Biology subjects

Zhang, X.

Publications and source records attributed to Zhang, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Dermal bone grows by cellular invasion and intercalary biomineralization

Dermal bone architecture bears great evolutionary significance for jawed vertebrates. Its thickness growth is believed to rely on matrix apposition by superficial osteoblasts. To test this hypothesis we employ genetic lineage mapping, conditional gene ablation, intravital matrix labelling and molecular 3D analysis at single cell resolution in vivo. We uncover an invasive mechanism incompatible with apposition. The pervasive arrangement of two layers sandwiching a third spongy layer develops from a molecularly defined bi-layer via a previously unknown developmental module: Outer layer osteoblasts form rosette-like assemblies around single invading cells. The latter bear unique molecular signatures and form de novo sheets inside the spongy layer, communicating with the layer below. The transcription factor Hand2 organizes this module by orchestrating rosette formation, their molecular heterogeneity, cellular invasion and spongy layer thickness growth. Surprisingly, invading osteoblasts secrete new biomineral matrix inside the older matrix, which keeps expanding. Such intercalary biomineralization provides new perspectives for bone biology and the evolution of endochondral ossification.

developmental biology↗

bioRxiv2026

A discrete Connexin26+ neural crest lineage emerges in mid-life and mediates enhanced central brainstem responses to elevated CO2 levels for deep breathing.

The precise control of breathing is fundamental to vertebrate survival. Increased PCO2 in blood and brain parenchyma causes an increase in both the frequency and volume of lung ventilation. We have previously demonstrated that CO2 directly binds to connexin26 (Cx26) hemichannels causing them to open and allow release ATP. We now document the role of Cx26 as a direct physiological CO2 sensor in vivo. Here we describe a unique Cx26+ neural crest cell lineage that populates the ventral brainstem in the vicinity of the PreBoetz nucleus/caudal CO2 chemosensory area during middle age and dies again in old age. Ablating Cx26 genetically specifically within this population of about 20 cells by two independent neural crest Cre-driver lines leads to a loss of local ATP release in the posterior chemosensory area as well as a 40% reduction in elevated tidal volume responses specifically in middle age, as measured by whole-body plethysmography. These in vivo effects change over a life-time: they directly mirror the arrival, wiring in middle age and later death of this cell population in old age. This is a first known example of a middle age change in cranial neural crest lineage composition and highlights the significant power of very few cells for global metabolism. Such lineage-dependent middle-age dynamics also impacts upon the evolution of eusociality: altricial naked pups of our common amniote/synapsid ancestors were heated by by the breath of their (middle-aged) carers, sensing elevated CO2 levels in hypercapnic burrows. Such mechanistic exaptation enabled small amniotes to sense and survive the lethal global CO2 spikes during the Permo-Triassic and other extinction events.

neuroscience↗

bioRxiv2026

A bipolar disorder-associated ultra-rare AKAP11 protein-truncating variant attenuates stimulus-dependent PKA activation and induces anxiety- and depression-related behaviors in mice

Rare genetic variants associated with psychiatric disorders are thought to have larger individual effect sizes and direct biological roles, offering a unique opportunity for understanding causal relationships between genetic architecture and disease phenotypes. A recent whole-exome sequencing meta-analysis identified an increased burden of ultra-rare protein-truncating variants (PTVs) in AKAP11 in patients with bipolar disorder and schizophrenia. AKAP11 encodes an A-kinase anchoring protein that mediates the association of protein kinase A (PKA) and its substrates. How ultra-rare AKAP11 variants contribute to psychiatric disorder pathogenesis remains unknown. Here, using CRISPR-Cas9 genome editing, we introduced into the mouse Akap11 locus an ultra-rare PTV identified in a human patient with bipolar disorder. This ultra-rare PTV reduced Akap11 mRNA and protein levels without producing stable novel mRNA isoforms, leading to Akap11 haploinsufficiency. Male heterozygous mice exhibited behavioral abnormalities consistent with anxiety- and depression-like phenotypes. Transcriptomic and proteomic profiling of the neocortex revealed molecular changes involving synaptic organization, synaptic signaling, and neurite morphogenesis. Notably, the heterozygous mutant showed selective elevation in type I, but not type II, PKA regulatory subunits, as well as PKA catalytic subunits, in the neocortex. Functionally, these changes were accompanied by reduced phosphorylation of PKA substrates, attenuated stimulus-dependent PKA activity in cortical excitatory neurons, and impaired nascent dendritic development in cortical neurons. Together, our study presents a clinically relevant and genetically precise mouse model that provides mechanistic insight into how ultra-rare PTVs may contribute to psychiatric disorder pathogenesis, and a platform for testing rational therapeutics.

