Biology subjects

Ai, H.

Publications and source records attributed to Ai, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Coupling Luciferase Activation and BRET Enables High-Contrast Red-Window Calcium Imaging

Genetically encoded bioluminescent calcium indicators enable excitation-free imaging of Ca2+ dynamics with low background, no photobleaching, and improved tissue penetration. However, many exhibit lower dynamic ranges than their fluorescent counterparts, motivating the development of new sensor configurations, particularly for imaging in the red optical window, where tissue absorption and scattering are reduced. Here, we developed HyBRIC and HyBRIC2, two hybrid indicators that integrate Ca2+-dependent luciferase activation and bioluminescence resonance energy transfer (BRET) within a single molecular architecture. When paired with pyCTZ luciferin, they exhibited an approximately 55-fold Ca2+-dependent signal increase in the red optical window in vitro. Compared with HyBRIC, HyBRIC2 showed improved brightness and responsiveness in mammalian cells. It robustly resolved induced Ca2+ dynamics in HEK293T and HeLa cells, with particularly strong red-window responses. Together, these findings expand the limited repertoire of responsive red-window bioluminescent calcium indicators and establish hybrid multiplicative sensing as a modular strategy for improving biosensor contrast.

biochemistry↗

bioRxiv2026

Exploring genetic, expression and regulatory patterns of parental alleles in Muscovy duck (Cairina moschata) using haplotype-resolved assemblies

BackgroundAlthough the biological mechanism for heterosis has been debated for a long time, heterosis is widely utilized to increase the global productivity of crops and livestock. Recently, the mechanism has been well characterized in crops and livestock with a male-heterogametic XY system due to genomic assembly advancements, especially the availability of haploid genomes. However, the biological mechanism for heterosis remains unclear in poultry possessing the female-heterogametic ZW system. ResultsHere, we assembled chromosome-level diploid and haploid genomes of the Muscovy duck. We developed an efficient and cost-effective method to assemble 12 variation graph-haploid Muscovy duck genomes from three full-sibling pairs with high quality using short-read Illumina sequences. We further characterized genetic, expression and regulatory patterns of parental alleles at multiple scales. We found that maternal haploid genomes generally had more open chromatin organization and higher accessibility, and higher levels of gene expression, while showing similar DNA methylation levels when compared to paternal haploid genomes. In contrast, the female paternal Z chromosome showed the most, and the male paternal Z chromosome presented more, relaxed chromatin organization and chromatin accessibility, and gene expression compared to the male maternal Z chromosome. Thus, the ZW system largely relies on compensation and balance to regulate gene expression on the sex Z chromosome. Moreover, we identified non-Mendelian regions covering 0.26% of the genome ([~]3.18 Mb). These regions contained lower gene density, GC content, and repeat sequence frequency, but were enriched for DNA motifs bound by transcription factors, likely leading to a compacted chromatin structure and lower chromatin accessibility. ConclusionsOur work here provides a comprehensive profile of parental alleles genetic, expression and regulatory patterns in the female-heterogametic ZW system, and might be useful for the utilization of heterosis in poultry.

genomics↗

bioRxiv2026

Hierarchical Anchoring Gating Dictates Specific H4K16 Acetylation by the Human MSL Complex

Histone acetylation is a versatile post-translational modification essential for diverse biological processes. While nearly all histone acetyltransferases (HATs) act promiscuously on multiple lysine residues, the human male-specific lethal (MSL) acetyltransferase complex mediates strictly site-specific acetylation of histone H4 at lysine 16 (H4K16ac), with its underlying mechanism remaining enigmatic. Here, we leveraged chemical protein synthesis to engineer a semisynthetic nucleosome probe that traps the MSL complex in a catalytically engaged state and determined its 3.12 [A] cryo-electron microscopy (cryo-EM) structure, which reveals a hierarchical anchoring-gating mechanism responsible for stringent H4K16 selectivity. The MSL complex adopts a dual-anchoring mode to bind nucleosomal DNA at superhelix location (SHL) 1.5 and the H2A-H2B acidic patch. Three spatially coordinated gating elements (the MSL1 anchor helix, KAT8 gating helix, and KAT8 gating hairpin) then conformationally constrain the H4 N-terminal tail, funneling H4K16 side chain exclusively into the active site while sterically occluding flanking lysines. Targeted mutations disrupting these interfaces impair H4K16ac in vitro and in cells, with concomitant defects in chromatin accessibility and transcription. Beyond the monomeric 1:1 complex, cryo-EM analysis identifies a 2:2 MSL-nucleosome assembly, wherein MSL3 dimerization mediates inter-nucleosomal contacts linked to genome-wide H4K16ac patterning. Collectively, our work delineates a residue-to-domain framework in which a chromatin acetylation writer hardwires specific catalysis into nucleosome engagement and scales this precision to higher-order chromatin architecture, providing a mechanistic foothold for understanding aberrant MSL-encoded chromatin programs in diseases.

