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Tian, X.

Publications and source records attributed to Tian, X..

At least 37 records · Page 2Linked to original sources
bioRxiv2026

Therapeutic Stress-induced Activation of PGCC Life Cycle Drives the Resistance Acquisition and Structured Tissue Differentiation

To elucidate how cancer cells generate resistance and defined forms of tissue structure in response to therapeutic stress, we tracked the temporal dynamics of life cycle of polyploid giant cancer cells (PGCCs) induced by the mitotic destabilizer vincristine (VCR). Live-cell fluorescence imaging revealed that VCR activated a distinct endoreplication-based life cycle, which replaced canonical mitosis. PGCCs exhibited reduced proliferative activity, enhanced epithelial-mesenchymal transition (EMT), progressive acquisition of blastomere-like features, and broad multilineage differentiation potential. Both PGCC populations and single PGCC-derived progeny displayed time- and dose-dependent acquisition of malignant traits in vitro and tumorigenic capacity in vivo. PGCC-derived spheroids exhibited ability to differentiate into the cells of origin from three germ layers. Importantly, pre-budding PGCCs induced by higher VCR concentrations exhibited enhanced ability for glandular structure formation and tissue differentiation. Morphologically, the nuclei of PGCC-derived spheroids underwent growth in size, formation of luminal structure, and followed by maturation of lumen. Mechanistically, PGCCs entered a senescent state characterized by elevated senescence-associated secretory phenotype (SASP)-manifested by rich proinflammatory cytokines. Notably, silencing IL1{beta}, IL6, or IL8, or pharmacological inhibition of their receptors, suppressed PGCC formation, budding, EMT, and blastomere-like reprogramming into structured tissue. Our studies provide novel mechanistic insights into early embryogenesis and tumorigenesis at the tissue structural developmental level.

cancer biology↗
bioRxiv2026

Dietary tryptophan mitigates lung ischemia-reperfusion injury via microbiota-derived indole-3-propionate and aryl hydrocarbon receptor signaling

BackgroundLung ischemia-reperfusion (IR) injury drives early morbidity after lung transplantation and cardiothoracic surgery, yet targeted preventive therapies are lacking. The gut-lung axis and microbiota-derived tryptophan metabolites, including indole-3-propionate (IPA), may regulate pulmonary immunity and inflammation. We investigated whether a tryptophan-rich (Trp-Rich) diet attenuates sterile lung IR injury by increasing microbiota-derived indole metabolites and reprogramming alveolar macrophage (AM) inflammatory responses. MethodsC57BL/6 mice received isocaloric tryptophan-standard (Trp-Std; 0.18%) or Trp-Rich (0.60%) diets for 14 days, then underwent unilateral left lung IR (60 min ischemia followed by 60 min reperfusion). Oxygen saturation, lung cytokines, and aryl hydrocarbon receptor (AhR) signaling readouts (Cyp1a1/Cyp1b1) were evaluated. Gut microbiota was profiled by 16S rRNA sequencing, and targeted metabolomics quantified tryptophan metabolites in feces, portal vein (PV) plasma, and lung tissue. To further assess inflammatory priming in vivo, mice were additionally challenged with intratracheal lipopolysaccharide (LPS). Mechanistic studies compared IPA with related indole metabolites in MH-S cells and primary human AMs, including ex vivo nutritional IR, LPS stimulation, and AhR stimulation and blockade using synthetic agonists and antagonists. ResultsTrp-Rich feeding improved post-IR oxygenation, reduced lung IL-1{beta}, and increased pulmonary Cyp1a1/Cyp1b1 gene expression. Trp-Rich diet remodeled the gut microbiota, including enrichment of Bifidobacterium and Lactobacillus, and increased IPA levels across feces, PV plasma, and lung tissue, with lower kynurenine/IPA ratios across matrices. In the LPS intratracheal challenge, Trp-Rich feeding reduced IL-6 levels in lung tissue and systemic plasma. Primary murine AMs isolated from Trp-Rich mice also showed reduced IL-1{beta} and IL-6 release in an ex vivo nutritional IR model. Among tested indole metabolites, IPA showed the strongest dose-dependent suppression of LPS-induced cytokines and chemokines in MH-S cells and primary human AMs, remained active in the ex vivo nutritional IR model, and its anti-inflammatory effect was abrogated by AhR blockade and enhanced by co-treatment with other indole metabolites. ConclusionsA Trp-Rich diet attenuated sterile lung IR injury, coinciding with gut microbiota remodeling, increased systemic and pulmonary IPA, reduced inflammatory priming, and reprogrammed AM responses. These data support diet- or microbiome-directed strategies targeting IPA-AhR signaling to mitigate perioperative lung IR injury. Caption for graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/714281v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1762c91org.highwire.dtl.DTLVardef@1681386org.highwire.dtl.DTLVardef@1da00aforg.highwire.dtl.DTLVardef@162f17f_HPS_FORMAT_FIGEXP M_FIG A tryptophan-rich diet remodels the gut microbiota and indole metabolite profiles, including IPA, enhances alveolar macrophage AhR signaling, and attenuates sterile lung ischemia-reperfusion injury. C_FIG

