ATP-elicited Cation Fluxes Promote Volume-regulated Anion Channel LRRC8/VRAC Transport cGAMP for Antitumor Immunity.

The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-ß response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-ß response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-ß response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.

Long-chain fatty acids - The turning point between 'mild' and 'severe' acute pancreatitis.

Acute pancreatitis (AP) is an inflammatory disease characterized by localized pancreatic injury and a systemic inflammatory response. Fatty acids (FAs), produced during the breakdown of triglycerides (TGs) in blood and peripancreatic fat, escalate local pancreatic inflammation to a systemic level by damaging pancreatic acinar cells (PACs) and triggering M1 macrophage polarization. This paper provides a comprehensive analysis of lipases' roles in the onset and progression of AP, as well as the effects of long-chain fatty acids (LCFAs) on the function of pancreatic acinar cells (PACs). Abnormalities in the function of PACs include Ca2+ overload, premature trypsinogen activation, protein kinase C (PKC) expression, endoplasmic reticulum (ER) stress, and mitochondrial and autophagic dysfunction. The study highlights the contribution of long-chain saturated fatty acids (LC-SFAs), especially palmitic acid (PA), to M1 macrophage polarization through the activation of the NLRP3 inflammasome and the NF-κB pathway. Furthermore, we investigated lipid lowering therapy for AP. This review establishes a theoretical foundation for pro-inflammatory mechanisms associated with FAs in AP and facilitating drug development.

A Microfluidic Chip-Based Automated System for Whole-Course Monitoring the Drug Responses of Organoids.

Tumor patients-derived organoids, as a promising preclinical prediction model, have been utilized to evaluate ex vivo drug responses for formulating optimal therapeutic strategies. Detecting adenosine triphosphate (ATP) has been widely used in existing organoid-based drug response tests. However, all commercial ATP detection kits containing the cell lysis procedure can only be applied for single time point ATP detection, resulting in the neglect of dynamic ATP variations in living cells. Meanwhile, due to the limited number of viable organoids from a single patient, it is impractical to exhaustively test all potential time points in search of optimal ones. In this work, a multifunctional microfluidic chip was developed to perform all procedures of organoid-based drug response tests, including establishment, culturing, drug treatment, and ATP monitoring of organoids. An ATP sensor was developed to facilitate the first successful attempt on whole-course monitoring the growth status of fragile organoids. To realize a clinically applicable automatic system for the drug testing of lung cancer, a microfluidic chip based automated system was developed to perform entire organoid-based drug response test, bridging the gap between laboratorial manipulation and clinical practices, as it outperformed previous methods by improving data repeatability, eliminating human error/sample loss, and more importantly, providing a more accurate and comprehensive evaluation of drug effects.

Erratum: Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.

This corrects the article 10.3791/66058.

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

[Application and Research Progress of Lung Cancer Organoid in Precision Medicine 
for Lung Cancer].

The continuous advancement of molecular detection technology has greatly propelled the development of precision medicine for lung cancer. However, tumor heterogeneity is closely associated with tumor metastasis, recurrence, and drug resistance. Additionally, different lung cancer patients with the same genetic mutation may exhibit varying treatment responses to different therapeutic strategies. Therefore, the development of modern precision medicine urgently requires the precise formulation of personalized treatment strategies through personalized tumor models. Lung cancer organoid (LCO) can highly simulate the biological characteristics of tumor in vivo, facilitating the application of innovative drugs such as antibody-drug conjugate in precision medicine for lung cancer. With the development of co-culture model of LCO with tumor microenvironment and tissue engineering technology such as microfluidic chip, LCO can better preserve the biological characteristics and functions of tumor tissue, further improving high-throughput and automated drug sensitivity experiment. In this review, we combine the latest research progress to summarize the application progress and challenges of LCO in precision medicine for lung cancer.
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Ferroptosis exacerbates hyperlipidemic acute pancreatitis by enhancing lipid peroxidation and modulating the immune microenvironment.

Abnormal activation of ferroptosis worsens the severity of acute pancreatitis and intensifies the inflammatory response and organ damage, but the detailed underlying mechanisms are unknown. Compared with other types of pancreatitis, hyperlipidemic acute pancreatitis (HLAP) is more likely to progress to necrotizing pancreatitis, possibly due to peripancreatic lipolysis and the production of unsaturated fatty acids. Moreover, high levels of unsaturated fatty acids undergo lipid peroxidation and trigger ferroptosis to further exacerbate inflammation and worsen HLAP. This paper focuses on the malignant development of hyperlipidemic pancreatitis with severe disease combined with the core features of ferroptosis to explore and describe the mechanism of this phenomenon and shows that the activation of lipid peroxidation and the aberrant intracellular release of many inflammatory mediators during ferroptosis are the key processes that regulate the degree of disease development in patients with HLAP. Inhibiting the activation of ferroptosis effectively reduces the intensity of the inflammatory response, thus reducing organ damage in patients and preventing the risk of HLAP exacerbation. Additionally, this paper summarizes the key targets and potential therapeutic agents of ferroptosis associated with HLAP deterioration to provide new ideas for future clinical applications.

