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Biology subjects

Miao, X.

Publications and source records attributed to Miao, X..

At least 19 recordsLinked to original sources

Platycodin D2 Targets Vimentin to Suppress Fibroblast-Myofibroblast Transition: A Novel Therapeutic Strategy Against Pulmonary Fibrosis

Background Pulmonary fibrosis is a progressive lung disease characterized by excessive extracellular matrix deposition and irreversible decline in lung function with limited effective therapeutic targets. Vimentin, a type III intermediate filament protein, plays a critical role in fibroblast activation and fibrogenesis, representing a promising drug target. Methods Novel vimentin ligands were screened via virtual screening. SPR assay verified compound-vimentin binding affinity. CETSA and CD spectroscopy analyzed protein structural changes after compound binding. In vitro FMT model and in vivo bleomycin-induced pulmonary fibrosis model were used to assess antifibrotic effects, and mutagenesis assays identified key binding amino acid residues. Results Platycodin D2 (PD2) from Platycodon grandiflorus was identified as a novel high-affinity vimentin ligand (Kd = 392.8 nM). PD2 binds vimentin directly, alters its secondary structure and induces insoluble aggregation. It effectively inhibits TGF-{beta}1-induced FMT in vitro and mitigates bleomycin-induced pulmonary fibrosis in vivo, reducing collagen deposition and improving animal survival. Critically, mutagenesis validation confirmed that key residues (Glu138 in chain A, Glu132 and Glu143 in chain B, as well as Glu50 and Arg54 in chain C) are essential for this interaction. Conclusions Our findings establish PD2 as a novel pharmacological inducer of aberrant vimentin aggregation, presenting a distinct target-driven strategy for antifibrotic drug development.

cell biology↗

Singlet-states-mediated highly efficient two-photon fluorescence in organic dyes

This study reports a brand-new continuous-wave-excited (CW-excited) two-photon fluorescence emission mechanism in indocyanine green (ICG), an organic fluorescent dye widely used in clinical practice. This mechanism is based on the excited state absorption (ESA) process of the first singlet excited state. Intramolecular electrons sequentially absorb two photons to reach a high-energy singlet excited state, followed by direct radiative transition to the ground state to generate fluorescence. The entire process is exclusively mediated by singlet states. We further summarize the essential requirements for organic dyes to realize this luminescence mechanism. First, the dye must possess at least two well-separated singlet excited states with distinct energies, corresponding to two absorption peaks at different wavelengths in the absorption spectrum. The wavelength of the high-energy singlet excited state is about half that of the first singlet excited state. Second, the peak in the absorption spectrum corresponding to the transition from the ground state to the first singlet excited state has a sufficiently large molar extinction coefficient. Third, the first singlet excited state exhibits the capability of ESA. Fourth, electrons at the high-energy singlet excited state can directly transit to the ground state and emit fluorescence. We validated this mechanism in a variety of organic dyes satisfying the above conditions, confirming its universality. Using CW laser as the excitation source, we achieved two-photon fluorescence imaging of mouse cerebral blood vessels at a depth of 400 m, which clearly resolves three-dimensional vascular networks with high resolution. We also performed two-photon fluorescence imaging on human gastric cancer tissue samples at a depth of around 150 m, which provides a low-cost strategy for clinicians to rapidly acquire high-contrast tumor tissue images.

bioengineering↗

Estimating uncertainty in family-based GWAS

Standard genome-wide association studies (GWASs) are vulnerable to confounding factors, including stratification, assortative mating, and dynastic effects. Family studies such as sibling-based GWAS (sib-GWAS) mitigate such confounding and are becoming the tool of choice for teasing apart direct genetic effects---causal effects of one's genotype on one's own phenotype---from other factors. However, due in part to their smaller sample sizes, sib-GWAS allelic effect estimates are much noisier than standard (i.e., population-based) GWAS estimates. The quantification of this estimation uncertainty is essential for many uses of sib-GWAS, including polygenic scoring, causal inference (e.g., Mendelian randomization), disentangling direct from indirect familial effects, and measuring assortative mating. Here, we investigate sources of uncertainty in sib-GWAS allelic effect estimators. We study their impacts on the biases of three uncertainty measurement methods, including two that are commonly used and a new resampling-based approach we propose. We find that heterogeneity in allelic effects or heteroskedasticity across families (e.g., due to variation in genetic backgrounds or environments) can bias existing methods, and that this bias is more severe for small samples and rare variants. In contrast, the resampling-based approach we propose is approximately unbiased under all scenarios we considered. We validate our theoretical predictions, as well as the importance of effect heterogeneity and heteroskedasticity, using simulations and empirical analysis in the UK Biobank. In sum, this study helps understand the sources of uncertainty in family-based genotype-phenotype association studies and provides a robust method to estimate uncertainty.

