Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

Assembly and recognition of keratins: A structural perspective.

Keratins are one of the major components of cytoskeletal network and assemble into fibrous structures named intermediate filaments (IFs), which are important for maintaining the mechanical properties of cells and tissues. Over the past decades, evidence has shown that the functions of keratins go beyond providing mechanical support for cells, they interact with multiple cellular components and are widely involved in the pathways of cell proliferation, differentiation, motility and death. However, the structural details of keratins and IFs are largely missing and many questions remain regarding the mechanisms of keratin assembly and recognition. Here we briefly review the current structural models and assembly of keratins as well as the interactions of keratins with the binding partners, which may provide a structural view for understanding the mechanisms of keratins in the biological activities and the related diseases.

Recent Advances in Low-Temperature Liquid Electrolyte for Supercapacitors.

As one of the key components of supercapacitors, electrolyte is intensively investigated to promote the fast development of the energy supply system under extremely cold conditions. However, high freezing point and sluggish ion transport kinetics for routine electrolytes hinder the application of supercapacitors at low temperatures. Resultantly, the liquid electrolyte should be oriented to reduce the freezing point, accompanied by other superior characteristics, such as large ionic conductivity, low viscosity and outstanding chemical stability. In this review, the intrinsically physical parameters and microscopic structure of low-temperature electrolytes are discussed thoroughly, then the previously reported strategies that are used to address the associated issues are summarized subsequently from the aspects of aqueous and non-aqueous electrolytes (organic electrolyte and ionic liquid electrolyte). In addition, some advanced spectroscopy techniques and theoretical simulation to better decouple the solvation structure of electrolytes and reveal the link between the key physical parameters and microscopic structure are briefly presented. Finally, the further improvement direction is put forward to provide a reference and guidance for the follow-up research.

Acylcarnitines promote gallbladder cancer metastasis through lncBCL2L11-THOC5-JNK axis.

BACKGROUND: The progression of gallbladder cancer (GBC) is accompanied by abnormal fatty acid ß-oxidation (FAO) metabolism. Different types of lipids perform various biological functions. This study aimed to determine the role of acyl carnitines in the molecular mechanisms of GBC progression. METHODS: Distribution of lipids in GBC was described by LC-MS-based lipidomics. Cellular localization, expression level and full-length of lncBCL2L11 were detected using fluorescence in situ hybridization (FISH) assays, subcellular fractionation assay and 5' and 3' rapid amplification of the cDNA ends (RACE), respectively. In vitro and in vivo experiments were used to verify the biological function of lncBCL2L11 in GBC cells. Methylated RNA Immunoprecipitation (MeRIP) was performed to detect the methylation levels of lncBCL2L11. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were used to identify lncBCL2L11 interacting proteins. Co-Immunoprecipitation (Co-IP) and Western blot assay were performed to validate the regulatory mechanism of lncBCL2L11 and THO complex. RESULTS: Acylcarnitines were significantly up-regulated in GBC tissues. High serum triglycerides correlated to decreased survival in GBC patients and promoted tumor migration. LncBCL2L11 was identified in the joint analysis of highly metastatic cells and RNA sequencing data. LncBCl2L11 prevented the binding of THOC6 and THOC5 and causes the degradation of THOC5, thus promoting the accumulation of acylcarnitines in GBC cells, leading to the malignant progression of cancer cells. In addition, highly expressed acylcarnitines stabilized the expression of lncBCL2L11 through N6-methyladenosine methylation (m6A), forming a positive feedback regulation in tumor dissemination. CONCLUSIONS: LncBCL2L11 is involved in gallbladder cancer metastasis through FAO metabolism. High lipid intake is associated with poor prognosis of GBC. Therefore, targeting lncBCL2L11 and its pathway-related proteins or reducing lipid intake may be significant for the treatment of GBC patients.

Nicotinamide N -methyltransferase promotes M2 macrophage polarization by IL6 and MDSC conversion by GM-CSF in gallbladder carcinoma.