Mechanism of ameliorating cerebral ischemia/reperfusion injury by antioxidant inhibition of autophagy based on network pharmacology and experimental verification.

Cerebral ischemia-reperfusion injury (CIRI) is one of the most difficult challenges in cerebrovascular disease research. It is primarily caused by excessive autophagy induced by oxidative stress. Previously, a novel compound X5 was found, and the excellent antioxidant activity of it was verified in this study. Moreover, network pharmacological analysis suggested that compound X5 was closely associated with autophagy and the mTOR pathway. In vitro, X5 could significantly inhibit the expression of autophagy proteins Beclin-1 and LC3-ß, which are induced by H2O2, and promote the expression of SIRT1. In vivo, compound X5 significantly reduced the infarct size and improved the neurological function scores in the middle cerebral artery occlusion (MCAO) model of rats. In conclusion, ROS-induced autophagy is closely related to mTOR, SIRT1 and others, and X5 holds promise as a candidate for the treatment of CIRI.

Identification of Novel Cuproptosis-Related Genes Mediating the Prognosis and Immune Microenvironment in Cholangiocarcinoma.

BACKGROUND: Cuproptosis is a novel type of mediated cell death strongly associated with the progression of several cancers and has been implicated as a potential therapeutic target. However, the role of cuproptosis in cholangiocarcinoma for prognostic prediction, subgroup classification, and therapeutic strategies remains largely unknown. METHODS: A systematic analysis was conducted among 146 cuproptosis-related genes and clinical information based on independent mRNA and protein datasets to elucidate the potential mechanisms and prognostic prediction value of cuproptosis-related genes. A 10-cuproptosis-related gene prediction model was constructed, and its effects on cholangiocarcinoma prognosis were significantly connected to poor patient survival. Additionally, the expression patterns of our model included genes that were validated with several cholangiocarcinoma cancer cell lines and a normal biliary epithelial cell line. RESULTS: First, a 10-cuproptosis-related gene signature (ADAM9, ADAM17, ALB, AQP1, CDK1, MT2A, PAM, SOD3, STEAP3, and TMPRSS6) displayed excellent predictive performance for the overall survival of cholangiocarcinoma. The low-cuproptosis group had a significantly better prognosis than the high-cuproptosis group with transcriptome and protein cohorts. Second, compared with the high-risk and low-risk groups, the 2 groups displayed distinct tumor microenvironments, reduced proportions of endothelial cells, and increased levels of cancer-associated fibroblasts based on CIBERSORTx and EPIC analyses. Third, patients' sensitivities to chemotherapeutic drugs and immune checkpoints revealed distinctive differences between the 2 groups. Finally, in replicating the expression patterns of the 10 genes, these results were validated with quantitative real-time polymerase chain reaction results validating the abnormal expression pattern of the target genes in cholangiocarcinoma. CONCLUSIONS: Collectively, we established and verified an effective prognostic model that could separate cholangiocarcinoma patients into 2 heterogeneous cuproptosis subtypes based on the molecular or protein characteristics of 10 cuproptosis-related genes. These findings may provide potential benefits for unveiling molecular characteristics and defining subgroups could improve the early diagnosis and individualized treatment of cholangiocarcinoma patients.

Clonal expansion of shared T cell receptors reveals the existence of immune commonality among different lesions of synchronous multiple primary lung cancer.

The increase in the detection rate of synchronous multiple primary lung cancer (MPLC) has posed remarkable clinical challenges due to the limited understanding of its pathogenesis and molecular features. Here, comprehensive comparisons of genomic and immunologic features between MPLC and solitary lung cancer nodule (SN), as well as different lesions of the same patient, were performed. Compared with SN, MPLC displayed a lower rate of EGFR mutation but higher rates of BRAF, MAP2K1, and MTOR mutation, which function exactly in the upstream and downstream of the same signaling pathway. Considerable heterogeneity in T cell receptor (TCR) repertoire exists among not only different patients but also among different lesions of the same patient. Invasive lesions of MPLC exhibited significantly higher TCR diversity and lower TCR expansion than those of SN. Intriguingly, different lesions of the same patient always shared a certain proportion of TCR clonotypes. Significant clonal expansion could be observed in shared TCR clonotypes, particularly in those existing in all lesions of the same patient. In conclusion, this study provided evidences of the distinctive mutational landscape, activation of oncogenic signaling pathways, and TCR repertoire in MPLC as compared with SN. The significant clonal expansion of shared TCR clonotypes demonstrated the existence of immune commonality among different lesions of the same patient and shed new light on the individually tailored precision therapy for MPLC.