biochemistry↗

bioRxiv2025

Steric Gating Dictates Selective Activation of BIRC6 by UBA6 Over UBA1

The giant E2-E3 chimera BIRC6 is specifically activated by the ubiquitin-activating enzyme UBA6, but not by UBA1. This specificity allows BIRC6 to ubiquitinate downstream substrates (e.g., caspases) and thereby regulate apoptosis. However, the molecular mechanism that underlies UBA6mediated specific activation of BIRC6 remains elusive. Here, we used a UbDha (Ub residue 76 dehydroalanine) probe to achieve one-step, activity-based trapping of the transient transthioesterification intermediate, and resolved the cryo-EM structures of two key complexes: 1) the doubly loaded UBA6-BIRC6UBC-UbT-UbA complex (3.4 [A] resolution) and 2) the singly loaded UBA6-BIRC6UBC-UbT complex (3.3 [A] resolution). Structural analysis reveals that the specific BIRC6-UBA6 pairing is dictated by steric compatibility between an insertion loop (residues 4649- 4653) in BIRC6s UBC domain and UBA6s gate helix. Unlike UBA1, UBA6 avoids steric clash with this insertion loop via two key conformational changes: a ~30{degrees} clockwise rotation and a 19 [A] displacement of its gate helix. These conformational changes collectively create a compatible cavity to accommodate the insertion loop. Biochemical validation confirms two key findings: 1) truncating BIRC6s insertion loop rescues UBA1-mediated ubiquitin charging of BIRC6UBC; 2) swapping UBA6s gate helix with that of UBA1 abolishes UBA6s transthioesterification activity towards BIRC6UBC. Our study provides key structural insights into the molecular basis of E1-E2 pairing specificity and advances our mechanistic understanding of apoptotic signalling.

biochemistry↗

bioRxiv2025

hpGRISZ: a high-performance fluorescent biosensor for in vivo imaging of synaptic zinc dynamics

Despite the crucial role of synaptic Zn2+ in neurotransmission and neural processing, direct in vivo measurement of Zn2+ transients has remained challenging due to the limited responsiveness of existing fluorescent indicators. Here we report hpGRISZ (high-performance green indicator for synaptic Zn2+), an ultraresponsive turn-on green fluorescent biosensor engineered through iterative linker optimization, mutagenesis, and directed evolution. hpGRISZ exhibits exceptional brightness, thermostability, and a large fluorescence response (F/F0 {approx} 25) with micromolar affinity suitable for detecting extracellular synaptic Zn2+ release. We comprehensively characterized hpGRISZ in vitro, in mammalian cells, and in vivo. A membrane-anchored version of hpGRISZ traffics robustly to the cell surface under physiological conditions, where it retains strong responsiveness and supports wide-field, two-photon, and fiber photometry recordings in awake mice. These assays revealed synaptic Zn2+ dynamics across distinct brain regions and neuronal circuits. Together, our findings establish hpGRISZ as a powerful tool for dissecting zinc signaling in neural circuits and as a prototype for next-generation genetically encoded biosensors for in vivo imaging.

neuroscience↗

bioRxiv2025

A Cryptic Interfacial Pocket Uncovered in Full CRL4CRBN-IKZF3 Ubiquitylation Complex Enhances IMiD Efficacy