BACKGROUND AND AIMS: Nicotinamide N -methyltransferase (NNMT), an enzyme responsible for the methylation of nicotinamide, is involved in many metabolic pathways in adipose tissue and the liver. However, the role of NNMT in editing the tumor immune microenvironment is not well understood. APPROACH AND RESULTS: Here, we identified that NNMT can promote IL6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by decreasing the tri-methyl-histone H3 levels on the promoters of IL6 and CSF2 (encoding GM-CSF) and CCAAT/Enhancer Binding Protein, an essential transcription factor for IL6 expression, thus promoting differentiation of macrophages into M2 type tumor-associated macrophages and generation of myeloid-derived suppressor cells from peripheral blood mononuclear cells. Treatment of xenografted tumor models overexpressing NNMT gallbladder carcinoma (GBC) cells with the NNMT inhibitor JBSNF-000088 resulted in compromised tumor development and decreased expression levels of IL6, GM-CSF, tumor-associated macrophage marker CD206, and myeloid-derived suppressor cell marker CD33 but increased expression levels of CD8. In addition, elevated expression of NNMT in tumors of patients with GBC was correlated with increased expression levels of CD206 and CD33 but with decreased levels of CD8 and survival of patients. CONCLUSIONS: These data highlight the critical role of NNMT in GBC progression. Inhibition of NNMT by JBSNF-000088 is a potential molecular target for GBC immunotherapy.

Arginine Methylation of MDH1 by CARM1 Inhibits Glutamine Metabolism and Suppresses Pancreatic Cancer.

Distinctive from their normal counterparts, cancer cells exhibit unique metabolic dependencies on glutamine to fuel anabolic processes. Specifically, pancreatic ductal adenocarcinoma (PDAC) cells rely on an unconventional metabolic pathway catalyzed by aspartate aminotransferase, malate dehydrogenase 1 (MDH1), and malic enzyme 1 to rewire glutamine metabolism and support nicotinamide adenine dinucleotide phosphate (NADPH) production. Here, we report that methylation on arginine 248 (R248) negatively regulates MDH1. Protein arginine methyltransferase 4 (PRMT4/CARM1) methylates and inhibits MDH1 by disrupting its dimerization. Knockdown of MDH1 represses mitochondria respiration and inhibits glutamine metabolism, which sensitizes PDAC cells to oxidative stress and suppresses cell proliferation. Meanwhile, re-expression of wild-type MDH1, but not its methylation-mimetic mutant, protects cells from oxidative injury and restores cell growth and clonogenic activity. Importantly, MDH1 is hypomethylated at R248 in clinical PDAC samples. Our study reveals that arginine methylation of MDH1 by CARM1 regulates cellular redox homeostasis and suppresses glutamine metabolism of pancreatic cancer.

Fatty Acid Binding Protein 1 is an Independent Prognostic Biomarker for Gallbladder Cancer with Direct Hepatic Invasion.

Background: Direct liver invasion (DI) is a predominant pathway of gallbladder cancer (GBC) metastasis, but the molecular alterations associated with DI remain addressed. This study identified specific genes correlated with DI, which may offer a potential biomarker for the diagnosis and prognosis of advanced GBC. Methods: RNA samples from 3 patients with DI of GBC were used for RNA-seq analysis. Differentially expressed genes and metabolic pathways between primary tumor (T) and DI tissue was used to analyze aberrant gene expressions. Immunohistochemistry (IHC) of fatty acid binding protein 1 (FABP1) in 62 patients with DI was engaged to evaluate its association with clinicopathological characteristics and prognosis. IHC of CD3+ and CD8+ T cells was analyzed for their correlation with FABP1 expression, clinicopathological features and prognosis. Univariate and multivariate Cox hazards regression analyses were performed to identify independent prognostic factors for disease-free survival (DFS) and overall survival (OS). Results: FABP1 mRNA levels were significantly upregulated in DI region compared to T tissue. IHC results showed identical results with elevated FABP1 (p < 0.0001). Expression of FABP1 in DI region was significantly associated with lymph node metastasis (P = 0.028), reduced DFS (P = 0.013) and OS (P = 0.022); in contrast, its expression in T region was not associated with clinicopathological characteristics and prognosis (P > 0.05). The density of CD8+ T cells in DI region with higher FABP1 expression was significantly lower than that with lower FABP1 expression (p = 0.0084). Multivariate analysis unveiled those hepatic metastatic nodules (HR = 3.35, 95%CI: 1.37-8.15, P = 0.008) and FABP1 expression in DI region (HR = 2.01, 95%CI: 1.05-3.88, P = 0.036) were high risk factors for OS, and FABP1(HR = 2.05, 95%CI: 1.04-4.06, P = 0.039) was also a high risk factor for DFS. Conclusions: Elevated expression of FABP1 in DI region serves as a potential prognostic biomarker for advanced GBC with DI.