immunology↗
bioRxiv2026

A synonymous mutation in MSMEG_4729 occurs at a high frequency in spontaneous D29-resistant mutants of Mycobacterium smegmatis

Compassionate use of mycobacteriophage therapy highlights the promising potential of phage therapy as an alternative treatment option for antibiotic-resistant infections when conventional treatments fail. However, realizing the full potential of phage therapy requires addressing key challenges, including host immune responses, the limited arsenal of therapeutically-useful mycobacteriophages, and the emergence of phage resistance. Dissecting the mechanisms of phage resistance is critical for ensuring the effectiveness and sustainability of phage therapy. In this study, we demonstrate that exposure to the lytic mycobacteriophage D29 triggers diverse genetic changes in Mycobacterium smegmatis. A synonymous mutation in MSMEG_4729 arises frequently but is insufficient to confer D29 resistance on its own. Instead, we identified possible Lsr2-independent activation of the lipooligosaccharide (LOS) biosynthesis cluster in a D29-resistant mutant harboring this mutation. We have also detected the possible activity of MSMEG_3213, a type II methyltransferase associated with m6A modifications in M. smegmatis. Finally, we isolated defense escape mutants (DEMs) of D29 capable of overcoming resistance in a strain with the MSMEG_4729 synonymous mutation. This profiling of M. smegmatiss likely defensive arsenal against the therapeutically-useful mycobacteriophage D29 provides a roadmap for further investigations and rational engineering of next-generation mycobacteriophages to combat drug-resistant mycobacterial infections. Impact statementInterest in phage therapy has been gaining traction recently, which is largely due to the serious threat of antimicrobial resistance. However, the efficacy and sustainability of phage therapy is threatened by certain challenges, which includes the ever existent threat of phage resistance. In this study, we identified several likely factors involved in D29 interaction with the model mycobacterium M. smegmatis. These findings set a roadmap for future investigations that would guide rational phage engineering to expand the currently limited arsenal of therapeutically useful mycobacteriophages as well as improve the efficiency of existing ones.

microbiology↗
bioRxiv2026

Likely role of promoter reconstitution in Mpr-mediated D29 resistance by Mycobacterium smegmatis

The multi-copy phage resistance gene (mpr) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr. Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter (rcp) than the original promoter (wtp) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp, implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp-driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis, adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

microbiology↗
bioRxiv2026

A reparative neutrophil subpopulation promotes spinal cord regeneration in zebrafish by controlling macrophage inflammation via Il-4

In mammals, a dysregulated immune response is detrimental to spinal cord repair. In zebrafish, which are capable of spinal cord regeneration, the immune response promotes regeneration. Neutrophils are the first immune cells to arrive at a spinal cord injury site, but their role in successful regeneration is not fully understood. Here we show that ablating neutrophils, including a subpopulation that expresses the cytokine il4, increases expression of il1b (coding for Il-1{beta}) in macrophages/microglia and impairs anatomical and functional recovery after a spinal cord injury in larval zebrafish. Regeneration is fully rescued by over-expression of il4 alone or experimentally reducing Il-1{beta} levels. Disruption of il4 mimics the detrimental effect of neutrophil ablation for axonal regeneration and is also rescued by reducing Il-1{beta} levels. Hence, after spinal cord injury, a pro-regenerative neutrophil subpopulation promotes spinal cord regeneration in larval zebrafish by controlling expression of il1b in macrophages/microglia. For this neutrophil action, il4 expression is necessary and sufficient. HIGHLIGHTS- Neutrophil ablation impairs spinal cord repair in zebrafish - The neutrophil response can be replaced by reducing Il-1{beta} levels - A pro-regenerative subpopulation of neutrophils expresses il4 - il4 overexpression fully rescues effects of neutrophil ablation

neuroscience↗
bioRxiv2026

Environmental temperature is a strong driver of subspecies competition in the Drosophila microbiome

