DNA-based ForceChrono probes for deciphering single-molecule force dynamics in living cells.

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

The gut-to-brain axis for toxin-induced defensive responses.

After ingestion of toxin-contaminated food, the brain initiates a series of defensive responses (e.g., nausea, retching, and vomiting). How the brain detects ingested toxin and coordinates diverse defensive responses remains poorly understood. Here, we developed a mouse-based paradigm to study defensive responses induced by bacterial toxins. Using this paradigm, we identified a set of molecularly defined gut-to-brain and brain circuits that jointly mediate toxin-induced defensive responses. The gut-to-brain circuit consists of a subset of Htr3a+ vagal sensory neurons that transmit toxin-related signals from intestinal enterochromaffin cells to Tac1+ neurons in the dorsal vagal complex (DVC). Tac1+ DVC neurons drive retching-like behavior and conditioned flavor avoidance via divergent projections to the rostral ventral respiratory group and lateral parabrachial nucleus, respectively. Manipulating these circuits also interferes with defensive responses induced by the chemotherapeutic drug doxorubicin. These results suggest that food poisoning and chemotherapy recruit similar circuit modules to initiate defensive responses.

AMPK directly phosphorylates TBK1 to integrate glucose sensing into innate immunity.

Nutrient sensing and damage sensing are two fundamental processes in living organisms. While hyperglycemia is frequently linked to diabetes-related vulnerability to microbial infection, how body glucose levels affect innate immune responses to microbial invasion is not fully understood. Here, we surprisingly found that viral infection led to a rapid and dramatic decrease in blood glucose levels in rodents, leading to robust AMPK activation. AMPK, once activated, directly phosphorylates TBK1 at S511, which triggers IRF3 recruitment and the assembly of MAVS or STING signalosomes. Consistently, ablation or inhibition of AMPK, knockin of TBK1-S511A, or increased glucose levels compromised nucleic acid sensing, while boosting AMPK-TBK1 cascade by AICAR or TBK1-S511E knockin improves antiviral immunity substantially in various animal models. Thus, we identify TBK1 as an AMPK substrate, reveal the molecular mechanism coupling a dual sensing of glucose and nuclei acids, and report its physiological necessity in antiviral defense.

Mechanistic formulation of inorganic membranes at the air-liquid interface.

Freestanding functional inorganic membranes, beyond the limits of their organic and polymeric counterparts1, may unlock the potentials of advanced separation2, catalysis3, sensors4,5, memories6, optical filtering7 and ionic conductors8,9. However, the brittle nature of most inorganic materials, and the lack of surface unsaturated linkages10, mean that it is difficult to form continuous membranes through conventional top-down mouldings and/or bottom-up syntheses11. Up to now, only a few specific inorganic membranes have been fabricated from predeposited films by selective removal of sacrificial substrates4-6,8,9. Here we demonstrate a strategy to switch nucleation preferences in aqueous systems of inorganic precursors, resulting in the formation of various ultrathin inorganic membranes at the air-liquid interface. Mechanistic study shows that membrane growth depends on the kinematic evolution of floating building blocks, which helps to derive the phase diagram based on geometrical connectivity. This insight provides general synthetic guidance towards any unexplored membranes, as well as the principle of tuning membrane thickness and through-hole parameters. Beyond understanding  a complex dynamic system, this study comprehensively expands the traditional notion of membranes in terms of composition, structure and functionality.

Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

A mitochondrial EglN1-AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress.

Mitochondria play essential roles in cancer cell adaptation to hypoxia, but the underlying mechanisms remain elusive. Through mitochondrial proteomic profiling, we here find that the prolyl hydroxylase EglN1 (PHD2) accumulates on mitochondria under hypoxia. EglN1 substrate-binding region in the ß2ß3 loop is responsible for its mitochondrial translocation and contributes to breast tumor growth. Furthermore, we identify AMP-activated protein kinase alpha (AMPKα) as an EglN1 substrate on mitochondria. The EglN1-AMPKα interaction is essential for their mutual mitochondrial translocation. After EglN1 prolyl-hydroxylates AMPKα under normoxia, they rapidly dissociate following prolyl-hydroxylation, leading to their immediate release from mitochondria. In contrast, hypoxia results in constant EglN1-AMPKα interaction and their accumulation on mitochondria, leading to the formation of a Ca2+ /calmodulin-dependent protein kinase 2 (CaMKK2)-EglN1-AMPKα complex to activate AMPKα phosphorylation, ensuring metabolic homeostasis and breast tumor growth. Our findings identify EglN1 as an oxygen-sensitive metabolic checkpoint signaling hypoxic stress to mitochondria through its ß2ß3 loop region, suggesting a potential therapeutic target for breast cancer.

Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Hydrogel-based molecular tension fluorescence microscopy for investigating receptor-mediated rigidity sensing.

