Biology subjects

Zhang, G.

Publications and source records attributed to Zhang, G..

At least 199 records · Page 11Linked to original sources

bioRxiv2024

AMHY and sex determination in egg-laying mammals

The sex chromosomes of egg-laying mammals (monotremes), which lack the sex determining gene SRY1, evolved independently to those of all therian mammals2,3. Here we characterise the candidate monotreme sex determining gene, the Y-localised anti-Mullerian hormone gene (AMHY)4,3 and trace its expression during the period of sexual differentiation. Monotreme AMHX and AMHY gametologues have significant sequence divergence at the promoter, gene and protein level, likely following an original allele inversion in the common monotreme ancestor but retain conserved features of TGF-{beta} molecules. Expression of sexual differentiation genes in the echidna fetal gonad were significantly different from that of therian mammals. AMHY expression was seen exclusively in the male gonad during sexual differentiation, whereas AMHX was expressed in both sexes. Experimental ectopic expression of platypus AMHX or AMHY in the chicken embryo did not masculinise the female urogenital system, a possible result of mammalian specific changes to AMH proteins preventing function in the chicken. Our results provide fundamental insight into the first steps of monotreme sex chromosome evolution and sex determination with developmental expression data strongly supporting AMHY as the primary male sex determination gene.

evolutionary biology↗

bioRxiv2024

GCI: a continuity inspector for complete genome assembly

MotivationRecent advances in long-read sequencing technologies have significantly facilitated the production of high-quality genome assembly. The telomere-to-telomere (T2T) gapless assembly has become the new golden standard of genome assembly efforts. Several recent efforts have claimed to produce T2T level reference genomes. However, a universal standard is still missing to qualify a genome assembly to be at T2T standard. Traditional genome assembly assessment metrics (N50 and its derivatives) have no capacity in differentiate between nearly T2T assembly and the truly T2T assembly in continuity either globally and locally. Also these metrics are independent of raw reads, which make them inflated easily by artificial operations. Therefore a gaplessness evaluation tool at single nucleotide resolution to reflect true completeness is urgently needed in the era of complete genomes. ResultsHere, we present a tool called Genome Continuity Inspector (GCI) to assess genome assembly continuity at the single base resolution, that can evaluate how close a genome assembly is close to T2T level. GCI utilized multiple aligners to map long reads from multiple platforms back to the assembly. By incorporating curated mapping coverage of high-confidence read alignments, GCI identifies potential assembly issues. Meanwhile, it also reports GCI scores to quantify the assembly overall continuity in the whole genome or chromosome scale. Availability and implementationThe open-source GCI code is freely available on Github (https://github.com/yeeus/GCI) under the MIT license.

bioinformatics↗

bioRxiv2024

Comparative genomics of macaques and integrated insights into genetic variation and population history

The crab-eating macaques (Macaca fascicularis) and rhesus macaques (M. mulatta) are widely studied nonhuman primates in biomedical and evolutionary research. Despite their significance, the current understanding of the complex genomic structure in macaques and the differences between species requires substantial improvement. Here, we present a complete genome assembly of a crab-eating macaque and 20 haplotype-resolved macaque assemblies to investigate the complex regions and major genomic differences between species. Segmental duplication in macaques is [~]42% lower, while centromeres are [~]3.7 times longer than those in humans. The characterization of [~]2 Mbp fixed genetic variants and [~]240 Mbp complex loci highlights potential associations with metabolic differences between the two macaque species (e.g., CYP2C76 and EHBP1L1). Additionally, hundreds of alternative splicing differences show post-transcriptional regulation divergence between these two species (e.g., PNPO). We also characterize 91 large-scale genomic differences between macaques and humans at a single-base-pair resolution and highlight their impact on gene regulation in primate evolution (e.g., FOLH1 and PIEZO2). Finally, population genetics recapitulates macaque speciation and selective sweeps, highlighting potential genetic basis of reproduction and tail phenotype differences (e.g., STAB1, SEMA3F, and HOXD13). In summary, the integrated analysis of genetic variation and population genetics in macaques greatly enhances our comprehension of lineage-specific phenotypes, adaptation, and primate evolution, thereby improving their biomedical applications in human diseases.

genomics↗

bioRxiv2024

Discovery of orally bioavailable SARS-CoV-2 papain-like protease inhibitor as a potential treatment for COVID-19