Immunomodulatory imide drugs (IMiDs) redirect the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ligase to ubiquitylate and degrade disease-linked proteins, but the full picture of how IMiDs glue the neosubstrate within the whole CRL4CRBN complex remains unknown. We determined cryo-electron microscopy structures of eight approved or clinical IMiDs in full CRL4CRBN ubiquitylation complexes at resolutions up to 3.4 [A], and revealed how these structurally distinct IMiDs exploit the structural plasticity of CRL4CRBN to organize conformationally conserved and compact active ubiquitylation assemblies with the neosubstrate IKZF3. Four "next-generation" IMiDs were found to additionally engage a cryptic gluing-driven interfacial (GDI) pocket at the non-degron zinc finger 3 (ZF3) of IKZF3, which contributes to their enhanced efficacy and neosubstrate specificity. The identification of cryptic gluing-driven pockets formed only in full ubiquitylation complexes provides new structure-directed design opportunities for IMiDs to improve therapeutic efficacy.

biochemistry↗

bioRxiv2025

Unique Gluing Effect of ASXL1 K351 Monoubiquitination Stimulates PR-DUB-Mediated Nucleosomal H2AK119Ub Deubiquitination

Protein ubiquitination plays a critical role in regulating fundamental biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for its role in targeting proteins for degradation, ubiquitination can also modulate enzymatic activity. However, all of the known mechanisms of such modulation involve spatially constrained mechanisms that take place near the enzyme-substrate interfaces. Here, we report an unprecedented regulatory paradigm mediated by ubiquitination that is located distal to the enzyme-substrate interface but can still activate the polycomb repressive deubiquitinase (PR-DUB) complex. Using cryo-electron microscopy, molecular dynamics (MD) simulations, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and kinetics assays, we determined that the ASXL1 K351 monoubiquitination promotes PR-DUB-mediated nucleosomal H2AK119Ub deubiquitination by stabilizing the catalytic pocket of PR-DUB, causing significant increase in maximal catalytic velocity (Vmax) but almost no change in the substrate binding affinity (Km). Structural analysis revealed that the ubiquitin moiety at ASXL1 K351 bridges the BAP1 and ASXL1 subunits, acting as an intramolecular "glue" that constrains the conformational dynamics of these subunits without altering the nucleosome binding interface. Further MD and HDX-MS results demonstrated the monoubiquitination of ASXL1 K351 enables PR-DUB to effectively maintain a spatial arrangement of the catalytic conformation that closely aligns with substrate cleavage. This study expands our understanding of the mechanisms of ubiquitin function to include intramolecular fastening with a molecular gluing effect and provides a deeper mechanistic understanding of PR-DUB activation in nucleosomal H2AK119Ub removal.

biochemistry↗

bioRxiv2025

7-ketocholesterol contributes to microglia-driven increases in astrocyte reactive oxygen species in Alzheimer's disease

Oxidative stress is a prominent feature of Alzheimers disease. Within this context, cholesterol undergoes oxidation, producing the pro-inflammatory product 7-ketocholesterol (7-KC). In this study, we observe elevated levels of 7-KC in the brains of the 3xTg mouse model of AD. To further understand the contribution of 7-KC on the oxidative environment, we developed a method to express a genetically encoded fluorescent hydrogen peroxide (H2O2) sensor in astrocytes, the primary source of cholesterol in the brain. With this sensor, we discovered that 7-KC increases H2O2 levels in astrocytes in vivo, but not when directly applied to astrocytes in vitro. Interestingly, when 7-KC was applied to a microglia cell line alone or mixed astrocyte and microglia cultures, it resulted in microglia activation and increased oxidative stress in astrocytes. Depletion of microglia from 3xTg mice resulted in reduced 7-KC in the brains of these mice. Taken together, these findings suggest that 7-KC, acting through microglia, contributes to increased astrocyte oxidative stress in AD. This study sheds light on the complex interplay between cholesterol oxidation, microglia activation, and astrocyte oxidative stress in the pathogenesis of AD.

neuroscience↗

bioRxiv2024

Pathway enrichment-based unsupervised learning identifies novel subtypes of cancer-associated fibroblasts in pancreatic ductal carcinoma

