CTSS (Cathepsin S) Modulates Stress-Related Carotid Artery Thrombosis in a Mouse FeCl3 Model.

BACKGROUND: Exposure to chronic psychological stress is a risk factor for metabolic cardiovascular disease. Given the important role of lysosomal CTSS (cathepsin S) in human pathobiology, we examined the role of CTSS in stress-related thrombosis, focusing on inflammation, oxidative stress, and apoptosis. METHODS: Six-week-old wild-type mice (CTSS+/+) and CTSS-deficient mice (CTSS-/-) randomly assigned to nonstress and 2-week immobilization stress groups underwent iron chloride3 (FeCl3)-induced carotid thrombosis surgery for morphological and biochemical studies. RESULTS: On day 14 poststress/surgery, stress had increased the lengths and weights of thrombi in the CTSS+/+ mice, plus harmful changes in the levels of PAI-1 (plasma plasminogen activation inhibitor-1), ADAMTS13 (a disintegrin-link and metalloproteinase with thrombospondin type 13 motifs), and vWF (von Willebrand factor) and arterial tissue CTSS expression. Compared to the nonstressed CTSS+/+ mice, the stressed CTSS-/- mice had decreased levels of PAI-1, vWF, TNF (tumor necrosis factor)-α, interleukin-1ß, toll-like receptor-4, cleaved-caspase 3, cytochrome c, p16INK4A, gp91phox, p22phox, ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein-1), MyD88 (myeloid differentiation primary response 88), and MMP (matrix metalloproteinase)-2/-9 and increased levels of ADAMTS13, SOD (superoxide dismutase)-1/-2, eNOS (endothelial NO synthase), p-Akt (protein kinase B), Bcl-2 (B-cell lymphoma-2), p-GSK3α/ß (glycogen synthase kinases alpha and beta), and p-Erk1/2 (extracellular signal-regulated kinase 1 and 2) mRNAs and/or proteins. CTSS deletion also reduced the arterial thrombus area and endothelial loss. A pharmacological inhibition of CTSS exerted a vasculoprotective action. In vitro, CTSS silencing and overexpression, respectively, reduced and increased the stressed serum and oxidative stress-induced apoptosis of human umbilical vein endothelial cells, and they altered apoptosis-related proteins. CONCLUSIONS: CTSS inhibition appeared to improve the stress-related thrombosis in mice that underwent FeCl3-induction surgery, possibly by reducing vascular inflammation, oxidative stress, and apoptosis. CTSS could thus become a candidate therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disease.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity.

BACKGROUND: Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. RESULTS: Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain. CONCLUSIONS: The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Proper RPA acetylation promotes accurate DNA replication and repair.

The single-stranded DNA (ssDNA) binding protein complex RPA plays a critical role in promoting DNA replication and multiple DNA repair pathways. However, how RPA is regulated to achieve its functions precisely in these processes remains elusive. Here, we found that proper acetylation and deacetylation of RPA are required to regulate RPA function in promoting high-fidelity DNA replication and repair. We show that yeast RPA is acetylated on multiple conserved lysines by the acetyltransferase NuA4 upon DNA damage. Mimicking constitutive RPA acetylation or blocking its acetylation causes spontaneous mutations with the signature of micro-homology-mediated large deletions or insertions. In parallel, improper RPA acetylation/deacetylation impairs DNA double-strand break (DSB) repair by the accurate gene conversion or break-induced replication while increasing the error-prone repair by single-strand annealing or alternative end joining. Mechanistically, we show that proper acetylation and deacetylation of RPA ensure its normal nuclear localization and ssDNA binding ability. Importantly, mutation of the equivalent residues in human RPA1 also impairs RPA binding on ssDNA, leading to attenuated RAD51 loading and homologous recombination repair. Thus, timely RPA acetylation and deacetylation likely represent a conserved mechanism promoting high-fidelity replication and repair while discriminating the error-prone repair mechanisms in eukaryotes.

Heterojunction Engineering Enhanced Self-Polarization of PVDF/CsPbBr3 /Ti3 C2 Tx Composite Fiber for Ultra-High Voltage Piezoelectric Nanogenerator.

Piezoelectric nanogenerator (PENG) for practical application is constrained by low output and difficult polarization. In this work, a kind of flexible PENG with high output and self-polarization is fabricated by constructing CsPbBr3 -Ti3 C2 Tx heterojunctions in PVDF fiber. The polarized charges rapidly migrate to the electrodes from the Ti3 C2 Tx nanosheets by forming heterojunctions, achieving the maximum utilization of polarized charges and leading to enhanced piezoelectric output macroscopically. Optimally, PVDF/4wt%CsPbBr3 /0.6wt%Ti3 C2 Tx -PENG exhibits an excellent voltage output of 160 V under self-polarization conditions, which is higher than other self-polarized PENG previously. Further, the working principle and self-polarization mechanism are uncovered by calculating the interfacial charge and electric field using first-principles calculation. In addition, PVDF/4wt%CsPbBr3 /0.6wt%Ti3 C2 Tx -PENG exhibits better water and thermal stability attributed to the protection of PVDF. It is also evaluated in practice by harvesting the energy from human palm taps and successfully lighting up 150 LEDs and an electronic watch. This work presents a new idea of design for high-performance self-polarization PENG.