The RNA-dependent RNA polymerase (RdRp), 3C-like protease (3CLpro), and papain-like protease (PLpro) are pivotal components in the viral life cycle of SARS-CoV-2, presenting as promising therapeutic targets. Currently, all FDA-approved antiviral drugs against SARS-CoV-2 are RdRp or 3CLpro inhibitors. However, the mutations causing drug resistance have been observed in RdRp and 3CLpro from SARS-CoV-2, which makes it necessary to develop antivirals with novel mechanisms. Through the application of a structure-based drug design (SBDD) approach, we discovered a series of novel potent non-covalent PLpro inhibitors with remarkable in vitro potency and in vivo PK properties. The co-crystal structures of PLpro with leads revealed that the residues D164 and Q269 around the S2 site are critical for improving the inhibitors potency. The lead compound GZNL-P36 not only inhibited SARS-CoV-2 and its variants at the cellular level with EC50 ranging from 58.2 nM to 306.2 nM, but also inhibited HCoV-NL63 and HCoV-229E with EC50 of 81.6 nM and 2.66 M, respectively. Oral administration of the compound resulted in significantly improved survival and notable reductions in lung viral loads and lesions in SARS- CoV-2 infection mouse model, consistent with RNA-seq data analysis. Our results indicate that PLpro inhibitor is a promising SARS-CoV-2 therapy.

microbiology↗

bioRxiv2024

Bacteria adapt nutrient deprivation by reducing vacant 70S ribosomes via the silencing factor RsfS

Bacteria rapidly remodel protein synthesis to survive nutrient limitation, yet how inactive ribosomes are selectively regulated remains incompletely understood. Ribosome silencing factor S (RsfS) is the only known bacterial factor that selectively dissociates vacant 70S ribosomes, but its physiological function in bacterial adaptation has remained elusive. Here, we demonstrate that RsfS promotes bacterial survival during nutrient starvation by selectively dismantling non-translating vacant 70S ribosomes while sparing translating ribosomes. Biochemical analyses show that RsfS efficiently dissociates vacant 70S ribosomes but has little effect on programmed ribosomes in the pre-translocation or post-translocation states, thereby preserving ongoing protein synthesis. Ribosome profiling further reveals that loss of RsfS results in a global reduction in translational efficiency and widespread translational reprogramming during starvation. Mechanistically, dissociation of vacant ribosomes by RsfS suppresses futile EF-G-dependent GTP hydrolysis and prevents unnecessary energy expenditure associated with inactive ribosome cycling. Consistent with this mechanism, RsfS enhances bacterial adaptation to nutrient deprivation without compromising active translation. Together, our findings identify RsfS as a unique regulator of bacterial ribosome homeostasis that conserves cellular energy through selective dissociation of vacant 70S ribosomes, revealing a previously unrecognized strategy by which bacteria optimize translation under nutrient-limited conditions.

microbiology↗

bioRxiv2024

Evolutionary trajectory of TRPM2 channel activation by adenosine diphosphate ribose and calcium

Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca2+-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins.

evolutionary biology↗

bioRxiv2024

Novel Ser74 of NF-kappaB/CgIkappaBalpha Phosphorylated by MAPK/ERK Regulates Temperature Adaptation in Oysters

Phosphorylation of I{kappa}B at Ser32 and Ser36 by IKKs during biotic stress triggers its ubiquitin-proteasome degradation, causing to the nuclear translocation of REL, representing a key cascade mechanism in metazoans conserved and immune core signaling pathway, NF-{kappa}B. However, studies on its response to abiotic stress and signal transduction by phosphorylation in mollusks are lacking. Here, we firstly report a novel heat-induced phosphorylation site (Ser74) at the major NF-{kappa}B/CgI{kappa}B of oysters, phosphorylated by MAPK/CgERK1/2, which independently mediated the subsequent ubiquitin-proteasome degradation without phosphorylation at Ser32 and Ser36 and decreased thermal stability. The degradation of CgI{kappa}B promoted CgREL nuclear translocation, which stimulated cell survival related gene expression to defend against thermal stress. The MAPK and NF-{kappa}B pathways exhibited stronger activation patterns in higher environmental temperature and in the warm-adapted Crassostrea angulata than those in the cold-adapted C. gigas-two allopatric congeneric oyster species with differential habitat temperatures. These findings unveil the complex and unique phosphorylation-mediated signal transduction mechanisms in marine invertebrates, and further expand our understanding of the evolution and function of established classical pathway crosstalk mechanisms.