Existing single-cell clustering methods are based on gene expressions that are susceptible to dropout events in single-cell RNA sequencing (scRNA-seq) data. To overcome this limitation, we proposed a pathway-based clustering method for single cells (scPathClus). scPathClus first transforms single-cell gene expression matrix into pathway enrichment matrix and generates its latent feature matrix. Based on the latent feature matrix, scPathClus clusters single cells using the method of community detection. Applying scPathClus to PDAC scRNA-seq datasets, we identified two types of cancer-associated fibroblasts (CAFs), termed csCAFs and gapCAFs, which highly expressed complement system and gap junction-related pathways, respectively. Spatial transcriptome analysis revealed that gapCAFs and csCAFs are located at cancer and non-cancer regions, respectively. Pseudotime analysis suggest a potential differentiation trajectory from csCAFs to gapCAFs. Bulk transcriptome analysis showed that gapCAFs-enriched tumors are more endowed with tumor-promoting characteristics and worse clinical outcomes, while csCAFs-enriched tumors confront stronger antitumor immune responses. Compared to established CAF subtyping methods, this method displays better prognostic relevance.

bioinformatics↗

bioRxiv2024

Mechanistic Insights into the Stimulation of the Histone H3K9 Methyltransferase Clr4 by Proximal H3K14 Ubiquitination

H3K9 methylation is an evolutionarily conserved hallmark of heterochromatin and plays crucial roles in chromosome segregation, genome stability, and gene expression regulation. Clr4 is the sole histone methyltransferase responsible for catalyzing H3K9 methylation in Schizosaccharomyces pombe. Clr4 K455/K472 automethylation and histone H3K14 ubiquitination (H3K14Ub) are vital activators of the catalytic activity of Clr4, ensuring appropriate heterochromatin deposition and preventing deleterious gene silencing. While the mechanism by which automethylation activates Clr4 was recently elucidated, the mechanism of the significantly pronounced stimulatory effect of H3K14Ub on Clr4 remains unclear. Here we determined the crystal structures of Clr4 bound to ubiquitinated and unmodified H3 peptides at resolutions of 2.60 [A] and 2.39 [A], respectively. Our structures reveal a synergistic mechanism underlying the stronger stimulatory effect by H3K14Ub compared to automethylation: site-specific ubiquitination constrained by the H3K14 linkage increases substrate affinity through multivalent interactions between ubiquitin and Clr4. Additionally, H3K14Ub facilitates the allosteric transition of Clr4 from an inactive apo conformation to a hyperactive "catalyzing state", accompanied by conformational changes in the C-SET-insertion (SI) region, complete release of the autoregulatory loop (ARL), and retraction of the {beta}9/10 loop. Finally, we propose a structural model for the Clr4 catalytic-regulatory cycle, depicting varying levels of conformational regulation mediated by automethylation and ubiquitination. This work provides structural insights into the interplay between different histone modifications and their collective impact on epigenetic regulation.

molecular biology↗

bioRxiv2024

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally-defined K63-Polyubiquitylated Linker Histone H1

The chemical synthesis of histones with homogeneous modifications is a potent approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). Here, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine-Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on ubiquitylated chromatosomes with different ubiquitin lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally-defined polyubiquitylated H1.0 by CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

biochemistry↗

bioRxiv2024

ATP-Independent Water-Soluble Luciferins Enable Non-Invasive High-Speed Video-Rate Bioluminescence Imaging of Mice

NanoLuc luciferase and its derivatives are attractive bioluminescent reporters recognized for their efficient photon production and ATP independence. However, utilizing them for in vivo imaging poses notable challenges. Low substrate solubility has been a prominent problem, limiting in vivo brightness, while substrate instability hampers consistent results and handling. To address these issues, we developed a range of caged PEGylated luciferins with improved stability and water solubility of up to 25 mM, resulting in substantial bioluminescence increases in mouse models. This advancement has created the brightest and most sensitive luciferase-luciferin combination, enabling high-speed video-rate imaging of freely moving mice with brain-expressed luciferase. Furthermore, we developed a bioluminescent Ca2+ indicator with exceptional sensitivity to physiological Ca2+ changes and paired it with a new substrate to showcase non-invasive, video-rate imaging of Ca2+ activity in a defined brain region in awake mice. These innovative substrates and the Ca2+ indicator are poised to become invaluable resources for biological and biomedical fields.