Most microbiome research focusses on the taxonomic composition at the species level to understand the impact of environmental factors, but intraspecific diversity has largely been ignored. To address this significant knowledge gap, we took advantage of the simple, culturable microbiome of Drosophila. First, we documented that natural populations of D. simulans harbor three diverged clades of Lactiplantibacillus plantarum, a key nutritional symbiont. We studied the distinct ecological roles of these three clades by exposing flies with their native microbiome to two temperature regimes in the laboratory. Tracking the three clades within the complete Drosophila microbiome over a period of more than 10 years at two temperatures, we identified strikingly distinct dynamics in response to the selection regime. We confirmed the functional differentiation of the three clades using in vitro growth measurements and in vivo mono-association assays. Our results highlight that environmental selection operates at the subspecies level. Therefore, we conclude that the functional diversification of the microbiome can only be understood when intra- and interspecific diversity is considered.

microbiology↗
bioRxiv2026

Identification and characterization of ethR and ethA genes impacting the sensitivity of Mycobacterium abscessus to ethionamide

Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium with rising global incidence. This pathogen exhibits intrinsic resistance to most antibiotics, presenting a major public health threat. Ethionamide (ETH) requires bioactivation by monooxygenase EthA to form the active ETH-NAD adduct. We previously identified MAB_3513 (NudC) as a phosphohydrolase that confers intrinsic resistance to M. abscessus by hydrolyzing this adduct. However, deletion of nudC results in only partial susceptibility to ETH, indicating the existence of additional resistance mechanisms. This study identified MAB_0984 as the EthR regulator in M. abscessus. Deletion of ethR in a nudC knockout background ({Delta}{Delta}ethR) significantly enhanced ETH-NAD adduct accumulation, leading to hypersusceptibility to ETH. Notably, the {Delta}ethR mutant exhibited higher susceptibility than {Delta}nudC, demonstrating that EthR is a more dominant mediator of ETH resistance than NudC. Furthermore, MAB_0985 (EthA1) and MAB_0103 (EthA2) were identified as the primary EthAs in M. abscessus. Deletion of either gene alone or in combination in the {Delta}nudC reduced adduct formation and increased resistance, while the triple mutant {Delta}{Delta}ethA1{Delta}ethA2 restored wild-type resistance. Using an intergenic region-eGFP reporter system and quantitative reverse transcription-PCR, we demonstrated that EthR confers resistance by specifically suppressing ethA1 expression in M. abscessus. The Mycobacterium tuberculosis EthR inhibitor BDM31343 could boost the efficacy of ETH against M. abscessus by inhibiting EthR. Collectively, this study identified the ethR and ethA genes in M. abscessus for the first time and elucidated their role in mediating resistance to ETH. Therefore, EthR is a promising target for potentiating the efficacy of ETH against M. abscessus. Impact statementMycobacterium abscessus constitutes an escalating global health threat, primarily due to its intrinsic resistance to most antibiotics. This study identifies MAB_0984 (EthR) as a dominant resistance determinant that exerts a more profound impact on ETH susceptibility than the previously characterized NudC. We demonstrate that EthR mediates resistance by specifically repressing the expression of EthA1 (MAB_0985), one of the primary monooxygenases responsible for ETH bioactivation. The EthRMtb inhibitor BDM31343 potentiated the activity of ETH against M. abscessus through inhibition of the EthR. These findings elucidated the mechanism of ETH resistance in M. abscessus, identifying EthR as a promising target for boosting the efficacy of ETH against M. abscessus.