cell biology↗

bioRxiv2024

Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy

A fundamental challenge in fluorescence microscopy is the defocused background caused by scattering light, optical aberration, or limited axial resolution. Severe defocus backgrounds will submerge the in-focus information and cause artifacts in the following processing. Here, we leverage a priori knowledge about dark channels of biological structures and dual frequency separation to develop a single-frame defocus removal algorithm. It stably improves the signal-to-background ratio and structural similarity index measure of images by approximately 10-fold, and recovers in-focus signal with 85% accuracy, even when the defocus background is 50 times larger than in-focus information. Our Dark-based optical sectioning approach (Dark sectioning) is fully compatible with various microscopy techniques, such as wide-filed microscopy, polarized microscopy, laser-scanning / spinning-disk confocal microscopy, stimulated emission depletion microscopy, lightsheet microscopy, and light-field microscopy. It also complements reconstruction or processing algorithms such as deconvolution, structure illumination microscopy, and super-resolution optical fluctuation imaging.

bioinformatics↗

bioRxiv2024

Efficient cloning of genes for wheat yield component traits from QTLs via sequencing of the RIL population

In wheat (Triticum aestivum L.), yield component traits (YCTs) are the most important yield traits. Only several genes for YCTs have been originally cloned. The efficient cloning of genes for YCTs directly from wheat remains a challenge. Here, we proposed a strategy for cloning genes from quantitative trait loci (QTLs) by sequencing of recombinant inbred lines (RILs) (QTL-Seq-RIL). Using the TN18 x LM6 RIL population as an example, we identified 138 candidate unigenes (CUGs) for YCTs from 77 stable QTLs. The average of CUGs per QTL was 1.8, which enabled us to confirm the CUGs directly. We have confirmed seven CUGs, TaIFABPL, TaDdRp, TaRLK, TaTD, TaTFC3, TaKMT and TaSPL17, via the CRISPR/Cas9 system. Of these, six genes were found firstly to regulate YCTs in crops except for TaSPL17. Five CUGs (include TaSPL17) for which orthologous genes have been cloned previously with the same or similar agronomic functions. It is to say, 11 CUGs were preliminarily validated using a single RIL population. QTL-Seq-RIL provides an efficient method for rapid gene cloning using existing RIL populations.

plant biology↗

bioRxiv2024

An axonal brake on striatal dopamine output by cholinergic interneurons

Depolarisation of distal axons is necessary for neurons to translate somatic action potentials into neurotransmitter release. Studies have shown that striatal cholinergic interneurons (ChIs) can directly drive ectopic action potentials in dopamine (DA) axons and trigger DA release. However, here we show that this action occurs within a broader context of axonal signal integration whereby activation of ChIs and depolarisation of DA axons by nicotinic receptors (nAChRs) limits the subsequent depolarisation and release of DA in response to ensuing activity. We demonstrate that activation of ChIs and nAChRs in ex vivo mouse striatum, even when it does not trigger DA release that is detectable by fast-scan cyclic voltammetry, limits DA release for [~]100 ms by depressing subsequent axonal depolarisation and calcium summation. This axonal brake on DA release is stronger in dorsal than ventral striatum, and is unrelated to DA depletion. In vivo, antagonism of nAChRs in dorsal striatum elevated extracellular DA levels and promoted conditioned place-preference, underscoring its physiological relevance. Our findings reveal that under physiological conditions in vivo, ChIs acting via nAChRs dynamically attenuate DA output driven by DA neuron activity, leading to a predominantly inverse relationship between ACh and DA signalling that varies continuously with ChI activity.

neuroscience↗

bioRxiv2024

Natural variation in infection specificityof Caenorhabditis briggsae isolates by two RNA viruses

Antagonistic relationships such as host-virus interactions potentially lead to rapid evolution and specificity in interactions. The Orsay virus is so far the only horizontal virus naturally infecting the nematode C. elegans. In contrast, several related RNA viruses infect its congener C. briggsae, including Santeuil (SANTV) and Le Blanc (LEBV) viruses. Here we focus on the hosts intraspecific variation in sensitivity to these two intestinal viruses. Many temperate-origin C. briggsae strains, including JU1264 and JU1498, are sensitive to both, while many tropical strains, such as AF16, are resistant to both. Interestingly, some C. briggsae strains exhibit a specific resistance, such as the HK104 strain, specifically resistant to LEBV. The viral sensitivity pattern matches the strains geographic and genomic relationships. The heavily infected strains mount a seemingly normal small RNA response that is insufficient to suppress viral infection, while the resistant strains show no small RNA response, suggesting an early block in viral entry or replication. We use a genetic approach from the host side to map genomic regions participating in viral resistance polymorphisms. Using Advanced Intercrossed Recombinant Inbred Lines (RILs) between virus-resistant AF16 and SANTV-sensitive HK104, we detect Quantitative Trait Loci (QTLs) on chromosomes IV and III. Building RILs between virus-sensitive JU1498 and LEBV-resistant HK104 followed by bulk segregant analysis, we identify a chromosome II QTL. In both cases, further introgressions of the regions confirmed the QTLs. This diversity provides an avenue for studying virus entry, replication, and exit mechanisms, as well as host-virus specificity and the host response to a specific virus infection.