[Relationship between Bacteria in the Lower Respiratory Tract/Lung Cancer 
and the Development of Lung Cancer as well as Its Clinical Application].

Due to the advancement of 16S rRNA sequencing technology, the lower respiratory tract microbiota, which was considered non-existent, has been revealed. The correlation between these microorganisms and diseases such as tumor has been a hot topic in recent years. As the bacteria in the surrounding can infiltrate the tumors, researchers have also begun to pay attention to the biological behavior of tumor bacteria and their interaction with tumors. In this review, we present the characteristic of the lower respiratory tract bacteria and summarize recent research findings on the relationship between these microbiota and lung cancer. On top of that, we also summarize the basic feature of bacteria in tumors and focus on the characteristic of the bacteria in lung cancer. The relationship between bacteria in lung cancer and tumor development is also been discussed. Finally, we review the potential clinical applications of bacterial communities in the lower respiratory tract and lung cancer, and summarize key points of sample collection, sequencing, and contamination control, hoping to provide new ideas for the screening and treatment of tumors.
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Contrastive Masked Autoencoders are Stronger Vision Learners.

Masked image modeling (MIM) has achieved promising results on various vision tasks. However, the limited discriminability of learned representation manifests there is still plenty to go for making a stronger vision learner. Towards this goal, we propose Contrastive Masked Autoencoders (CMAE), a new self-supervised pre-training method for learning more comprehensive and capable vision representations. By elaboratively unifying contrastive learning (CL) and masked image model (MIM) through novel designs, CMAE leverages their respective advantages and learns representations with both strong instance discriminability and local perceptibility. Specifically, CMAE consists of two branches where the online branch is an asymmetric encoder-decoder and the momentum branch is a momentum updated encoder. During training, the online encoder reconstructs original images from latent representations of masked images to learn holistic features. The momentum encoder, fed with the full images, enhances the feature discriminability via contrastive learning with its online counterpart. To make CL compatible with MIM, CMAE introduces two new components, i.e., pixel shifting for generating plausible positive views and feature decoder for complementing features of contrastive pairs. Thanks to these novel designs, CMAE effectively improves the representation quality and transfer performance over its MIM counterpart. CMAE achieves the state-of-the-art performance on highly competitive benchmarks of image classification, semantic segmentation and object detection. Notably, CMAE-Base achieves 85.3% top-1 accuracy on ImageNet and 52.5% mIoU on ADE20k, surpassing previous best results by 0.7% and 1.8% respectively.

Adjuvant crizotinib treatment selected by patient-derived organoids in a patient with stage IIIA adenocarcinoma with novel LRRTM4-ALK fusion: a case report.

Background: Over 90 different anaplastic lymphoma kinase (ALK) fusions have been reported, and patients with different ALK fusion partners exhibit different responses to targeted therapy. Patient-derived organoid (PDO), a kind of 3-dimensional culture, is a promising model for drug-sensitivity testing for personalized treatment decision-making. It further has the potential to provide treatment strategy for patients with novel mutations, rare mutations, and concomitant mutations, serving as a supplement to evidence-based medicine. Case Description: We report a case in which a man with stage IIIA adenocarcinoma had pleural effusion 1 month after surgery. A novel leucine-rich repeat transmembrane neuronal protein 4 (LRRTM4)-ALK fusion was unveiled by next-generation sequencing (NGS), and PDOs were used in drug-sensitivity testing to select a proper adjuvant therapy for this patient. We chose crizotinib based on result of the test and drugs' availability in China and helped the patient achieve a more than 3-year-long disease-free survival (DFS). Higher variant allele frequencies (VAFs) of the driver mutation were also found in PDOs and their waste culture medium, indicating that the PDO model could filter out cells with driver genes or stemness and help us to identify the critical cancer cell colony in treatment decision-making. Conclusions: For the first time, we report the case of a LRRTM4-ALK fusion. The patient achieved a more than 3-year long-term DFS under crizotinib treatment, which was selected by an emerging PDO drug-sensitivity test model. We also discovered the enrichment of a low-abundance driver mutation in PDO and its waste culture medium, providing a new direction for future research.

TREX2 enables efficient genome disruption mediated by paired CRISPR-Cas9 nickases that generate 3'-overhanging ends.