microbiology↗
bioRxiv2026

Glucose is a ligand-like trigger for secretion of postprandial GLP-1

Glucagon-like peptide 1 (GLP-1) is secreted from the intestinal L cells in a glucose-dependent manner. However, the mechanism underlying how glucose stimulates the secretion remains unclear. Here, we found that glucose at postprandial concentrations near the Km of GK for glucose ([~]10 mM), as well as nonmetabolizable glucose analogs, triggers GLP-1 secretion by intestinal L cells through directly binding to glucokinase (GK), without increasing intracellular ATP levels. We also show that in postprandially high glucose, glucose-bound GK interacts and inhibits KATP, as assessed by immunoprecipitation and FRET-FLIM analyses, as well as patch clamping. In addition, we have utilized GK mutants identified in diabetics, demonstrating that those mutants defective in binding to glucose impair GLP-1 secretion in however high glucose, whereas mutants constitutively bound to glucose with defects in catalysis, promote GLP-1 secretion even in low glucose. Furthermore, the KATP mutant unable to bind to GK blocks GLP-1 secretion in high glucose. We have also provided evidence showing that sodium-glucose cotransporter 1 (SGLT1) plays an important role in the basal secretion of GLP-1, but not in high glucose. We have thus revealed that glucose the molecule itself and GK act as a ligand and the receptor, respectively, to induce GLP-1 secretion in response to high glucose, providing new therapeutic avenues targeting metabolic diseases.

biochemistry↗
bioRxiv2025

Fast and slow architectures of cortical dynamics in human brain

The human brain operates across a vast temporal range, from fast perception and action to slow physiological regulation. The capacity has attributed to a unitary cortical gradient of intrinsic timescales, yet such a unidimensional model cannot explain how local circuits simulateously support both rapid external behavior and slow internal body-coupled dynamics. Using SPLIT (spectral piecewise-linear inference of timescales) on a large-scale intracranial stereo-electroencephalography (8,619 contacts, 185 individuals), we identified dissociable fast (~10-100 Hz) and slow (~1-10 Hz) temporal components. Only the fast-component timescales followed the canonical sensorimotor-to-association cortical hierarchy. Slow-component timescales showed no hierarchical gradient, but were instead covaried with heart rate and enriched at site with heart-related neural responses. This dual temporal architecture persisted across wakefulness, rest, sleep, and anesthesia, challenging the unitary view of brain timescales and revealing an intrinsic bipartite organization that is associated with cortical hierarchy or neurophysiology.

neuroscience↗
bioRxiv2025

CGAS (Chloroplast Genome Analysis Suite): An Automated Python Pipeline for Comprehensive Comparative Chloroplast Genomics

BackgroundChloroplast genome analysis underpins plant phylogenetics, comparative genomics, and molecular marker development. Although high-quality chloroplast genomes are now routinely generated, downstream comparative analyses still rely on fragmented toolchains requiring separate tools for annotation, codon analysis, and SNP detection, extensive manual curation, and ad hoc scripting, which limit reproducibility and scalability. ResultsWe present the Chloroplast Genome Analysis Suite (CGAS), an automated Python-based pipeline designed as a comprehensive solution for comparative chloroplast genomics. CGAS integrates nine core analytical modules into a single, reproducible workflow operating directly on annotated GenBank files and pre-aligned FASTA datasets. The pipeline performs automated gene content analysis, publication-ready gene table generation, chloroplast structural characterization (LSC/SSC/IR boundaries and GC content), codon usage and amino acid composition analyses enabling batch processing of 10-50+ genomes, SNP detection with transition/transversion statistics, intron feature characterization, simple sequence repeat identification, and nucleotide diversity profiling. Unlike existing tools, CGAS emphasizes biologically informed handling of edge cases, including trans-spliced genes (e.g., rps12), IR-mediated gene duplication, and annotation artifacts. All outputs are generated in standardized, publication-ready Excel and Word formats with timestamped provenance. ConclusionsCGAS provides a unified, automated, and biologically robust framework for comparative chloroplast genome analysis. By minimizing manual intervention and emphasizing batch processing, the suite substantially accelerates methodologically consistent analyses. The pipeline is openly available at https://github.com/abdullah30/Chloroplast-Genome-Analysis-Suite-CGAS and readily extensible, supporting its adoption as a standardized analytical backend for chloroplast comparative genomics.