genetics↗

bioRxiv2024

Functional architecture of intracellular oscillations in hippocampal dendrites

Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions and report an advancing gradient of dendritic theta phase along the basal-tuft axis, then describe a pre-dominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.

neuroscience↗

bioRxiv2024

Metagenomic, metabolomic, and lipidomic shifts associated with fecal microbiota transplantation for recurrent Clostridioides difficile infection

Recurrent C. difficile infection (rCDI) is an urgent public health threat for which the last resort and lifesaving treatment is a fecal microbiota transplant (FMT). However, the exact mechanisms which mediate a successful FMT are not well understood. Here we use longitudinal stool samples collected from patients undergoing FMT to evaluate changes in the microbiome, metabolome, and lipidome after successful FMTs. We show changes in the abundance of many lipids, specifically acylcarnitines and bile acids, in response to FMT. These changes correlate with Enterobacteriaceae, which encode carnitine metabolism genes, and Lachnospiraceae, which encode bile salt hydrolases and baiA genes. LC-IMS-MS revealed a shift from microbial conjugation of primary bile acids pre-FMT to secondary bile acids post-FMT. Here we define the structural and functional changes in successful FMTs. This information will help guide targeted Live Biotherapeutic Product development for the treatment of rCDI and other intestinal diseases.

microbiology↗

bioRxiv2024

XKidneyOnco: An Explainable Framework to Classify Renal Oncocytoma and Chromophobe Renal Cell Carcinoma with a Small Sample Size

Renal oncocytoma and chromophobe renal cell carcinoma are two kidney cancer types that present a diagnostic challenge to pathologists and other clinicians due to their microscopic similarities. While RO is a benign renal neoplasm, ChRCC is considered malignant. Therefore, the differentiation between the two is crucial. In this study, we introduce an explainable framework to accurately differentiate ChRCC from RO, histologically. Our approach examined H&E-stained images of 656 ChRCC and 720 RO, and achieved a diagnostic accuracy of 88.2%, the sensitivity of 87%, and 100% specificity for explainable AI, which either outperforms or operate on par with convolutional neural network (CNN) models. Besides, we enrolled 44 pathology experts (including pathologists and pathology trainees) to differentiate the two tumors. The average accuracy of pathologists was 73%, which is 15.2% lower than our framework. These results indicate that the combination of human expert along with explainable AI achieve higher accuracy in differentiating the two tumors, while it reduces the workload of experts and offers the desired explainability for the medical experts.

pathology↗

bioRxiv2024

Transposon-mediated genic rearrangements underlie variation in small RNA pathways

Transposable elements (TEs) are parasitic DNA sequences that insert into the host genome and can cause alterations in host gene structure and expression. Host organisms cope with the often detrimental consequences caused by recent transposition and develop mechanisms that repress TE activities. In the nematode Caenorhabditis elegans, a small interfering RNA (siRNA) pathway dependent on the helicase ERI-6/7 primarily silences long terminal repeat retrotransposons and recent genes of likely viral origin. By studying gene expression variation among wild C. elegans strains, we discovered that structural variants and transposon remnants at the eri-6/7 locus alter its expression in cis and underlie a trans-acting expression quantitative trait locus affecting non-conserved genes and pseudogenes. Multiple insertions of the Polinton DNA transposon (also known as Mavericks) reshuffled the eri-6/7 locus in different configurations, separating the eri-6 and eri-7 exons and causing the inversion of eri-6 as seen in the reference N2 genome. In the inverted configuration, gene function was previously shown to be repaired by unusual trans-splicing mediated by direct repeats flanking the inversion. We show that these direct repeats originated from terminal inverted repeats specific to C. elegans Polintons. This trans-splicing event occurs infrequently compared to cis-splicing to novel downstream exons, thus affecting the production of ERI-6/7. Diverse Polinton-induced structural variations display regulatory effects within the locus and on targets of ERI-6/7-dependent siRNA pathways. Our findings highlight the role of host-transposon interactions in driving rapid host genome diversification among natural populations and shed light on evolutionary novelty in genes and splicing mechanisms.

genomics↗

bioRxiv2024

Balancing stability, dynamics and kinetics in phase separation of intrinsically disordered proteins