Phase-insensitive amplifier gain estimation at Cramér-Rao bound for two-mode squeezed state of light.

Phase-insensitive amplifiers (PIAs), as a class of important quantum devices, have found significant applications in the subtle manipulation of multiple quantum correlation and multipartite quantum entanglement. Gain is a very important parameter for quantifying the performance of a PIA. Its absolute value can be defined as the ratio of the output light beam power to the input light beam power, while its estimation precision has not been extensively investigated yet. Therefore, in this work, we theoretically study the estimation precision from the vacuum two-mode squeezed state (TMSS), the estimation precision of the coherent state, and the bright TMSS scenario, which has the following two advantages: it has more probe photons than the vacuum TMSS and higher estimation precision than the coherent state. The advantage in terms of estimation precision of the bright TMSS compared with the coherent state is researched. We first simulate the effect of noise from another PIA with gain M on the estimation precision of the bright TMSS, and we find that a scheme in which the PIA is placed in the auxiliary light beam path is more robust than two other schemes. Then, a fictitious beam splitter with transmission T is used to simulate the noise effects of propagation loss and imperfect detection, and the results show that a scheme in which the fictitious beam splitter is placed before the original PIA in the probe light beam path is the most robust. Finally, optimal intensity difference measurement is confirmed to be an accessible experimental technique to saturate estimation precision of the bright TMSS. Therefore, our present study opens a new avenue for quantum metrology based on PIAs.

ATF1 promotes the malignancy of lung adenocarcinoma cells by transcriptionally regulating ZNF143 expression.

The clinical oncogenic functions and mechanisms of activating transcription factor 1 (ATF1) in the progression of lung adenocarcinoma have not been completely elucidated. In this study, by employing human lung adenocarcinoma tissues and cells, we detect the correlation of ATF1 expression with the clinicopathological features and prognosis of patients with lung adenocarcinoma and find that ATF1 promotes lung adenocarcinoma cell proliferation and migration by transcriptionally enhancing zinc finger protein 143 (ZNF143) expression. ATF1 and ZNF143 are strongly expressed in lung adenocarcinoma tissues compared with those in the adjacent normal tissues, and high ATF1 and ZNF143 expressions are related to poor disease-free survival of lung adenocarcinoma patients. ATF1 overexpression results in increased proliferation and migration of lung adenocarcinoma cells, whereas knockdown of ATF1 inhibits cell proliferation and migration. Furthermore, ATF1 transcriptionally regulates the expression of ZNF143, and ATF1 and ZNF143 expressions are positively correlated in lung adenocarcinoma tissues. ZNF143 knockdown blocks lung adenocarcinoma cell migration, which is mediated by ATF1 upregulation. Hence, this study provides a potential therapeutic candidate for the treatment of lung adenocarcinoma.

Contaminant containment for sustainable remediation of persistent contaminants in soil and groundwater.

Contaminant containment measures are often necessary to prevent or minimize offsite movement of contaminated materials for disposal or other purposes when they can be buried or left in place due to extensive subsurface contamination. These measures can include physical, chemical, and biological technologies such as impermeable and permeable barriers, stabilization and solidification, and phytostabilization. Contaminant containment is advantageous because it can stop contaminant plumes from migrating further and allow for pollutant reduction at sites where the source is inaccessible or cannot be removed. Moreover, unlike other options, contaminant containment measures do not require the excavation of contaminated substrates. However, contaminant containment measures require regular inspections to monitor for contaminant mobilization and migration. This review critically evaluates the sources of persistent contaminants, the different approaches to contaminant remediation, and the various physical-chemical-biological processes of contaminant containment. Additionally, the review provides case studies of contaminant containment operations under real or simulated field conditions. In summary, contaminant containment measures are essential for preventing further contamination and reducing risks to public health and the environment. While periodic monitoring is necessary, the benefits of contaminant containment make it a valuable remediation option when other methods are not feasible.

Aristolochic acid I exposure triggers ovarian dysfunction by activating NLRP3 inflammasome and affecting mitochondrial homeostasis.