genetics↗

A Novel Glycoproteomics Platform for High-Throughput Identification of Disease-Associated Glycoforms

Glycosylation is a critical post-translational modification, and its aberrant forms are potent disease biomarkers; however, the comprehensive, site-specific identification of all glycosites and glycoforms across an entire proteome remains prohibitively slow and computationally demanding. To address this bottleneck and accelerate biomarker discovery, we introduce the Glycoproteomics Data Analysis Software (GDAS), a novel, high-throughput platform designed to provide confident, proteome-scale identification of disease-specific glycoforms. GDAS streamlines the analysis through a core, multi-step workflow: it initially employs an ultrafast open search (e.g., MSFragger-Glyco) on mass spectrometry data to rapidly screen and statistically reduce the vast proteome database to a manageable subset of significantly regulated glycoproteins, conserving computational resources for subsequent, in-depth, targeted N- and O-glycosylation analysis using specialized tools (e.g., GlycReSoft and O-Pair). Furthermore, a unique Final Analysis Module utilizes an advanced statistical and machine learning pipeline (incorporating Bootstrap/Bayesian methods, XGBoost, and Random Forest) to integrate quantitative results and generate a robust, comprehensive glycosylation score. We demonstrate GDASs power to recognize biologically relevant glycosylation changes in targeted proteins by validating it using published Alzheimers disease data. GDAS can be downloaded from https://github.com/yang-lab/GDAS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/708406v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@189b270org.highwire.dtl.DTLVardef@1221045org.highwire.dtl.DTLVardef@15a3f50org.highwire.dtl.DTLVardef@1f2d43f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

Wake consolidates event memory, sleep binds it with context

Sleep is thought to be superior to wakefulness in consolidating memory, due to a hippocampus-driven process enhancing the contextual integration of events. We compared recall of associative memory in different contexts, and found that memory is also consolidated during wakefulness, although in a qualitatively distinct way. Healthy participants performed a cued stimulus-response (S-R) learning task requiring speeded object classification. Learning was followed by 2 hours of sleep or wakefulness. Reaction times revealed stronger S-R memory after sleep than wakefulness with retrieval in the same context, and this benefit was linked to increased sleep spindle activity. In contrast, S-R memory was stronger after wakefulness than sleep when tested in a different context. This wake-dependent enhancement had a fast onset and was reversed by delayed sleep. We conclude that, unlike sleep strengthening the binding of an event with its context, wakefulness stabilizes event representations itself, i.e., not bound to a specific context.