neuroscience↗

bioRxiv2026

Distinct Roles of Working Memory and Inhibitory Control in Speech-in-Noise Perception

Difficulty perceiving speech in noise is the primary complaint of patients seeking hearing healthcare, even absent overt losses in audiometric thresholds. Yet current clinical practice largely focuses on sensory function while ignoring the potentially outsized effects of executive function on everyday suprathreshold listening. Speech perception in noise depends on the dynamic interplay between the fidelity of sensory coding along the auditory pathway and higher-order top-down cognitive processes that support goal-directed listening, both of which can independently change across the lifespan. Here we examined how two subcomponents of executive function, working memory and inhibitory control, vary across the adult lifespan and contribute to speech-in-noise performance. Two hundred participants aged 18 to 73 with normal hearing thresholds were tested on a validated online auditory psychophysics platform. Working memory was assessed with an n-back task and inhibitory control with an auditory Stroop task. Participants then performed two speech-in-noise tasks: a modified rhyme test emphasizing sensory coding through energetic masking at the cochlea, and a matrix sentence task emphasizing executive function through informational masking. Age significantly affected performance on both tasks. Aging was associated with reduced inhibitory control (increased Stroop cost) but no significant effect on working memory. Regression analyses revealed that both working memory and inhibitory control predicted performance on the informational masking task, whereas only inhibitory control predicted performance on the energetic masking task. These findings underscore the critical role of executive function in everyday suprathreshold listening and support including cognitive measures in clinical assessments of hearing difficulties.

neuroscience↗

bioRxiv2026

Superficial spinal Tac1-lineage neurons are polymodal nociceptive

Tac1-lineage neurons in the spinal dorsal horn have been implicated in coping behavior in response to sustained noxious stimuli, but their developmental origin, cellular heterogeneity, and functional contribution remain incompletely understood. Here, we characterized the molecular identity, developmental trajectory, electrophysiological properties, and sensory responses of spinal Tac1-lineage neurons. In adults, Tac1-lineage neurons comprised a heterogeneous population of excitatory and inhibitory interneurons and excitatory projection neurons distributed across the superficial dorsal horn. In contrast, at embryonic day 16.5, Tac1-lineage neurons in laminae I/IIo were exclusively projection neurons, revealing a marked developmental transition in the composition of the Tac1 lineage. Electrophysiological recordings from spinal cord slices further demonstrated substantial functional heterogeneity, with most Tac1-lineage neurons exhibiting phasic or single-spike firing and smaller populations displaying tonic or reluctant firing. In vivo calcium imaging in anesthetized mice revealed prominent responses to noxious mechanical and thermal stimulation. Interestingly, Tac1-lineage neurons used distinct strategies to encode cold and heat intensity. Together, these findings reveal pronounced developmental and functional heterogeneity within the Tac1 lineage and demonstrate that adult Tac1-lineage neurons are predominately polymodal nociceptive.

neuroscience↗

bioRxiv2026

Dermal bone remodelling by invasive osteoblasts evolved in stem gnathostomes

The evolutionary origin of the human face can be traced to the earliest vertebrates with a dermal skeleton in which canonical skeletal Issues and cell types arose. To elucidate its developmental evolution we performed genetic mosaic labelling, 3D single cell analysis and fluorescent matrix birthdating in vivo. We discover a common lineage origin of endothelial cells and osteoblasts and de novo vasculogenesis (not angiogenesis) as the dominant mechanism. Inside the spongy (cancellous) layer, invasive OPN+/RUNX2+ osteoblasts establish two orthogonal collagen sca[ff]olds, remodel them by directional secretion of matrix metalloproteinases and employ controllers of hydroxyapatite crystal resorption previously considered to be osteoclast- specific. Such intercalary biomineralization, comprising cellular sheet and volumetric biomineral expansion, renders all layers malleable even during postnatal stages. We trace this mechanism to bone ontogenies of the earliest skeletonizing vertebrates and resolve cell/Issue homologies controversial for almost two centuries. Our new model also has implications for bone matrix bioengineering and cancer osteomimicry that might replay some of these ontogenetic processes.