Aristolochic acids are widely distributed in the plants of Aristolochiaceae family and Asarum species. Aristolochic acid I (AAI) is the most frequent compound of aristolochic acids, which can accumulate in the soil, and then contaminates crops and water and enters the human body. Research has shown that AAI affects the reproductive system. However, the mechanism of AAI's effects on the ovaries at the tissue level still needs to be clarified. In this research, we found AAI exposure reduced the body and ovarian growth in mice, decreased the ovarian coefficient, prevented follicular development, and increased atretic follicles. Further experiments showed that AAI upregulated nuclear factor-κB and tumor necrosis factor-α expression, activated the NOD-like receptor protein 3 inflammasome, and led to ovarian inflammation and fibrosis. AAI also affected mitochondrial complex function and the balance between mitochondrial fusion and division. Metabolomic results also showed ovarian inflammation and mitochondrial dysfunction due to AAI exposure. These disruptions reduced the oocyte developmental potential by forming abnormal microtubule organizing centers and expressing abnormal BubR1 to destroy spindle assembly. In summary, AAI exposure triggers ovarian inflammation and fibrosis, affecting the oocyte developmental potential.

ETDA as a legacy soil chelatant: a comparative study to a more environmentally sensitive alternative for metal removal by Pistia stratiotes L.

The accuracy of environmental risk assessment depends upon selecting appropriate matrices to extract the most risk-relevant portion of contaminant(s) from the soil. Here, we applied the chelatants EDTA and tartaric acid to extract a metal-contaminated soil. Pistia stratiotes was applied as an indicator plant to measure accumulation from the metal-laden bulk solutions generated, in a hydroponic experiment lasting 15 days. Speciation modeling was used to elucidate key geo-chemical mechanisms impacting matrix and metal-specific uptake revealed by experimental work. The highest concentrations of soil-borne metals were extracted from soil by EDTA (7.4% for Cd), but their uptake and translocation to the plant were restricted due to the formation of stable metal complexes predominantly with DOC. Tartaric acid solubilized metals to a lesser extent (4.6% for Cd), but a higher proportion was plant available due to its presence mainly in the form of bivalent metal cations. The water extraction showed the lowest metal extraction (e.g., 3.9% for Cd), but the metal species behaved similarly to those extracted by tartaric acid. This study demonstrates that not all extractions are equal and that metal-specific speciation will impact accurate risk assessment in soil (water)-plant systems. In the case of EDTA, a deleterious impact on DOC leaching is an obvious drawback. As such, further work should now determine soil and not only metal-specific impacts of chelatants on the extraction of environmentally relevant portions of metal(loid)s.

EZH2 regulates pancreatic cancer cells through E2F1, GLI1, CDK3, and Mcm4.

Pancreatic cancer (PC) is one of the most common malignant tumors in digestive tract. To explore the role of epigenetic factor EZH2 in the malignant proliferation of PC, so as to provide effective medical help in PC. Sixty paraffin sections of PC were collected and the expression of EZH2 in PC tissues was detected by immunohistochemical assay. Three normal pancreas tissue samples were used as controls. The regulation of EZH2 gene on proliferation and migration of normal pancreatic cell and PC cell were determined by MTS, colony forming, Ki-67 antibody, scratch and Transwell assays. Through differential gene annotation and differential gene signaling pathway analysis, differentially expressed genes related to cell proliferation were selected and verified by RT-qPCR. EZH2 is mainly expressed in the nuclei of pancreatic tumor cells, but not in normal pancreatic cells. The results of cell function experiments showed that EZH2 overexpression could enhance the proliferation and migration ability of PC cell BXPC-3. Cell proliferation ability increased by 38% compared to the control group. EZH2 knockdown resulted in reduced proliferation and migration ability of cells. Compared with control, proliferation ability of cells reduced by 16%-40%. The results of bioinformatics analysis of transcriptome data and RT-qPCR demonstrated that EZH2 could regulate the expression of E2F1, GLI1, CDK3 and Mcm4 in normal and PC cells. The results revealed that EZH2 might regulate the proliferation of normal pancreatic cell and PC cell through E2F1, GLI1, CDK3 and Mcm4.

Overview of multifunctional cysteinyl cathepsins in atherosclerosis-based cardiovascular disease: from insights into molecular functions to clinical implications.

Cysteinyl cathepsins (CTSs) are widely known to have a proteolysis function that mediates recycling of unwanted proteins in endosomes and lysosomes, and investigation of CTSs has greatly improved with advances in live-imaging techniques both in vivo and in vitro, leading to three key findings. (1) CTSs are relocated from the lysosomes to other cellular spaces (i.e., cytosol, nucleus, nuclear membrane, plasma membrane, and extracellular milieu). (2) In addition to acidic cellular compartments, CTSs also exert biological activity in neutral environments. (3) CTSs also exert multiple nontraditional functions in, for example, extracellular matrix metabolism, cell signaling transduction, protein processing/trafficking, and cellular events. Various stimuli regulate the expression and activities of CTSs in vivo and vitro-e.g., inflammatory cytokines, oxidative stress, neurohormones, and growth factors. Accumulating evidence has confirmed the participation of CTSs in vascular diseases characterized by atherosclerosis, plaque rupture, thrombosis, calcification, aneurysm, restenosis/in-stent-restenosis, and neovasel formation. Circulating and tissue CTSs are promising as biomarkers and as a diagnostic imaging tool in patients with atherosclerosis-based cardiovascular disease (ACVD), and pharmacological interventions with their specific and non-specific inhibitors, and cardiovascular drugs might have potential for the therapeutic targeting of CTSs in animals. This review focuses on the update findings on CTS biology and the involvement of CTSs in the initiation and progression of ACVD and discusses the potential use of CTSs as biomarkers and small-molecule targets to prevent deleterious nontraditional functions in ACVD.