Structure of cell-cell adhesion mediated by the Down syndrome cell adhesion molecule.

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.

Microscopic-Level Insights into Solvation Chemistry for Nonsolvating Diluents Enabling High-Voltage/Rate Aqueous Supercapacitors.

Localized "water-in-salt" (LWIS) electrolytes are promising candidates for the next generation of high-voltage aqueous electrolytes with low viscosity/salt beyond high-salt electrolytes. An effective yet high-function diluent mainly determines the properties of LWIS electrolytes, being a key issue. Herein, the donor number of solvents is identified to serve as a descriptor of interaction intensity between solvents and salts to screen the organic diluents having few impacts on the solvation microenvironment and intrinsic properties of the original high-salt electrolyte, further leading to the construction of a novel low-viscosity electrolyte with a low dosage of the LiNO3 salt and well-kept intrinsic Li+-NO3--H2O clusters. Nonsolvating diluents, especially acetonitrile (AN) that has never been reported previously, are presented with the capability of constructing a LWIS electrolyte with nonflammability, electrode-philic features, lower viscosity, decreased salt dosage, and a greatly enhanced ion diffusion coefficient by about 280 times. This strongly relies on a huge difference of about 5000 times in coordination and solubility between AN and H2O toward LiNO3 (0.05 vs 25 mol kgsolvent-1) and the moderate interaction between AN and H2O. Multi-spectroscopic techniques and molecular dynamics simulations uncover the solvation chemistry at the microscopic level and the interplay among cations, anions, and H2O without/with AN. The identified unique diluting and nonsolvating effects of AN reveal well-maintained cation-anion-H2O clusters and enhanced intermolecular hydrogen bonding between AN and H2O, further reinforcing the H2O stability and expanding the voltage window up to 3.28 V. This is a breakthrough that is far beyond high-viscosity/salt electrolytes for high-voltage and high-rate aqueous supercapacitors.

Edge Length-Programmed Single-Stranded RNA Origami for Predictive Innate Immune Activation and Therapy.

Ligands targeting nucleic acid-sensing receptors activate the innate immune system and play a critical role in antiviral and antitumoral therapy. However, ligand design for in situ stability, targeted delivery, and predictive immunogenicity is largely hampered by the sophisticated mechanism of the nucleic acid-sensing process. Here, we utilize single-stranded RNA (ssRNA) origami with precise structural designability as nucleic acid sensor-based ligands to achieve improved biostability, organelle-level targeting, and predictive immunogenicity. The natural ssRNAs self-fold into compact nanoparticles with defined shapes and morphologies and exhibit resistance against RNase digestion in vitro and prolonged retention in macrophage endolysosomes. We find that programming the edge length of ssRNA origami can precisely regulate the degree of macrophage activation via a toll-like receptor-dependent pathway. Further, we demonstrate that the ssRNA origami-based ligand elicits an anti-tumoral immune response of macrophages and neutrophils in the tumor microenvironment and retards tumor growth in the mouse pancreatic tumor model. Our ssRNA origami strategy utilizes structured RNA ligands to achieve predictive immune activation, providing a new solution for nucleic acid sensor-based ligand design and biomedical applications.