synthetic biology↗

bioRxiv2024

Nine high-quality Anas genomes provide new insights into Anas evolution and domestication

The evolutionary origin and genetic architecture of domestic animals are becoming more tractable as the availability of more domestic species genomes. Evolutionary studies of wild and domestic organisms have yielded many fascinating discoveries, while the stories behind the species diversity of Anas or the domestication of duck were largely unknown. Here, we assembled eight chromosome-level Anas genomes. Together with our recently available Pekin duck genome, we investigated Anas phylogeny, genetic differentiation, and gene flow. Extensive phylogenetic inconsistencies were observed in Anas genomes, particularly two phylogeny conflicts between autosome and Z-chromosome. However, the Z chromosome was less impacted by introgression and more suitable to elucidate phylogenetic relationships than autosomes. From the Z-chromosome perspective, we found that the speciation of Anas platyrhynchos and Anas zonorhyncha accompanied with female-biased gene flow, and remodeled duck domestication history. Moreover, we constructed an Anas pan-genome and identified several differentiated SVs between domestic and wild ducks. These SVs might act as repressors/enhancers to regulate their neighboring genes (i.e., GHR and FER), which represented the promising "domestication genes". Additionally, Anas genomes were found being presented LTR retrotransposon bursts, which might largely contribute to functional shifts of genes involved in duck domestication (i.e., MITF and IGF2BP1). This study opens a new window to unravel avian speciation and domestication from Z chromosome.

genomics↗

bioRxiv2024

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/581057v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ffeb68org.highwire.dtl.DTLVardef@b83871org.highwire.dtl.DTLVardef@81b34corg.highwire.dtl.DTLVardef@1224b0b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Capturing Snapshots of Nucleosomal H2A K13/K15 Ubiquitination Mediated by the Monomeric E3 Ligase RNF168

AbstractsThe DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, plays a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent Lys13 and Lys15 sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to ubiquitinate H2AK13/15 site-specifically has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c[~]Ub in complex with nucleosomes. Here, we developed mechanism-based chemical trapping strategies and determined the cryo-EM structures of the RNF168/UbcH5c-Ub/NCP complex captured in transient H2AK13/15 monoubiquitination and adjacent dual-monoubiquitination reactions. Our structural analysis revealed that RNF168 stably binds to the nucleosomal H2A-H2B acidic patch through a basic helix with multiple interactions, which positions the UbcH5c active centre directly over the H2A N-terminus, providing a "helix-anchoring" mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the "compass-binding" mode of dimeric E3 ligases. Furthermore, our chemically synthesized ubiquitinated histones have enabled the elucidation of the efficiency of Ub installation and the interplay between the initial and subsequent Ub modifications on the adjacent H2A K13 and K15 sites. Overall, our work not only provides structural snapshots of H2A K13/K15 site-specific monoubiquitination and adjacent dual-monoubiquitination, but also offers a near-atomic resolution structural framework for understanding how pathogenic mutations or physiological modifications affect the molecular function of RNF168 in H2A K13/15 ubiquitination.

biochemistry↗

bioRxiv2023

Mesoscale functional organization and connectivity of color, disparity, and naturalistic texture in human second visual area

Although parallel processing has been extensively studied in the low-level geniculostriate pathway and the high-level dorsal and ventral visual streams, less is known at the intermediate-level visual areas. In this study, we employed high-resolution fMRI at 7 Tesla to investigate the columnar and laminar organizations for color, disparity, and naturalistic texture in the human secondary visual cortex (V2), and their informational connectivity with lower and higher order visual areas. Although fMRI activations in V2 showed reproducible interdigitated color-selective thin and disparity-selective thick "stripe" columns, we found no clear evidence of columnar organization for naturalistic textures. Cortical depth-dependent analyses revealed the strongest color-selectivity in the superficial layers of V2, along with both feedforward and feedback informational connectivity with V1 and V4. Disparity selectivity was similar across different cortical depths of V2, which showed significant feedforward and feedback connectivity with V1 and V3ab. Interestingly, the selectivity for naturalistic texture was strongest in the deep layers of V2, with significant feedback connectivity from V4. Thus, while local circuitry within cortical columns is crucial for processing color and disparity information, feedback modulations from V4 play a dominant role in processing naturalistic textures in area V2.