evolutionary biology↗

bioRxiv2026

Risk factors and immunological characteristics of SARS-CoV-2 RNA re-positivity in convalescent COVID-19 Patients: A retrospective study

Purpose: To investigate the incidence, clinical features, and risk factors for SARS-CoV-2 nucleic acid re-positivity in recovered COVID-19 patients. Methods: We enrolled 441 convalescent COVID-19 patients from the rehabilitation ward of the Fifth Hospital of Shijiazhuang between January and April 2021. Based on nucleic acid test results, patients were categorized into repositive (n = 69) and non-repositive (n = 372) groups. Demographic, clinical, laboratory, and imaging data were compared between groups. Logistic regression was used to identify factors associated with re-positivity. Results: The overall re-positivity rate was 15.65%. Compared with the non-repositive group, the repositive group presented with a significantly higher proportion of moderate disease severity (72.46% vs. 50.54%) and a lower proportion of patients under 14 years old (7.25% vs. 23.66%). Additionally, multi-lobar involvement ([≥] 3 lobes) during the acute phase was more common in the repositive group (43.48% vs. 26.61%), with all differences being statistically significant (P < 0.001). Laboratory analysis revealed that repositive patients had significantly lower lymphocyte and eosinophil counts, as well as reduced albumin (ALB), aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, and albumin/globulin (A/G) ratio. Furthermore, circulating CD3+, CD4+, and CD8+ T-cell counts were markedly decreased. In contrast, the neutrophil-to-lymphocyte ratio (NLR), immunoglobulin M (IgM), immunoglobulin A (IgA), and neutralizing antibody (NAb) levels were significantly elevated in re-positive patients (all P < 0.05). Multivariate logistic regression analysis identified the CD8+ as independent risk factors for re-positivity. Conclusion: In convalescent patients, SARS-CoV-2 RNA re-positivity correlates with distinct clinical and immunological characteristics. The CD8+ T-cell level is an independent predictor, highlighting that dysregulated immune and inflammatory responses contribute to delayed viral clearance.

immunology↗

bioRxiv2026

DiffDomain-Spectrum identifies structurally reorganized TADs from sparse aggregated single-cell Hi-C contact maps

Structurally reorganized topologically associating domains (TADs) capture condition- or cell-type-specific remodeling of chromatin contacts and are important for understanding genome organization in health and disease. Emerging single-cell Hi-C (scHi-C) technologies enable such comparisons across heterogeneous cell populations, but aggregated scHi-C contact maps remain sparse at biologically meaningful 25 kb resolution, limiting reliable TAD reorganization detection. Here we present DiffDomain-Spectrum, a spectral statistical framework for identifying reorganized TADs between conditions or cell types from aggregated raw scHi-C contact maps. It tests normalized TAD-level difference matrices without separately normalizing sparse maps or enhancing individual scHi-C contact maps. Comparison with a semicircle-law null integrates evidence across the full eigenvalue spectrum. Across multiple scHi-C platforms, DiffDomain-Spectrum balances false positive control and detection sensitivity relative to alternative bulk callers, and detects a substantially higher proportion of reference TADs as reorganized than the boundary-focused single-cell method scHiCluster. Detected TADs show coherent aggregate contact patterns and CTCF binding changes and are enriched for differentially expressed genes, supporting biological relevance. Together, these results establish DiffDomain-Spectrum as a statistically principled framework for comparative domain-level analysis of sparse aggregated scHi-C contact maps without single-cell map enhancement.

genetics↗

bioRxiv2026

Genetic regulation of circulating metabolome in cattle

The circulating metabolome is a vital intermediate layer linking genetics to complex phenotypes, yet existing genome-wide studies-even in humans-rarely capture dynamic physiological contexts or cellular regulatory mechanisms. Here, we present the Cattle Metabolome Atlas (https://cattlema.farmgtex.org/), a comprehensive resource of 3,436 plasma metabolites and 4,851 serum metabolites from 4,651 animals with matched sequence-level genotypes across highly dynamic parity and lactation stages. We mapped 728 plasma and 622 serum metabolite quantitative trait loci (mQTL), revealing widespread context-dependent regulatory architectures. Integrating these mQTL with the multi-tissue expression quantitative trait loci (eQTL) and single-cell atlas of 59 tissues demonstrates that 74-79% of mQTL colocalize with eQTL across 29 tissues, prioritizing the liver as the primary systemic hub and resolving metabolic programs at single-cell resolution. Furthermore, we charted 538 causal gene-tissue-metabolite-trait cascades across 11 complex traits in cattle. Finally, cross-species analyses demonstrate partial evolutionary conservation of genetic metabolism between cattle and humans, establishing this atlas as a powerful asset for cattle genetics and genomics, selective breeding, and comparative biology.

