintains Cardiac Mitochondrial Homeostasis

AimsMitochondria play a vital role in cellular metabolism and energetics and support normal cardiac function. Disrupted mitochondrial function and homeostasis cause a variety of heart diseases. Fam210a (family with sequence similarity 210 member A), a novel mitochondrial gene, is identified as a hub gene in mouse cardiac remodeling by multi-omics studies. Human FAM210A mutations are associated with sarcopenia. However, the physiological role and molecular function of FAM210A remain elusive in the heart. We aim to determine the biological role and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health in vivo. Methods and ResultsTamoxifen-induced MHCMCM-driven conditional knockout of Fam210a in the mouse cardiomyocytes induced progressive dilated cardiomyopathy and heart failure, ultimately causing mortality. Fam210a deficient cardiomyocytes exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Furthermore, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in cardiomyocytes at the early stage before contractile dysfunction and heart failure. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response (ISR), resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of heart failure. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodeling and damage in ischemia-induced heart failure. ConclusionThese results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal cardiomyocyte contractile function. This study also offers a new therapeutic target for treating ischemic heart disease. Translational PerspectiveMitochondrial homeostasis is critical for maintaining healthy cardiac function. Disruption of mitochondrial function causes severe cardiomyopathy and heart failure. In the present study, we show that FAM210A is a mitochondrial translation regulator required for maintaining cardiac mitochondrial homeostasis in vivo. Cardiomyocyte-specific FAM210A deficiency leads to mitochondrial dysfunction and spontaneous cardiomyopathy. Moreover, our results indicate that FAM210A is downregulated in human and mouse ischemic heart failure samples and overexpression of FAM210A protects hearts from myocardial infarction induced heart failure, suggesting that FAM210A mediated mitochondrial translation regulatory pathway can be a potential therapeutic target for ischemic heart disease.

pathology↗

bioRxiv2023

Guide to the construction and use of an adaptive optics two-photon microscope with direct wavefront sensing

Two-photon microscopy, combined with appropriate optical labeling, has enabled the study of structure and function throughout nervous systems. This methodology enables, for example, the measurement and tracking of sub-micrometer structures within brain cells, the spatio-temporal mapping of spikes in individual neurons, and the spatio-temporal mapping of transmitter release in individual synapses. Yet the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300 to 400 {micro}m of neocortex. To obviate this limitation, we measure the wavefront at the focus of the excitation beam and utilize adaptive optics that alters the incident wavefront to achieve an improved focal volume. We describe the constructions, calibration, and operation of a two-photon microscopy that incorporates adaptive optics to restore diffraction-limited resolution throughout the nearly 900 {micro}m depth of mouse cortex. Our realization utilizes a guide star formed by excitation of red-shifted dye within the blood serum to directly measure the wavefront. We incorporate predominantly commercial optical, optomechanical, mechanical, and electronic components; computer aided design models of the exceptional custom components are supplied. The design is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in mouse neocortex by imaging the morphology of somatostatin-expressing neurons at 700 {micro}m beneath the pia, calcium dynamics of layer 5b projection neurons, and glutamate transmission to L4 neurons.

neuroscience↗

bioRxiv2022

RNA binding protein PRRC2B mediates translation of specific proteins and regulates cell cycle progression

Accumulating evidence suggests that posttranscriptional control of gene expression, including RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and sequencing (PAR-CLIP-seq), we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites near the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by polysome-associated RNA-seq, resulting in reduced G1/S phase transition and cell proliferation. Antisense oligonucleotides blocking PRRC2B interactions with CCND2 mRNA decreased its translation, thus inhibiting G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome analysis revealed RNA-independent interactions with eukaryotic translation initiation factors 3 (eIF3) and 4G2 (eIF4G2). The interaction with translation initiation factors is essential for PRRC2B function since the eIF3/eIF4G2-interacting defective mutant, unlike wild-type PRRC2B, failed to rescue the translation deficiency or cell proliferation inhibition caused by PRRC2B knockdown. Altogether, our findings reveal that PRRC2B is essential for efficiently translating specific proteins required for cell cycle progression and cell proliferation. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/520836v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@cc9385org.highwire.dtl.DTLVardef@e92f55org.highwire.dtl.DTLVardef@1228c7corg.highwire.dtl.DTLVardef@1352179_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

A novel PLS1 c.981+1G>A variant causes autosomal-dominant hereditary hearing loss in a family via up-regulation of the PI3K-Akt signaling pathway

BackgroundThe fimbrin protein family contains a variety of proteins, of which Plastin1 (PLS1) is an important member. The latest researches show that variations in the coding region of PLS1 gene is related to the development of deafness. However, it remains unknown about the molecular mechanism of deafness caused by PLS1 gene variants. Methodswhole exome sequencing was performed on the affected family members and healthy ones to identify the pathogenic variants, followed by sanger sequencing. Minigene assay was conducted to investigate the impact of the variant on PLS1 mRNA splicing. The pathogenicity of the variant was further investigated in zebrafish. RNA-sequencing (RNA-seq) was done for analyzing the dysregulated downstream signaling pathways caused by knockdown of PLS1 expression. ResultsWe identified a novel variant PLS1 c.981+1G>A in a large Chinese family with hearing loss and demonstrated that the variant is responsible for the occurrence of hearing loss by inducing Exon8 skipping or deletion of Exon8 (47bp). The variant caused abnormal inner ear phenotypes, characterized by decreased mean otolith distance, anterior otolith diameter, posterior otolith diameter, and cochlear diameter, swimming speed and distance in zebrafish. Furthermore, silencing PLS1 expression significantly upregulated genes in the PI3K-Akt signaling pathway, including Col6a3, Spp1, Itgb3 and Hgf. ConclusionsPLS1 c.981+1G>A is a novel pathogenic variant for hearing loss by inducing Exon8 skipping or deletion of Exon8 (47bp). Up-regulation of the PI3K-Akt signaling pathway plays an important role in the pathogenesis of PLS-1 gene.

genetics↗

bioRxiv2022

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFRs nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

neuroscience↗