bioinformatics↗
bioRxiv2025

A division-associated envelope protein, MAB_2363, drives intrinsic resistance and virulence in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to conventional antibiotics, significantly limiting treatment options. Our previous studies established that MAB_2362 (SteA) is a key regulator of cell division that contributes to intrinsic resistance and virulence. Considering that SteA-like proteins often act alongside SteB counterparts, we hypothesized that the adjacent gene MAB_2363 encodes the corresponding SteB-like division regulator. In this study, we found that deletion of MAB_2363 significantly increased susceptibility to multiple antibiotics and disrupted cell wall permeability. Microscopy revealed striking cell division defects in the mutant, including elongated cell morphology and multiple septa. Subcellular localization of a GFP-MAB_2363 fusion protein demonstrated its enrichment at division septa, confirming its direct involvement in cell division. Deletion of MAB_2363 resulted in attenuated virulence, as evidenced by reduced bacterial survival in macrophages and murine infection models. To assess its functional relation with MAB_2362, we compared the single-deletion mutant of MAB_2363 with the single-deletion mutant of MAB_2362 and the double-deletion mutant of MAB_2362-MAB_2363. Notably, the phenotypes of the MAB_2363 mutant, including cell division defects, antibiotic susceptibility, and virulence, were markedly milder than those of the other two mutants. Collectively, these findings indicate that MAB_2363 functions as a secondary but essential division-associated factor that operates during cell division, thereby influencing intrinsic resistance and virulence in M. abscessus.

microbiology↗
bioRxiv2025

Automatic binding of basic sensory features requires consciousness

Conscious awareness requires establishing coherent percepts. Yet, whether consciousness is necessary for initiating the integration of basic sensory features remains unclear. Competing theories implicate distinct functional regimes of consciousness in the process of feature binding and creating conscious percepts. We used a novel multi-feature oddball paradigm with intracranial stereo-electroencephalography (sEEG) recordings in awake and anesthetized states to investigate the functional boundary of consciousness. In the awake state, the auditory attributes of loudness and tone, as well as the binding of the two features, were automatically encoded without attention to the stimuli in a functionally localized sensory cortical network. In the anesthetized state, the cortical registration of single attributes was preserved, whereas the binding was abolished. Moreover, anesthesia mostly influenced later cortical processes after stimuli offset. These results reveal the borderline of exertion of consciousness between the encoding and manipulation of basic sensory features in local cortical circuits - the functional boundary of consciousness constrains the feedforward binding and recurrent process directly at local rather than global level computations.

neuroscience↗
bioRxiv2025

Characterization of Endogenous and Epitope-Tagged Ubiquitin Expression and Ubiquitination Patterns at Drosophila Neuromuscular Junctions

Ubiquitination is a highly regulated post-translational modification that controls protein stability, activity, localization, and interactions. Disruption of this process contributes to a broad range of human diseases, including cancer, neurodegeneration, and immune dysfunction. The genetic model organism Drosophila melanogaster offers powerful tools to study ubiquitination in vivo, where versatile systems such as GAL4/UAS, Split-GAL4, GeneSwitch, and MARCM can be combined with imaging and biochemical approaches to achieve precise spatial and temporal analyses. To fully exploit these advantages, experimental designs often require epitope-tagged UAS-Ubiquitin transgenes--both modified and unmodified--that accurately recapitulate the behavior of endogenous Ubiquitin. Here, we report the generation and validation of a panel of epitope-tagged UAS-Ubiquitin transgenes. We first characterize the endogenous ubiquitination pattern at larval neuromuscular junctions (NMJs), then compare that with transgenically expressed Ubiquitin in muscle. Our analysis showed that transgenic His-Myc-, HA-, and His-Biotin-tagged Ubiquitin proteins accurately reproduce endogenous postsynaptic ubiquitination patterns. These validated transgenes provide versatile resources for investigating ubiquitination dynamics across diverse cellular and developmental contexts.

genetics↗
bioRxiv2025

PRISM: ancestry-aware integration of tissue-specific genomic annotations enhances the transferability of polygenic scores