Paired SpCas9 nickases (SpCas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired SpCas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired SpCas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired SpCas9n that potentially generate short 3'-overhanging ends at overlapping SpCas9n target sites, suggesting sequential nicking of overlapping target sites by SpCas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to SpCas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to SpCas9n. SpCas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired SpCas9n, allowing practical use of this TREX2-based strategy in genome editing.

Self-Assembled Amphiphilic Chitosan Nanomicelles: Synthesis, Characterization and Antibacterial Activity.

Although amphiphilic chitosan has been widely studied as a drug carrier for drug delivery, fewer studies have been conducted on the antimicrobial activity of amphiphilic chitosan. In this study, we successfully synthesized deoxycholic acid-modified chitosan (CS-DA) by grafting deoxycholic acid (DA) onto chitosan C2-NH2, followed by grafting succinic anhydride, to prepare a novel amphiphilic chitosan (CS-DA-SA). The substitution degree was 23.93% for deoxycholic acid and 29.25% for succinic anhydride. Both CS-DA and CS-DA-SA showed good blood compatibility. Notably, the synthesized CS-DA-SA can self-assemble to form nanomicelles at low concentrations in an aqueous environment. The results of CS, CS-DA, and CS-DA-SA against Escherichia coli and Staphylococcus aureus showed that CS-DA and CS-DA-SA exhibited stronger antimicrobial effects than CS. CS-DA-SA may exert its antimicrobial effect by disrupting cell membranes or forming a membrane on the cell surface. Overall, the novel CS-DA-SA biomaterials have a promising future in antibacterial therapy.

TMB+-mediated etching of urchin-like gold nanostructures for colorimetric sensing.

The morphology-dependent localized surface plasmon resonance of gold nanostructures has been widely utilized for designing sensors. One method relies on the color change of gold nanoparticles upon etching. In previous work, TMB2+oxidized from 3,3',5,5'-tetramethylbenzidine (TMB) was found to etch gold nanorods (AuNRs), leading to a spectrum of different colors. However, the preparation of TMB2+needs the addition of a strong acid and other harsh conditions. Herein, a new colorimetric biosensing platform was developed using urchin-like gold nanoparticles (AuNUs). Compared with AuNRs, the etching of AuNUs can happen under mild conditions by TMB+at pH 6, protecting enzymes and proteins from denaturation. The role of CTAB surfactant was dissected, and its bromide ions were found to be involved in the etching process. Based on these observations, a one-step colorimetric detection of H2O2was realized by using horseradish peroxidase and H2O2to oxidize TMB. Within 30 min, this system achieved a detection limit of 80 nM H2O2. This work offered fundamental insights into the etching of anisotropic gold nanostructures and optimized the etching conditions. These advancements hold promise for broader applications in biosensing and analytical chemistry.

Primary choledocholithiasis occurrence and recurrence is synergetcally modulated by the bile microbiome and metabolome alternations.

AIMS: Primary choledocholithiasis is a common digestive disease with high morbidity and relapse. However, the compositions and functions of the bile microbial ecosystem and the pathogenesis of microfloral regulation of host metabolism resulting in stone formation are poorly understood. MAIN METHODS: Biliary samples collected from patients with acute cholangitis induced by benign biliary stricture (nonlithiasis group, n = 17) and primary choledocholithiasis (lithiasis group, n = 33) were subjected to multiomics analyses. Furthermore, clinicopathological features collected over a 24-month follow-up period were examined to evaluate the predictive value of candidate microbes. KEY FINDINGS: Five alpha diversity indices of the bile microbiome were significantly decreased in the lithiasis group. Furthermore, we identified 49 differential bile flora between the two groups, and the relative abundances of 6 bacteria, Actinobacteria, Actinobacteriota, Staphylococcales, Micrococcales, Altererythrobacter and Carnobacteriaceae, were associated with primary choledocholithiasis relapse conditions. Multiomics analyses showed that specific changes in disease-related bacterial taxa were closely related to metabolite variation (low-molecular weight carboxylic acids, sterol liquid and acylcarnitine), which might reflect disease prognosis. According to microbiomic and metabolomic pathway analyses, we revealed that bacterial infections, microbiota-derived amino acid metabolites and secondary bile acid-related pathways were significantly enriched in the stone-formation group, suggesting a novel host-microbial metabolic mechanism of primary choledocholithiasis. SIGNIFICANCE: Our study first indicates bile host-microbial dysbiosis modulates the abnormal accumulation of metabolites might further disrupt calcium homeostasis and generate insoluble saponification. Additionally, we determined the predictive value of Actinomycetes phylum reduction for recurrence in primary common bile duct stone patients.

A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells.

Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.