neuroscience↗

SARS-CoV-2 S protein activates the HIV latent reservoir through the mTOR pathway

During the global COVID-19 pandemic, mRNA vaccines using the S protein as antigen were widely used.Vaccine-induced S proteins can persist in vivo for weeks, triggering low-level immune activation. HIV latent reservoir maintenance is a major challenge for ART therapy, especially when immune pressure is waning. This then raises critical questions for HIV-infected patients: does prolonged exposure to S proteins affect HIV latent reservoir stability? Recent studies have pointed out that S proteins may activate the mTOR signaling pathway, which in turn affects the immune response and metabolic processes of cells. And the mTOR pathway is closely related to the maintenance and activation of HIV latent reservoir. However, how S proteins affect the HIV latent reservoir and the mechanism of activation are unclear. To explore the mechanism of how SARS-CoV-2 S proteins regulate the HIV latent reservoir and to explore whether S proteins regulate the HIV latent reservoir through the mTOR pathway, we constructed an in vitro HIV latent reservoir model for our experiments.To evaluate the potential role of S protein in HIV latent reservoir activation, relevant markers of HIV latent reservoir activation were detected using ELISA, flow cytometry, and RT-qPCR; and the relationship between S protein and mTOR was also detected by WB, CO-IP, and IFC.It was found that S proteins activated the HIV latent reservoir while increasing mTOR expression. It was further observed that mTOR inhibitors significantly inhibited S protein-induced activation of the HIV latent reservoir, and mTOR activators reversed the inhibitory effect of mTOR inhibitors on HIV latent reservoir activation. In summary, we found that S proteins activated the HIV latent reservoir through the mTOR pathway. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/670887v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@f6d87aorg.highwire.dtl.DTLVardef@1fe4cc0org.highwire.dtl.DTLVardef@60eefeorg.highwire.dtl.DTLVardef@1ea9a34_HPS_FORMAT_FIGEXP M_FIG S protein interacts with mTOR and activates the mTOR-p-p70S6K-pS6 pathway, which promotes HIV transcription C_FIG

immunology↗

Saddle Curvature Association of nsP1 Facilitates the Replication Complex Assembly of Chikungunya Virus in Cells

The replication of positive-sense RNA viruses, including the pathogenic SARS-CoV-1 and -2, DENV, and ZIKV, often takes place in curved cellular membrane compartments of tens to hundreds of nanometers in diameter within host cells. The non-structural proteins (nsPs) of viruses are found to critically control the formation and maintenance of such unique nanoscale membrane structures. However, the molecular mechanism underlying the association and assembly of nsPs around curved membranes has remained elusive, due to the technical difficulties of imaging the nanoscale interaction in live cells. In this study, we leveraged vertically aligned nanostructures to define membrane curvatures in live cells and investigated the assembly of viral nsPs on these curved membranes. Using Chikungunya virus as a model system, we found that its nsP1 was preferentially bound to and stabilized around positively curved membrane sites and the preference became more apparent as the radius of curvature decreased to 150 nm or smaller. This preferential accumulation was mainly attributed to the hydrophobic residues on recently identified membrane association loops (MA loops) of each nsP1 monomer. Molecular dynamics simulations further confirmed the improved binding kinetics and stability of nsP1 on positively curved membranes, especially when a 12-mer ring was formed by the nsP1 dodecamer. More interestingly, a saddle curvature association was enabled by the 3D coordination of nsP1 monomers along the dodecamer ring, where the MA loops of individual nsP1 monomers sensed a wide range of the positively curved membrane in the x-z plane while the rigid nsP1 dodecamer ring stabilized the negative curvature in the x-y plane. Its strong coupling to the saddle curvature fulfilled the need to constrain the neck of the membrane spherule for viral replication. Strikingly, productive CHIKV replication exhibited strong enrichment on patterned nanostructure array, proving the effectiveness of membrane curvature-guided assembly of the functional replication complex. Our findings revealed that the cell membrane facilitated the local enrichment of viral nsPs in a curvature- dependent manner. It opens up membrane curvature modulation as a new dimension to dissect the formation and regulation of membrane compartments for viral replication.

biophysics↗

Red-shifted GRAB acetylcholine sensors for multiplex imaging in vivo

The neurotransmitter acetylcholine (ACh) is essential in both the central and peripheral nervous systems. Recent studies highlight the significance of interactions between ACh and various neuromodulators in regulating complex behaviors. The ability to simultaneously image ACh and other neuromodulators can provide valuable information regarding the mechanisms underlying these behaviors. Here, we developed a series of red fluorescent G protein-coupled receptor activation-based (GRAB) ACh sensors, with a wide detection range and expanded spectral profile. The high-affinity sensor, rACh1h, reliably detects ACh release in various brain regions, including the nucleus accumbens, amygdala, hippocampus, and cortex. Moreover, rACh1h can be co-expressed with green fluorescent sensors in order to record ACh release together with other neurochemicals in various behavioral contexts using fiber photometry and two-photon imaging, with high spatiotemporal resolution. These new ACh sensors can therefore provide valuable new insights regarding the functional role of the cholinergic system under both physiological and pathological conditions.

neuroscience↗

In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

neuroscience↗

The study on the impact of indoleacetic acid on enhancing the ability of the rumen's original microecology to degrade aflatoxin B1.