The limited transferability of polygenic scores (PGS) across populations constrains their clinical utility and risks exacerbating health disparities, given challenges in multi-ancestry training, fine-mapping, and variant prioritization using genomic annotations, particularly when biologically relevant reference resources are sparse or unavailable for the target population. Here, we introduce PRISM, a transfer learning approach that jointly addresses these challenges to enhance PGS transferability. Applying PRISM to 7352 fine-mapped variants, 414 ENCODE annotations, and 406,659 individuals from the UK Biobank, we demonstrate that ancestry-aware integration of tissue-specific annotations yields the largest gains in predictive performance for African ancestry, with an average improvement of 13.10% (p=1.6x10-5) over annotation-agnostic multi-ancestry PGS. Notably, the best-performing model uses 102-fold fewer annotations than non-specific models, with contributions from broad categories of annotations. Overall, PRISM complements ongoing data diversification efforts by providing an immediately applicable strategy based on the integration of biologically aligned, best-available resources to address genomic health equity.

genetics↗
bioRxiv2025

High-Power Dual-Channel Chamber for High-Frequency Magnetic Neuromodulation

ObjectiveSeveral novel methods, including magnetogenetics and magnetoelectric stimulation, use high frequency alternating magnetic fields to precisely manipulate neural activity. To quantify the behavioral effects of such interventions in a freely moving mouse, we developed a dual-channel magnetic chamber, specifically designed for rate-sensitive magnetothermal-genetic stimulation, and adaptable for other uses of alternating magnetic fields. ApproachThrough an optimized coil design, the system allows independent control of two spatially orthogonal uniform magnetic fields delivered at different frequencies within a 10 x 10 x 6 cm3 chamber suitable for mouse studies. The two channels have nominal frequencies of 50 and 550 kHz with peak magnetic field strengths of 88 and 12.5 mT, achieved with resonant coil drives having peak voltages of 1.6 and 1.8 kV and currents of 1.0 and 0.26 kA, respectively. Additionally, a liquid cooling system enables magnetic field generation for second-level durations, and an observation port and camera allow video capture of the animals behavior within the chamber. Main ResultsThe system generates high-amplitude magnetic fields across two widely separated frequency channels with negligible interference (< 1%). Relatively uniform magnetic field distribution ({+/-}10% across 94% of the chamber volume) is maintained throughout the chamber, and temperature increase of the inner side of the coil enclosure during the operation is limited to < 0.35 {degrees}C/s to ensure in vivo safety. Using cobalt-doped and undoped iron oxide nanoparticles, we demonstrate channel-specific heating rates of 3.5 {degrees}C/s and 1.5 {degrees}C/s, respectively, validating frequency-selectivity. Both channels can run continuously for 4 seconds stably. SignificanceWe present a novel magnetic stimulation platform that combines high-frequency, high-power capability with two independently-controlled channels generating different frequencies, along with a real-time behavioral observation system for freely moving animals. The system supports frequency-multiplexed stimulation strategies for precise modulation of neural activity, making it a versatile tool for advancing magnetogenetics, neural circuit interrogation, and noninvasive stimulation approaches in neuroscience and bioengineering.

bioengineering↗
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

Quantitative Multicolored Deep Imaging of Human Bones Reveals a Composite Osteo-Sinusoidal Niche for Mesenchymal Stromal Cells.

Human bone marrow mesenchymal stromal/stem cells (BM-MSCs) are widely utilized in clinical trials and tissue engineering; however, their native microenvironment remains poorly understood. Here, we introduced a tissue-clearing protocol for human bones and integrated it with simultaneous mRNA and protein detection. Using this protocol, named DeepBone, we spatially mapped BM-MSCs relative to key bone microenvironment components, including human blood capillaries, adipocytes, sinusoids, and bony trabeculae. Quantitative analysis revealed that the native microenvironment of human BM-MSCs in young bone is enriched in vasculature, sinusoids, bone matrix, and adipocytes. In contrast, in aged bone, BM-MSCs showed no preferential association with bone or adipocytes. Proliferative BM-MSCs were predominantly found along blood vessels. Moreover, we identified a specialized microenvironment for BM-MCs in young bone, characterized by sinusoids coiled around trabeculae and enriched by R-type vessels. These findings provide novel insights into the native niches of BM-MSCs, offering a foundation for the development of tissue-engineering strategies that mimic their physiological context.

cell biology↗