Extracellular matrix (ECM) rigidity serves as a crucial mechanical cue impacting diverse biological processes. However, understanding the molecular mechanisms of rigidity sensing has been limited by the spatial resolution and force sensitivity of current cellular force measurement techniques. Here we developed a method to functionalize DNA tension probes on soft hydrogel surfaces in a controllable and reliable manner, enabling molecular tension fluorescence microscopy for rigidity sensing studies. Our findings showed that fibroblasts respond to substrate rigidity by recruiting more force-bearing integrins and modulating integrin sampling frequency of the ECM, rather than simply overloading the existing integrin-ligand bonds, to promote focal adhesion maturation. We also demonstrated that ECM rigidity positively regulates the pN force of T cell receptor-ligand bond and T cell receptor mechanical sampling frequency, promoting T cell activation. Thus, hydrogel-based molecular tension fluorescence microscopy implemented on a standard confocal microscope provides a simple and effective means to explore detailed molecular force information for rigidity-dependent biological processes.

MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

The origin of different bending stiffness between double-stranded RNA and DNA revealed by magnetic tweezers and simulations.

The subtle differences in the chemical structures of double-stranded (ds) RNA and DNA lead to significant variations in their biological roles and medical implications, largely due to their distinct biophysical properties, such as bending stiffness. Although it is well known that A-form dsRNA is stiffer than B-form dsDNA under physiological salt conditions, the underlying cause of this difference remains unclear. In this study, we employ high-precision magnetic-tweezer experiments along with molecular dynamics simulations and reveal that the relative bending stiffness between dsRNA and dsDNA is primarily determined by the structure- and salt-concentration-dependent ion distribution around their helical structures. At near-physiological salt conditions, dsRNA shows a sparser ion distribution surrounding its phosphate groups compared to dsDNA, causing its greater stiffness. However, at very high monovalent salt concentrations, phosphate groups in both dsRNA and dsDNA become fully neutralized by excess ions, resulting in a similar intrinsic bending persistence length of approximately 39 nm. This similarity in intrinsic bending stiffness of dsRNA and dsDNA is coupled to the analogous fluctuations in their total groove widths and further coupled to the similar fluctuation of base-pair inclination, despite their distinct A-form and B-form helical structures.

Bone marrow-derived IGF-1 orchestrates maintenance and regeneration of the adult skeleton.

Insulin-like growth factor I (IGF-1) is a key regulator of tissue growth and development in response to growth hormone stimulation. In the skeletal system, IGF-1 derived from osteoblasts and chondrocytes are essential for normal bone development; however, whether bone marrow (BM)-resident cells provide distinct sources of IGF-1 in the adult skeleton remains elusive. Here, we show that BM stromal cells (BMSCs) and megakaryocytes/platelets (MKs/PLTs) express the highest levels of IGF-1 in adult long bones. Deletion of Igf1 from BMSCs by Lepr-Cre leads to decreased bone formation, impaired bone regeneration, and increased BM adipogenesis. Importantly, reduction of BMSC-derived IGF-1 contributes to fasting-induced marrow fat accumulation. In contrast, deletion of Igf1 from MKs/PLTs by Pf4-Cre leads to reduced bone formation and regeneration without affecting BM adipogenesis. To our surprise, MKs/PLTs are also an important source of systemic IGF-1. Platelet-rich plasma (PRP) from Pf4-Cre; Igf1f/fmice showed compromised osteogenic potential both in vivo and in vitro, suggesting that MK/PLT-derived IGF-1 underlies the therapeutic effects of PRP. Taken together, this study identifies BMSCs and MKs/PLTs as two important sources of IGF-1 that coordinate to maintain and regenerate the adult skeleton, highlighting reciprocal regulation between the hematopoietic and skeletal systems.

Universality in RNA and DNA deformations induced by salt, temperature change, stretching force, and protein binding.

Nucleic acid deformations play important roles in many biological processes. The physical understanding of nucleic acid deformation by environmental stimuli is limited due to the challenge in the precise measurement of RNA and DNA deformations and the complexity of interactions in RNA and DNA. Magnetic tweezers experiments provide an excellent opportunity to precisely measure DNA and RNA twist changes induced by environmental stimuli. In this work, we applied magnetic tweezers to measure double-stranded RNA twist changes induced by salt and temperature changes. We observed RNA unwinds when lowering salt concentration, or increasing temperature. Our molecular dynamics simulations revealed the mechanism: lowering salt concentration or increasing temperature enlarges RNA major groove width, which causes twist decrease through twist-groove coupling. Combining these results with previous results, we found some universality in RNA and DNA deformations induced by three different stimuli: salt change, temperature, and stretching force. For RNA, these stimuli first modify the major groove width, which is transduced into twist change through twist-groove coupling. For DNA, these stimuli first modify diameter, which is transduced into twist change through twist-diameter coupling. Twist-groove coupling and twist-diameter coupling appear to be utilized by protein binding to reduce DNA and RNA deformation energy cost upon protein binding.