Aflatoxin B1 (AFB1) poses a serious threat to the health of cattle and is strictly monitored in their diets. Rumen microorganisms in cattle have the ability to degrade AFB1, but this capability is limited. Enhancing the degradation capacity of the original rumen microecology for AFB1 is a novel approach. From a molecular perspective,indoleacetic-3-acid (IAA) promotes the expression of cytochromes, which can improve the degradation of AFB1. Therefore, this study aims to investigate the impact of different concentrations of IAA on the degradation of AFB1 by the rumen microecology. Experiments used rumen fluid from three adult cows as donors, and the cows were all fed the same total mixed ration. Rumen fluid was collected from these three cannulated cows before morning feeding to prepare in vitro fermentation fluid. The experiments used a completely randomized design, with each treatment repeated four times. The results showed that as the fermentation time increased, the content of AFB1 gradually decreased, with a degradation rate of up to 75.73% after 24 h. AFB1 altered the rumen fermentation pattern, with a significant reduction in the content of acetic acid (P<0.05) and a significant decrease in the acetic acid to propionic acid ratio (P<0.05). It also affected the rumen microecology, causing a significant reduction in the abundance of Ruminococcus amylophilus, Prevotella ruminicola, and Fusobacterium succinogenes (P<0.05). In addition, this study found that with the increase in the amount of IAA added, the content of AFB1 in the rumen gradually decreased. IAA enhances the degradation capacity of the original rumen microecology for AFB1, and the addition of IAA alleviates the impact of AFB1 on Ruminococcus amylophilus, Prevotella ruminicola, and Fusobacterium succinogenes in the rumen (P<0.05). Moreover, the addition of IAA can promote the stability of the rumen microecology, with significantly higher acetic acid and acetic acid to propionic acid ratios in the fermentation fluid compared to the non-added group (P<0.05). In summary, the addition of IAA can improve the degradation capacity of the rumen microecology for AFB1, providing a new solution for alleviating the impact of AFB1 on animal health.

microbiology↗

Wnt signaling dosage controlled by a Ctnnb1 enhancer balances homeostasis and tumorigenesis of intestinal epithelia

The {beta}-catenin-dependent canonical Wnt signaling is pivotal in organ development, tissue homeostasis, and cancer. Here we identified an upstream enhancer of Ctnnb1 - the coding gene for {beta}-catenin, named ieCtnnb1 (intestinal enhancer of Ctnnb1), which is crucial for intestinal homeostasis. ieCtnnb1 is predominantly active in the base of small intestinal crypts and throughout the epithelia of large intestine. Knockout of ieCtnnb1 led to a reduction in Ctnnb1 transcription, compromising the canonical Wnt signaling in intestinal crypts. Single-cell sequencing revealed that ieCtnnb1 knockout altered epithelial compositions and potentially compromised functions of small intestinal crypts. While deletion of ieCtnnb1 hampered epithelial turnovers in physiologic conditions, it prevented occurrence and progression of Wnt/{beta}-catenin-driven colorectal cancers. Human ieCTNNB1 drove reporter gene expression in a pattern highly similar to mouse ieCtnnb1. ieCTNNB1 contains a single-nucleotide polymorphism associated with CTNNB1 expression levels in human gastrointestinal epithelia. The enhancer activity of ieCTNNB1 in colorectal cancer tissues was stronger than that in adjacent normal tissues. HNF4 and phosphorylated CREB1 were identified as key trans-factors binding to ieCTNNB1 and regulating CTNNB1 transcription. Together, these findings unveil an enhancer-dependent mechanism controlling the dosage of Wnt signaling and homeostasis in intestinal epithelia.

cell biology↗

Enrichment of glycoRNAs using galactose oxidase, hydrazide chemistry, and glycosidase digestion