Liquid-liquid phase separation is a ubiquitous molecular phe-nomenon that plays crucial roles in a multitude of essential cellular activities. Intrinsically disordered proteins (IDPs), which lack well-defined three-dimensional structures, are prevalent participants in phase separation due to their inherent potential for promoting multivalent binding-the major driving force for this process. Understanding the underlying mechanisms of phase separation is challenging, as phase separation is a complex process, involving numerous molecules and various types of interactions. Here, we used a simplified coarse-grained model of IDPs to investigate the thermodynamic stability of the dense phase, conformational properties of IDPs, chain dynamics and kinetic rates of forming condensates. We focused on the IDP system, in which the oppositely charged IDPs are maximally segregated, inherently possessing a high propensity for phase separation. By varying interaction strengths, salt concentrations and temperatures, we observed that IDPs in the dense phase exhibited highly conserved conformational characteristics, which are more extended than those in the dilute phase. This implies that condensate formation acts as a protective shield, enabling IDPs to maintain conformational ensemble with high resistance to the changes in interactions and environmental conditions. Although the chain motions and global conformational dynamics of IDPs in the condensates are slow due to the high viscosity, local chain flexibility at the short timescales is largely preserved with respect to that at the free state. Strikingly, we observed a non-monotonic relationship between interaction strengths and kinetic rates for forming condensates. As strong interactions of IDPs result in high stable condensates, our results suggest that the thermodynamics and kinetics of phase separation are decoupled and optimized by the speed-stability balance through underlying molecular interactions. Our findings contribute to the molecular-level understanding of phase separation and offer valuable insights into the developments of engineering strategies for precise regulation of biomolecular condensates.

biophysics↗

bioRxiv2024

A Combinatorial Strategy for HRV 3C Protease Engineering to Achieve the N-terminal Free Cleavage

Human rhinovirus 3C protease (HRV 3C-P) has a high specificity against the substrate of LEVLFQ{downarrow}G at P1 site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3C-P, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3C-P engineering to expand its P1 specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1 specificity to recognize 20 different amino acids with the highest activity against LEVLFQ{downarrow}M (kcat/KM= 3.72 {+/-} 0.04 mM-1{middle dot}s-1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2 site, but with an expanded P1 specificity. Structural simulation of C3 indicated a reconstructed S1 binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3C-P, also provide new tools for other enzyme engineering. Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/574269v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b891eborg.highwire.dtl.DTLVardef@174151dorg.highwire.dtl.DTLVardef@1656f4org.highwire.dtl.DTLVardef@a6c1bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Integrated Analyses of Multi-omic Data Derived from Paired Primary Lung Cancer and Brain Metastasis Reveals the Metabolic Vulnerability as a Novel Therapeutic Target

Lung cancer brain metastases (LC-BrMs) are frequently associated with dismal mortality rates in patients with lung cancer; however, standard of care therapies for LC-BrMs are still limited in their efficacy. A deep understanding of molecular mechanisms and tumor microenvironment of LC-BrMs will provide us with new insights into developing novel therapeutics for treating patients with LC-BrMs. Here, we performed integrated analyses of genomic, transcriptomic, proteomic and metabolomic data which were derived from a total number of 174 patients with paired and unpaired primary lung cancer and LC-BrM, spanning four published and two newly generated patient cohorts on both bulk and single cell levels. We uncovered that LC-BrMs exhibited significantly higher intra-tumor heterogeneity. We also observed that mutations in a subset of genes were almost always shared by both primary lung cancers and LC-BrM lesions, including TTN, TP53, MUC16, LRP1B, RYR2, and EGFR. In addition, the genome-wide landscape of somatic copy number alterations was similar between primary lung cancers and LC-BrM lesions. Nevertheless, several regions of focal amplification were significantly enriched in LC-BrMs, including 5p15.33 and 20q13.33. Intriguingly, integrated analyses of transcriptomic, proteomic and metabolomic data revealed mitochondrial-specific metabolism was activated but tumor immune microenvironment was suppressed in LC-BrMs. Subsequently, we validated our results by conducting real-time quantitative reverse transcription PCR experiments, immunohistochemistry and multiplexed immunofluorescence staining of patients paired tumor specimens. Patients with a higher expression of mitochondrial metabolism genes but a lower expression of immune genes in their LC-BrM lesions tended to have a worse survival outcome. Therapeutically, targeting oxidative phosphorylation with gamitrinib in patient-derived organoids specific to LC-BrMs induced apoptosis and inhibited cell proliferation. The combination of gamitrinib plus anti-PD-1 immunotherapy significantly improved survival of mice bearing LC-BrMs. In conclusion, our findings not only provide comprehensive and integrated perspectives of molecular underpinnings of LC-BrMs but also contribute to the development of a potential, rationale-based combinatorial therapeutic strategy with the goal of translating it into clinical trials for patients with LC-BrMs.

cancer biology↗