Encoded Fusion-Mediated MicroRNA Signature Profiling of Tumor-Derived Extracellular Vesicles for Pancreatic Cancer Diagnosis.

MicroRNAs (miRNAs) in tumor-derived extracellular vesicles (tEVs) are important cancer biomarkers for cancer screening and early diagnosis. Multiplex detection of miRNAs in tEVs facilitates accurate diagnosis but remains a challenge. Herein, we propose an encoded fusion strategy to profile the miRNA signature in tEVs for pancreatic cancer diagnosis. A panel of encoded-targeted-fusion beads was fabricated for the selective recognition and fusion of tEVs, with the turn-on fluorescence signals of molecule beacons for miRNA quantification and barcode signals for miRNA identification using readily accessible flow cytometers. Using this strategy, six types of pancreatic-cancer-associated miRNAs can be profiled in tEVs from 2 µL plasma samples (n = 36) in an isolation-free and lysis-free manner with only 2 h of processing, offering a high accuracy (98%) to discriminate pancreatic cancer, pancreatitis, and healthy donors. This encoded fusion strategy exhibits great potential for multiplex profiling of miRNA in tEVs, offering new avenues for cancer diagnosis and screening.

The oncogenic role of NF1 in gallbladder cancer through regulation of YAP1 stability by direct interaction with YAP1.

BACKGROUND: Gallbladder cancer (GBC) is the most prevalent and invasive biliary tract malignancy. As a GTPase-activating protein, Neurofibromin 1 (NF1) is a tumor suppressor that negatively regulates the RAS signaling pathway, and its abnormality leads to neurofibromatosis type 1 (NF-1) disease. However, the role of NF1 playing in GBC and the underlying molecular mechanism has not been defined yet. METHODS: A combination of NOZ and EH-GB1 cell lines as well as nude mice, were utilized in this study. mRNA expression and protein levels of NF1 and YAP1 were evaluated by quantitative real-time PCR (qRT-PCR), western blot (WB), and immunohistochemistry (IHC). In vitro and in vivo assays were performed to explore the biological effects of NF1 in NOZ and EH-GB1 cells via siRNA or lv-shRNA mediated knockdown. Direct interaction between NF1 and YAP1 was detected by confocal microscopy and co-immunoprecipitation (Co-IP), and further confirmed by GST pull-down assay and isothermal titration calorimetry assay (ITC). The stability of proteins was measured by western blot (WB) in the presence of cycloheximide. RESULTS: This study showed that a higher level of NF1 and YAP1 was found in GBC samples than in normal tissues and associated with worse prognoses. The NF1 knockdown impaired the proliferation and migration of NOZ in vivo and in vitro by downregulating YAP1 expression. Moreover, NF1 co-localized with YAP1 in NOZ and EH-GB1 cells, and the WW domains of YAP1 specifically recognized the PPQY motif of NF1. The structural modeling also indicated the hydrophobic interactions between YAP1 and NF1. On the other hand, YAP1 knockdown also impaired the proliferation of NOZ in vitro, phenocopying the effects of NF1 knockdown. Overexpression of YAP1 can partially rescue the impaired proliferation in NF1 stably knockdown cells. In mechanism, NF1 interacted with YAP1 and increased the stability of YAP1 by preventing ubiquitination. CONCLUSIONS: Our findings discovered a novel oncogenic function of NF1 by directly interacting with YAP1 protein and stabilizing YAP1 to protect it from proteasome degradation in NOZ cells. NF1 may serve as a potential therapeutic target in GBC.

Six-Membered Aromatic Nitrogen Heterocyclic Anti-Tumor Agents: Synthesis and Applications.