genetics↗

bioRxiv2026

A T2T Benchmark Reveals How Reference Choice Shapes Human Genome Interpretation

The completion of telomere-to-telomere (T2T) human genomes has expanded the accessible landscape of human genetic variation, yet benchmark resources remain limited to conventional high-confidence regions defined by existing reference frameworks. Here, we generated a near-perfect diploid T2T genome (T2T-LIN) from a Chinese individual and established assembly-based truth sets by comparison with T2T-YAO, an ancestry-matched near-perfect T2T reference genome. The benchmark showed a heterozygous/homozygous SNV ratio of ~2, consistent with expectations under Hardy-Weinberg equilibrium, and enabled genome-wide evaluation of reference-dependent biases. We found that reference choice substantially influences genome interpretation: ancestry-matched linear T2T references provided the most faithful representation of individual genomic variation and enabled more accurate genome reconstruction than unmatched linear, diploid and graph-based references. Benchmarking previously inaccessible repetitive and structurally complex regions revealed substantial limitations of current variant callers that were masked by conventional metrics. The T2T-LIN and YAO-LIN benchmarks establish a T2T-era framework for evaluating reference-dependent genome interpretation and variant discovery across nearly the complete human genome.

genomics↗

bioRxiv2026

FFPERescuer: deep unsupervised domain adaptation for the reconstruction of gene expression profiles derived from formalin-fixed paraffin-embedded samples

Formalin-fixed paraffin-embedded (FFPE) tumor tissues often suffer from RNA degradation, posing a long-standing challenge for reliable transcriptomic profiling. Here, we propose FFPERescuer, a deep learning framework employing unsupervised domain adaptation, to rectify distorted gene expression data. FFPERescuer comprises a partial encoder that maps a small subset of genes to high-level representations and a decoder to reconstruct full gene expression profiles. On simulated data with varying noise levels, FFPERescuer faithfully recovered gene expression profiles, achieving high Pearson correlation coefficients (PCCs > 0.85) with the ground truth. In FF-FFPE-matched cohorts, FFPERescuer significantly enhanced expression profile concordance, with average PCCs increased by 23% (P < 0.05). Applying to cancer subtyping, FFPERescuer improved classification accuracy from 67% to 92%, recapitulated subtype-specific biological properties lost in the FFPE-derived data, and enhanced survival associations. Our studies provide a powerful framework for reliable transcriptomic profiling from FFPE-archived tumor samples that are widely available in the clinic.

bioinformatics↗

bioRxiv2026

Extrachromosomal Circular DNA Function as Mobile Elements to Alter the Mammalian Germline Genome

The viability of any species including the human requires that the germline genome is kept stable as it is transmitted across generations by the germ cells. Failure to safeguard the genome integrity and stability would lead to inherited diseases and infertility. Thus, a better understanding of the mechanisms that alter the germline genome is crucial to ensure human health and our continuation as a species. Here, we show that the extrachromosomal circular DNA (eccDNA) in the mouse and human male germline represents a new mechanism in altering the mammalian germline genome. To enable the tracking of germline eccDNA in vivo, we established a novel mouse model that allows the generation of a reporter eccDNA in a cell type-specific manner. Using this mouse model, we showed that eccDNA formed in the developing male germ cells can integrate into the germline genome. Using eccDNA-containing sperm for in vitro fertilization led to the eccDNA sequence being inherited by the embryos. By analyzing a large cohort of long-read whole genome sequencing data, we showed that eccDNA-mediated germline genome insertions represent an important source of human genome structural variations. Finally, by leveraging human sperm samples, we found that diabetes induces an increase in sperm eccDNA quantity, which is mediated at least in part through poly (ADP-ribose) polymerases. Together, our results provide new insights into how the mammalian germline genome can be altered, with important implications for human health and genome evolution.