Ribonucleic acid (RNA), essential for protein production and immune function, undergoes glycosylation, a process that attaches carbohydrates to RNA, creating unique glycoRNAs. These sugar-coated RNA molecules regulate immune responses and may be related to immune disorders. However, studying them is challenging due to RNAs fragility. Therefore, a robust method for identifying glycosylated RNA is important. To address this, we optimized methods for enriching and identifying glycoRNAs, opening doors to explore their potential interactions with immune receptors and tumor suppression. Our approach involved investigating factors such as preservation solutions, enzyme buffers, digestion temperature, and incubation time. We successfully achieved efficient digestion of both N-linked and O-linked glycoRNAs at room temperature using 25 mM ammonium bicarbonate, demonstrating the effectiveness of this method. Additionally, RNA preservation in RNAlater at -80{degrees}C allows controlled release of glycoRNAs within hours. While sequential digestion of different glycoRNA types is possible, significant degradation occurs after the first enzyme step. Thus, we recommend separate harvesting for each type of glycoRNA. These optimized protocols, utilizing SPCgRNA and TnORNA methods, pave the way for further research on N- and O-glycoRNAs in health and disease.

biochemistry↗

Membrane curvature catalyzes actin nucleation through nano-scale condensation of N-WASP-FBP17

Actin remodeling is spatiotemporally regulated by surface topographical cues on the membrane for signaling across diverse biological processes. Yet, the mechanism dynamic membrane curvature prompts quick actin cytoskeletal changes in signaling remain elusive. Leveraging the precision of nanolithography to control membrane curvature, we reconstructed catalytic reactions from the detection of nano-scale curvature by sensing molecules to the initiation of actin polymerization, which is challenging to study quantitatively in living cells. We show that this process occurs via topographical signal-triggered condensation and activation of the actin nucleation-promoting factor (NPF), Neuronal Wiskott-Aldrich Syndrome protein (N-WASP), which is orchestrated by curvature-sensing BAR-domain protein FBP17. Such N-WASP activation is fine-tuned by optimizing FBP17 to N-WASP stoichiometry over different curvature radii, allowing a curvature-guided macromolecular assembly pattern for polymerizing actin network locally. Our findings shed light on the intricate relationship between changes in curvature and actin remodeling via spatiotemporal regulation of NPF/BAR complex condensation.

molecular biology↗

multimedia: Multimodal Mediation Analysis of Microbiome Data

Mediation analysis has emerged as a versatile tool for answering mechanistic questions in microbiome research because it provides a statistical framework for attributing treatment effects to alternative causal pathways. Using a series of linked regressions, this analysis quantifies how complementary data relate to one another and respond to treatments. Despite these advances, existing softwares rigid assumptions often result in users viewing mediation analysis as a black box. We designed the multimedia R package to make advanced mediation analysis techniques accessible, ensuring that statistical components are interpretable and adaptable. The package provides a uniform interface to direct and indirect effect estimation, synthetic null hypothesis testing, bootstrap confidence interval construction, and sensitivity analysis, enabling experimentation with various mediator and outcome models while maintaining a simple overall workflow. The software includes modules for regularized linear, compositional, random forest, hierarchical, and hurdle modeling, making it well-suited to microbiome data. We illustrate the package through two case studies. The first re-analyzes a study of the microbiome and metabolome of Inflammatory Bowel Disease patients, uncovering potential mechanistic interactions between the microbiome and disease-associated metabolites, not found in the original study. The second analyzes new data about the influence of mindfulness practice on the microbiome. The mediation analysis highlights shifts in taxa previously associated with depression that cannot be explained indirectly by diet or sleep behaviors alone. A gallery of examples and further documentation can be found at https://go.wisc.edu/830110. IMPORTANCEMicrobiome studies routinely gather complementary data to capture different aspects of a microbiomes response to a change, such as the introduction of a therapeutic. Mediation analysis clarifies the extent to which responses occur sequentially via mediators, thereby supporting causal, rather than purely descriptive, interpretation. multimedia is a modular R package with close ties to the wider microbiome software ecosystem that makes statistically rigorous, flexible mediation analysis easily accessible, setting the stage for precise and causally informed microbiome engineering.

bioinformatics↗

Integrated Transcriptomic and Metabolomic Profiling of Sheep Ovarian Tissues Confer their association in Fecundity associated Pathways