Cancer stands as a serious malady, posing substantial risks to human well-being and survival. This underscores the paramount necessity to explore and investigate novel antitumor medications. Nitrogen-containing compounds, especially those derived from natural sources, form a highly significant category of antitumor agents. Among these, antitumor agents with six-membered aromatic nitrogen heterocycles have consistently attracted the attention of chemists and pharmacologists. Accordingly, we present a comprehensive summary of synthetic strategies and clinical implications of these compounds in this review. This entails an in-depth analysis of synthesis pathways for pyridine, quinoline, pyrimidine, and quinazoline. Additionally, we explore the historical progression, targets, mechanisms of action, and clinical effectiveness of small molecule inhibitors possessing these structural features.

A Gallbladder Cancer Survival Prediction Model Based on Multimodal Fusion Analysis.

BACKGROUND: Gallbladder cancer is the sixth most common malignant gastrointestinal tumor. Radical surgery is currently the only effective treatment, but patient prognosis is poor, with a 5-year survival rate of only 5-10%. Establishing an effective survival prediction model for gallbladder cancer patients is crucial for disease status assessment, early intervention, and individualized treatment approaches. The existing gallbladder cancer survival prediction model uses clinical data-radiotherapy and chemotherapy, pathology, and surgical scope-but fails to utilize laboratory examination and imaging data, limiting its prediction accuracy and preventing sufficient treatment plan guidance. AIMS: The aim of this work is to propose an accurate survival prediction model, based on the deep learning 3D-DenseNet network, integrated with multimodal medical data (enhanced CT imaging, laboratory test results, and data regarding systemic treatments). METHODS: Data were collected from 195 gallbladder cancer patients at two large tertiary hospitals in Shanghai. The 3D-DenseNet network extracted deep imaging features and constructed prognostic factors, from which a multimodal survival prediction model was established, based on the Cox regression model and incorporating patients' laboratory test and systemic treatment data. RESULTS: The model had a C-index of 0.787 in predicting patients' survival rate. Moreover, the area under the curve (AUC) of predicting patients' 1-, 3-, and 5-year survival rates reached 0.827, 0.865, and 0.926, respectively. CONCLUSIONS: Compared with the monomodal model based on deep imaging features and the tumor-node-metastasis (TNM) staging system-widely used in clinical practice-our model's prediction accuracy was greatly improved, aiding the prognostic assessment of gallbladder cancer patients.

Recognition of lipoproteins by scavenger receptor class A members.

Scavenger receptor class A (SR-A) proteins are type II transmembrane glycoproteins that form homotrimers on the cell surface. This family has five known members (SCARA1 to 5, or SR-A1 to A5) that recognize a variety of ligands and are involved in multiple biological pathways. Previous reports have shown that some SR-A family members can bind modified low-density lipoproteins (LDLs); however, the mechanisms of the interactions between the SR-A members and these lipoproteins are not fully understood. Here, we systematically characterize the recognition of SR-A receptors with lipoproteins and report that SCARA1 (SR-A1, CD204), MARCO (SCARA2), and SCARA5 recognize acetylated or oxidized LDL and very-low-density lipoprotein in a Ca2+-dependent manner through their C-terminal scavenger receptor cysteine-rich (SRCR) domains. These interactions occur specifically between the SRCR domains and the modified apolipoprotein B component of the lipoproteins, suggesting that they might share a similar mechanism for lipoprotein recognition. Meanwhile, SCARA4, a SR-A member with a carbohydrate recognition domain instead of the SRCR domain at the C terminus, shows low affinity for modified LDL and very-low-density lipoprotein but binds in a Ca2+-independent manner. SCARA3, which does not have a globular domain at the C terminus, was found to have no detectable binding with these lipoproteins. Taken together, these results provide mechanistic insights into the interactions between SR-A family members and lipoproteins that may help us understand the roles of SR-A receptors in lipid transport and related diseases such as atherosclerosis.

Structural basis of the bHLH domains of MyoD-E47 heterodimer.