developmental biology↗

bioRxiv2026

Unlocking Sensitive Data with SPHERE in the Age of AI

Sensitive human data underpin discoveries across medicine, biology and the social sciences, yet privacy regulation often prevents sharing them with collaborators or artificial intelligence (AI) systems. We introduce SPHERE, a model-free method that makes sensitive datasets directly usable by AI and shareable for open science as a synthetic twin, while the original records never leave the local environment. Across 33 datasets spanning five scientific domains, SPHERE protects individual privacy against adversarial re-identification attacks while preserving the data's statistical structure: means, variances and correlations are reproduced exactly, effect size and P value in linear statistical analysis is numerically identical, nonlinear machine-learning utility is retained, and each twin is generated in seconds on a laptop. Frontier AI agents running on the twin reach the same scientific conclusions as on the original records. Analyses of the twin reproduce genome- and proteome-wide results at UK Biobank scale and recover the findings of landmark studies across three independent cohorts and consortia. The approach also extends to deep-learning embeddings across language, vision and time-series, with minimal utility loss. We make the Stanford Alzheimer's Disease Research Center cohort openly available for the first time, as a SPHERE twin spanning nine modalities that any registered researcher can analyze without an approval process. We release SPHERE with certification of each twin's privacy and fidelity, and an AI agent that autonomously executes research tasks on sensitive data without ever accessing it. Sensitive datasets that are currently closed to research could thus become routine inputs to open science and AI to enable key discoveries.

bioinformatics↗

bioRxiv2026

Structural basis of endogenous inverse agonism and subtype selectivity at melanocortin receptors

Endogenous inverse agonists suppress constitutive G protein-coupled receptor (GPCR) signaling, but their mechanisms remain poorly understood. Here we report cryo-EM structures of melanocortin-1 receptor (MC1R) bound to its endogenous inverse agonist, agouti signaling protein (ASIP) and melanocortin-4 receptor (MC4R) bound to agouti-related protein (AgRP). Together with previously reported structures and those we determined using extracellular nanobodies developed here, these data delineate a conformational continuum underlying receptor activation and silencing. Both inverse agonists occlude the orthosteric pocket as molecular corks and drive a shared TM3-centered extracellular remodeling. Against this common mechanism, subtype selectivity is encoded not by the conserved orthosteric pocket but by the divergent extracellular receptor surface, engaged through an ASIP C-terminal loop-dependent clasp. These findings establish a mechanistic framework for endogenous inverse agonism and identify the receptor periphery as a tractable target for subtype-selective modulation.

biophysics↗

bioRxiv2026

Cortical Hierarchy Dynamically Organizes Large-Scale Neural Propagation

Flexible behaviour depends on the continuous coordination of sensory-driven and internally guided processing, yet whether the cortical hierarchy spanning lower-order sensory to higher-order association systems dynamically organizes large-scale cortical propagation over time remains unclear. Here we combined source-resolved magnetoencephalography with Riemannian cortical-flow modelling to derive hierarchy consistency, a moment-to-moment measure of the alignment between cortical propagation and the principal sensory-to-association functional gradient. We found that large-scale cortical propagation was dynamically organized by the cortical hierarchy. Hierarchy consistency exhibited a reproducible low-frequency periodic component that defined a characteristic timescale for the continuous updating of propagation direction. This dynamic organization was coordinated by a distributed cortical switchboard spanning the default-mode, salience, control and limbic systems, and was constrained by structural connectivity and network-control architecture. It flexibly adapted to behavioural demands, with hierarchy consistency increasing across both sensorimotor and working-memory states, while its characteristic periodicity shifted in a task-dependent manner. Moreover, hierarchy-related propagation dynamics were systematically reorganized across ageing and associated with higher-order cognitive function. Together, these findings establish the cortical hierarchy as a dynamic organizing principle that continuously shapes the direction and temporal evolution of large-scale cortical propagation to support adaptive behaviour.

neuroscience↗

bioRxiv2026

Kintsugi maps nucleus-poor RNA compartments in subcellular spatial transcriptomics