neuroscience↗

bioRxiv2023

SHRIMP: Genetically Encoded mScarlet-derived Red Fluorescent Hydrogen Peroxide Sensor with High Brightness and Minimal Photoactivation

Red fluorescent protein (RFP) derived indicators are popular due to advantages such as increased imaging depth and reduced autofluorescence and cytotoxicity. However, most RFP-based indicators have low brightness and are susceptible to blue-light-induced photoactivation. In this study, we aimed to overcome the limitations of existing red fluorescent indicators. We utilized mScarlet-I, a highly bright and robust monomeric RFP, to develop a circularly permuted variant called cpmScarlet. We further engineered cpmScarlet into a novel red fluorescent indicator specifically for hydrogen peroxide (H2O2), a crucial reactive oxygen species (ROS) involved in redox signaling and oxidative stress. The resultant indicator, SHRIMP (mScarlet-derived H2O2 Redox Indicator with Minimal Photoactivation), exhibited excitation and emission peaks at [~]570 and 595 nm, respectively, and demonstrated a maximum five-fold fluorescence turn-off response to H2O2. Importantly, SHRIMP was not susceptible to blue-light-induced photoactivation and showed high brightness both in its purified protein form and when expressed in mammalian cells. We successfully employed SHRIMP to visualize H2O2 dynamics in mammalian cells with exogenously added H2O2 and in activated macrophages. Additionally, we demonstrated its utility for multiparameter imaging by co-expressing SHRIMP with GCaMP6m, a green fluorescent calcium indicator, enabling simultaneous monitoring of H2O2 and calcium dynamics in mammalian cells in response to thapsigargin (TG) and epidermal growth factor (EGF) stimulation. Furthermore, we expressed SHRIMP in isolated primary mouse islet tissue, and SHRIMP exhibited excellent brightness and capability for effective detection of H2O2 production during streptozotocin (STZ)-induced {beta}-cell damage. This study successfully transformed mScarlet-I, a bright and robust monomeric RFP, into a circularly permuted variant (cpmScarlet) and developed the first cpmScarlet-based genetically encoded fluorescent indicator called SHRIMP. SHRIMP exhibits high brightness and insensitivity to photoactivation and is a valuable tool for real-time monitoring of H2O2 dynamics in various biological systems. Further research may yield an expanded family of cpmScarlet-based red fluorescent indicators with enhanced photophysical properties.

biochemistry↗

bioRxiv2023

Comprehensive catalogs for microbial genes and metagenome-assembled genomes of the swine lower respiratory tract microbiome identify the relationship of microbial species with lung lesions

Understanding the community structure and functional capacity of the lower respiratory tract microbiome is crucial for elucidating its roles in respiratory tract diseases. However, there are few studies about it owing to the difficulty in obtaining microbial samples from the lower respiratory tract. Here we collected 745 microbial samples from the porcine lower respiratory tract by harvesting 675 pigs, and constructed a gene catalog containing 10,337,194 nonredundant genes, of which only 30% could be annotated taxonomically. We obtained 397 metagenome-assembled genomes (MAGs) including 111 MAGs with high-quality. These 397 MAGs were further clustered into 292 species-level genome bins (SGBs), among which 56% SGBs are unknown with current databases. Combining with the lung lesion phenotype, we found that Mycoplasma hyopneumoniae strains and the adhesion-related virulence factors harboring in their genomes were significantly associated with porcine lung lesions, implying the role of adhesion and overgrowth of pathogenic M. hyopneumoniae in host lung diseases. This study provided important resources for the study of porcine lower respiratory tract microbiome and lung health.

microbiology↗