Impairments in endogenous AMPA receptor dynamics correlates with learning deficits in Alzheimer's disease model mice.

AMPA receptors (AMPARs) play a critical role in synaptic plasticity and learning and memory, and dysfunction or dysregulation of AMPARs could lead to various neurological and psychiatric disorders, such as Alzheimer's disease (AD). However, the dynamics and/or longitudinal changes of AMPARs in vivo during AD pathogenesis remain elusive. Here, employing 5xFAD SEP-GluA1 KI mice, we investigated endogenous AMPA receptor dynamics in a whisker deflection-associated Go/No-go learning paradigm. We found a significant increase in synaptosomal AMPA receptor subunits GluA1 in WT mice after learning, while no such changes were detected in 7-mo-old 5xFAD mice. Daily training led to an increase in endogenous spine surface GluA1 in Control mice, while this increase was absent in 5xFAD-KI mice which correlates with its learning defects in Go/No-go paradigm. Furthermore, we demonstrated that the onset of abnormal AMPAR dynamics corresponds temporally with microglia and astrocyte overactivation. Our results have shown that impairments in endogenous AMPA receptor dynamics play an important role in learning deficits in 5xFAD mice and AD pathogenesis.

CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice.

Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake.

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.

Calcium signals regulate the functional differentiation of thymic iNKT cells.

How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4+ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca2+ buffering in NKT2 cells. In addition, the low Ca2+ extrusion rate may also contribute to the higher Ca2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.

Event-driven acquisition for content-enriched microscopy.

A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition framework, in which neural-network-based recognition of specific biological events triggers real-time control in an instant structured illumination microscope. Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because event-driven acquisition allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content.

HDAC2 counteracts vascular calcification by activating autophagy in chronic kidney disease.

Vascular calcification is a major risk factor for cardiovascular disease mortality, with a significant prevalence in chronic kidney disease (CKD). Pharmacological inhibition of histone acetyltransferase has been proven to protect against from vascular calcification. However, the role of Histone Deacetylase 2 (HDAC2) and molecular mechanisms in vascular calcification of CKD remains unknown. An in vivo model of CKD was established using mouse fed with a high adenine and phosphate diet, and an in vitro model was produced using human aortic vascular smooth muscle cells (VSMCs) stimulated with ß-glycerophosphate (ß-GP). HDAC2 expression was found to be reduced in medial artery of CKD mice and ß-GP-induced VSMCs. Overexpression of HDAC2 attenuated OPN and OCN upregulation, α-SMA and SM22α downregulation, and calcium deposition in aortas of CKD. The in vitro results also demonstrated that ß-GP-induced osteogenic differentiation was inhibited by HDAC2. Furthermore, we found that HDAC2 overexpression caused an increase in LC3II/I, a decrease in p62, and an induction of autophagic flux. Inhibition of autophagy using its specific inhibitor 3-MA blocked HDAC2's protective effect on osteogenic differentiation in ß-GP-treated VSMCs. Taken together, these results suggest that HDAC2 may protect against vascular calcification by the activation of autophagy, laying out a novel insight for the molecular mechanism in vascular calcification of CKD.

NDRG1 Signaling Is Essential for Endothelial Inflammation and Vascular Remodeling.

BACKGROUND: NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. METHODS AND RESULTS: By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1ß (interleukin-1ß) and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. CONCLUSIONS: Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.

STGIC: A graph and image convolution-based method for spatial transcriptomic clustering.

Spatial transcriptomic (ST) clustering employs spatial and transcription information to group spots spatially coherent and transcriptionally similar together into the same spatial domain. Graph convolution network (GCN) and graph attention network (GAT), fed with spatial coordinates derived adjacency and transcription profile derived feature matrix are often used to solve the problem. Our proposed method STGIC (spatial transcriptomic clustering with graph and image convolution) is designed for techniques with regular lattices on chips. It utilizes an adaptive graph convolution (AGC) to get high quality pseudo-labels and then resorts to dilated convolution framework (DCF) for virtual image converted from gene expression information and spatial coordinates of spots. The dilation rates and kernel sizes are set appropriately and updating of weight values in the kernels is made to be subject to the spatial distance from the position of corresponding elements to kernel centers so that feature extraction of each spot is better guided by spatial distance to neighbor spots. Self-supervision realized by Kullback-Leibler (KL) divergence, spatial continuity loss and cross entropy calculated among spots with high confidence pseudo-labels make up the training objective of DCF. STGIC attains state-of-the-art (SOTA) clustering performance on the benchmark dataset of 10x Visium human dorsolateral prefrontal cortex (DLPFC). Besides, it's capable of depicting fine structures of other tissues from other species as well as guiding the identification of marker genes. Also, STGIC is expandable to Stereo-seq data with high spatial resolution.