The basic helix-loop-helix (bHLH) family is one of the most conserved transcription factor families that plays an important role in regulating cell growth, differentiation and tissue development. Typically, members of this family form homo- or heterodimers to recognize specific motifs and activate transcription. MyoD is a vital transcription factor that regulates muscle cell differentiation. However, it is necessary for MyoD to form a heterodimer with E-proteins to activate transcription. Even though the crystal structure of the MyoD homodimer has been determined, the structure of the MyoD heterodimer in complex with the E-box protein remains unclear. In this study, we determined the crystal structure of the bHLH domain of the MyoD-E47 heterodimer at 2.05 Å. Our structural analysis revealed that MyoD interacts with E47 through a hydrophobic interface. Moreover, we confirmed that heterodimerization could enhance the binding affinity of MyoD to E-box sequences. Our results provide new structural insights into the heterodimer of MyoD and E-box protein, suggesting the molecular mechanism of transcription activation of MyoD upon binding to E-box protein.

tRF-3013b inhibits gallbladder cancer proliferation by targeting TPRG1L.

BACKGROUND: tRNA-derived fragments (tRFs) are newly discovered noncoding RNAs and regulate tumor progression via diverse molecular mechanisms. However, the expression and biofunction of tRFs in gallbladder cancer (GBC) have not been reported yet. METHODS: The expression of tRFs in GBC was detected by tRF and tiRNA sequencing in GBC tissues and adjacent tissues. The biological function of tRFs was investigated by cell proliferation assay, clonal formation assay, cell cycle assay, and xenotransplantation model in GBC cell lines. The molecular mechanism was discovered and verified by transcriptome sequencing, fluorescence in situ hybridization (FISH), target gene site prediction, and RNA binding protein immunoprecipitation (RIP). RESULTS: tRF-3013b was significantly downregulated in GBC compared with para-cancer tissues. Decreased expression of tRF-3013b in GBC patients was correlated with poor overall survival. Dicer regulated the production of tRF-3013b, and its expression was positively correlated with tRF-3013b in GBC tissues. Functional experiments demonstrated that tRF-3013b inhibited GBC cell proliferation and induced cell-cycle arrest. Mechanically, tRF-3013b exerted RNA silencing effect on TPRG1L by binding to AGO3, and then inhibited NF-κB. TPRG1L overexpression could rescue the effects of tRF-3013b on GBC cell proliferation. CONCLUSIONS: This study indicated that Dicer-induced tRF-3013b inhibited GBC proliferation by targeting TPRG1L and repressed NF-κB, pointing to tRF-3013b as a novel potential therapeutic target of GBC.

Interactions of ferritin with scavenger receptor class A members.

Scavenger receptors are a superfamily of membrane-bound receptors that recognize both self and nonself targets. Scavenger receptor class A (SR-A) has five known members (SCARA1 to -5 or SR-A1 to -A5), which are type II transmembrane proteins that form homotrimers on the cell surface. SR-A members recognize various ligands and are involved in multiple biological pathways. Among them, SCARA5 can function as a ferritin receptor; however, the interaction between SCARA5 and ferritin has not been fully characterized. Here, we determine the crystal structures of the C-terminal scavenger receptor cysteine-rich (SRCR) domain of both human and mouse SCARA5 at 1.7 and 2.5 Å resolution, respectively, revealing three Ca2+-binding sites on the surface. Using biochemical assays, we show that the SRCR domain of SCARA5 recognizes ferritin in a Ca2+-dependent manner, and both L- and H-ferritin can be recognized by SCARA5 through the SRCR domain. Furthermore, the potential binding region of SCARA5 on the surface of ferritin is explored by mutagenesis studies. We also examine the interactions of ferritin with other SR-A members and find that SCARA1 (SR-A1, CD204) and MARCO (SR-A2, SCARA2), which are highly expressed on macrophages, also interact with ferritin. By contrast, SCARA3 and SCARA4, the two SR-A members without the SRCR domain, have no detectable binding with ferritin. Overall, these results provide a mechanistic view regarding the interactions between the SR-A members and ferritin that may help to understand the regulation of ferritin homeostasis by scavenger receptors.

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.