Subcellular spatial transcriptomics captures where RNA is in tissue, but a single location holds too few molecules of any one gene to estimate composition alone. Every current method fixes in advance where to borrow -- a smoothing scale, a cell outline or a factor model -- and the fixed choice shapes what is visible. Kintsugi removes the fixed choice and lets held-out molecules decide, gene by gene, how much to borrow from spatial neighbours and from other genes at the same location. On a lung section measured by both Xenium and Visium HD, the data-chosen allocation placed an epithelial programme where the Xenium molecules were, ahead of smoothing, cell segmentation and a factor model; the result replicated across tissues and against protein. Across a 45-core pulmonary fibrosis cohort, separating composition from captured amount shows that a fibroblastic focus is not a place with more RNA but a place with different RNA: 2.8-fold higher in activated-fibroblast composition while segmented nuclear density is at most 1.08-fold higher.

bioinformatics↗

bioRxiv2026

A Hymenoptera-restricted gene mediating ant castes co-opts deeply conserved machinery to control organ size

Lineage-specific genes are widespread and have been implicated as phenotypic innovation inducers, but how they acquire complex developmental functions remains poorly understood. Ant queens and workers develop dramatically different organ sizes from identical genomes under juvenile hormone (JH) control, yet the molecular effectors translating JH signalling into caste-specific organ growth remain unknown. Here we identify torch, a Hymenoptera-restricted gene, as the most consistently gyne-biased and JH-responsive gene across 68 ant species. Knockdown of torch in virgin queens of Monomorium pharaonis produces a worker-like, multi-organ growth-restricted phenotype. Mechanistically, torch harbours an E-box-like motif activated by the JH receptor Gce-Tai and acts as a GA-repeat-binding transcription factor that regulates Hippo signalling, the deeply conserved organ-size control pathway in animals. Expressing torch heterologously in mice and a growth-restricted Drosophila background shows that the gene retained its general growth-promoting activity across more than 700 million years of animal evolution in lineages that lack the gene, establishing that its function is mediated through conserved rather than ant-specific machinery. A lineage-specific gene can therefore acquire complex morphogenetic function by co-opting ancient organ-size circuitry, providing a general route by which novel genes can drive phenotypic innovation.

evolutionary biology↗

bioRxiv2026

Attenuated Salmonella-Mediated Delivery of GSDMD Potentiates PD-1 Blockade Therapy against Melanoma

Immunotherapy has emerged as a core therapeutic strategy for melanoma. Programmed death protein 1 (PD-1) is a critical immune checkpoint molecule that restrains host anti-tumor immunity, and therapeutic agents blocking the PD-1 signaling pathway have been widely deployed in clinical practice. Nevertheless, single-agent PD-1 blockade fails to elicit robust clinical responses in the majority of patients. Therefore, there is an urgent unmet need to develop combinatorial regimens capable of augmenting the anti-tumor efficacy of PD-1 inhibition. Gasdermin D (GSDMD), a pore-forming effector protein that orchestrates pyroptosis, exerts inherent anti-tumor activities upon overexpression. However, whether GSDMD can synergize with PD-1 blockade to enhance therapeutic outcomes against melanoma remains poorly defined. To address this question, we established an attenuated Salmonella engineered strain for targeted delivery of GSDMD, and further investigated the anti-melanoma therapeutic efficacy of combining this engineered bacterium with anti-PD-1 antibody via immunofluorescence staining, flow cytometry and other analytical approaches. Our in vivo results demonstrated that combinatorial treatment markedly suppressed melanoma progression in tumor-bearing mice relative to monotherapy with either GSDMD-expressing bacteria or anti-PD-1 antibody alone. Mechanistically, co-treatment upregulated intratumoral expression of GSDMD and the pro-apoptotic protein BAX, while simultaneously downregulating PD-1 expression. In addition, the GSDMD/anti-PD-1 combination significantly elevated the proportions of CD4 and CD8 T lymphocytes in both peripheral blood and splenic tissues, and facilitated robust tumor infiltration by these two T cell subsets. Compared with phosphate-buffered saline (PBS) and scramble control groups, combinatorial therapy promoted tumor infiltration of M1-type tumor-associated macrophages (TAMs) and repolarized TAMs away from the immunosuppressive M2 phenotype. Consistently, serum levels of the pro-inflammatory cytokines TNF- and IFN-{gamma} were markedly elevated following combined intervention. Collectively, this study verifies that attenuated Salmonella carrying GSDMD synergizes with anti-PD-1 antibody to elicit potent anti-tumor effects in melanoma-bearing mice by amplifying systemic and intratumoral anti-tumor immune responses, which provides a preclinical rationale for novel combinatorial therapeutic strategies against melanoma.

cancer biology↗