BackgroundLow fertility is considered the major constraint in sheep rearing industry depending on several factors like, estrus cycle, ovulation rate and litter size but fecundity of ewe plays a key role in sheep reproduction, influenced by several intrinsic and extrinsic factors. However, genetic improvements of traits associated with reproduction through conventional breeding is a very complex and slow process. In current study, we went through a comprehensive integration of high throughput transcriptomic and metabolomics approaches to understand the role of key regulatory genes and metabolites in fecundity of two different and widely raised sheep breeds (Small Tail Han & Dolang) in different regions of China. ResultUPLC/MS/MS system based metabolomic profiling of ovarian tissue from both breeds results into the identification of 1,423 metabolites, including 542 DEMs (379 upregulated and 163 downregulated). Integration of metabolomics and transcriptomics data identified 48 pathways contributed by 37 genes and 85 metabolites through regulatory network analysis. Functional enrichment analysis showed significantly enriched pathways associated with fecundity including Riboflavin metabolism, xenobiotics, bile acid biosynthesis, and Drug metabolism, which produces hormones for regulation of ovarian function, ovulation, and establishment of pregnancy. Further, analyzed two restrictive constrained plots analyzed via multivariate statistical analysis. In one plot complement component C3 associated with Leukotriene D4, and Uridine 5-diphosphate involved in the processes of Neuroactive legend receptor interaction pathway and in second plot IFNGR1 associated with Progesterone, Fumaric acid, and Cortisone involved in the processes of cancer pathway and any disruptions in hormonal balance may induce cancer, which can affect fertility, menstrual cycles, and overall reproductive health. ConclusionExpression profiling, functional enrichments, co-expression network analysis and integrated transcriptomemetabolome data showed gene-metabolite association in energy metabolism, Inflammation, and drug metabolism, all of which play a role in ovarian physiology and ovarian metabolic disorders. Identification and validation of genes, metabolites, and gene-metabolite interactions will help to elucidate the regulatory mechanisms and pathways underlying sheep fecundity and could be leveraged to improve reproductive traits. Graphical Abstract: Scheme of Study O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/572136v1_ufig1.gif" ALT="Figure 1"> View larger version (119K): org.highwire.dtl.DTLVardef@12a42b7org.highwire.dtl.DTLVardef@182169forg.highwire.dtl.DTLVardef@19b2ed4org.highwire.dtl.DTLVardef@11f9eab_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Improved dual-color GRAB sensors for monitoring dopaminergic activity in vivo

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a vast network of dopaminergic projections. To fully dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent GPCR activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity, and signal-to-noise properties with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in freely moving mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala, and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

neuroscience↗

Replication Protein A1 is essential for DNA damage repair during mammalian oogenesis

Persistence of unrepaired DNA damage in oocytes is detrimental and may cause genetic aberrations, miscarriage, and infertility. RPA, an ssDNA-binding complex, is essential for various DNA-related processes. Here we report that RPA plays a novel role in DNA damage repair during postnatal oocyte development after meiotic recombination. To investigate the role of RPA during oogenesis, we inactivated RPA1 (replication protein A1), the largest subunit of the heterotrimeric RPA complex, specifically in oocytes using two germline-specific Cre drivers (Ddx4-Cre and Zp3-Cre). We find that depletion of RPA1 leads to the disassembly of the RPA complex, as evidenced by the absence of RPA2 and RPA3 in RPA1-deficient oocytes. Strikingly, severe DNA damage occurs in RPA1-deficient GV-stage oocytes. Loss of RPA in oocytes triggered the canonical DNA damage response mechanisms and pathways, such as activation of ATM, ATR, DNA-PK, and p53. In addition, the RPA deficiency causes chromosome misalignment at metaphase I and metaphase II stages of oocytes, which is consistent with altered transcript levels of genes involved in cytoskeleton organization in RPA1-deficient oocytes. Absence of the RPA complex in oocytes severely impairs folliculogenesis and leads to a significant reduction in oocyte number and female infertility. Our results demonstrate that RPA plays an unexpected role in DNA damage repair during mammalian folliculogenesis.

developmental biology↗

Monitoring norepinephrine release in vivo using next-generation GRABNE sensors

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter, yet researches using prototype NE sensors were limited by their low sensitivities. Here, we developed next-generation versions of GPCR activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response, high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

neuroscience↗