Reactive Magnesium Nitride Additive: A Drop-in Solution for Lithium/Garnet Wetting in All-Solid-State Batteries.

Garnet oxides such as Li6.4 La3 Zr1.4 Ta0.6 O12 (LLZTO) are promising solid electrolyte materials for all-solid-state lithium-metal batteries because of high ionic conductivity, low electronic leakage, and wide electrochemical stability window. While LLZTO has been frequently discussed to be stable against lithium metal anode, it is challenging to achieve and maintain good solid-on-solid wetting at the metal/ceramic interface in both processing and extended electrochemical cycling. Here we address the challenge by a powder-form magnesium nitride additive, which reacts with the lithium metal anode to produce well-dispersed lithium nitride. The in situ formed lithium nitride promotes reactive wetting at the Li/LLZTO interface, which lowers interfacial resistance, increases critical current density (CCD), and improves cycling stability of the electrochemical cells. The additive recipe has been diversified to titanium nitride, zirconium nitride, tantalum nitride, and niobium nitride, thus supporting the general concept of reactive dispersion-plus-wetting. Such a design can be extended to other solid-state devices for better functioning and extended cycle life.

The potential of biochar as a microbial carrier for agricultural and environmental applications.

Biochar can be an effective carrier for microbial inoculants because of its favourable properties promoting microbial life. In this review, we assess the effectiveness of biochar as a microbial carrier for agricultural and environmental applications. Biochar is enriched with organic carbon, contains nitrogen, phosphorus, and potassium as nutrients, and has a high porosity and moisture-holding capacity. The large number of active hydroxyl, carboxyl, sulfonic acid group, amino, imino, and acylamino hydroxyl and carboxyl functional groups are effective for microbial cell adhesion and proliferation. The use of biochar as a carrier of microbial inoculum has been shown to enhance the persistence, survival and colonization of inoculated microbes in soil and plant roots, which play a crucial role in soil biochemical processes, nutrient and carbon cycling, and soil contamination remediation. Moreover, biochar-based microbial inoculants including probiotics effectively promote plant growth and remediate soil contaminated with organic pollutants. These findings suggest that biochar can serve as a promising substitute for non-renewable substrates, such as peat, to formulate and deliver microbial inoculants. The future research directions in relation to improving the carrier material performance and expanding the potential applications of this emerging biochar-based microbial immobilization technology have been proposed.

Practical approach of As(V) adsorption by fabricating biochar with low basicity from FeCl3 and lignin.

One of the main challenges of biochar application for environmental cleanup is rise of pH in water or soil due to high ash and alkali metal contents in the biochar. While this intrinsic property of biochar is advantageous in alleviating soil and water acidity, it severely impairs the affinity of biochar toward anionic contaminants such as arsenic. This study explored a technical approach that can reduce the basicity of lignin-based biochar by utilizing FeCl3 during production of biochar. Three types of biochar were produced by co-pyrolyzing feedstock composed of different combinations of lignin, red mud (RM), and FeCl3, and the produced biochar samples were applied to adsorption of As(V). The biochar samples commonly possessed porous carbon structure embedded with magnetite (Fe3O4) particles. The addition of FeCl3 in the pyrolysis feedstock had a notable effect on reducing basicity of the biochar to yield significantly lower solution pH values than the biochar produced without FeCl3 addition. The extent of As(V) removal was also closely related to the final solution pH and the greatest As(V) removal (>77.6%) was observed for the biochar produced from co-pyrolysis of lignin, RM, and FeCl3. The results of adsorption kinetics and isotherm experiments, along with x-ray spectroscopy (XPS), strongly suggested adsorption of As(V) occurred via specific chemical reaction (chemisorption) between As(V) and Fe-O functional groups on magnetite. Thus, the overall results suggest the use of FeCl3 is a feasible practical approach to control the intrinsic pH of biochar and impart additional functionality that enables effective treatment of As(V).

Triple-gene deletion for osteocalcin significantly impairs the alignment of hydroxyapatite crystals and collagen in mice.

Osteocalcin (Ocn), also known as bone Gla protein, is synthesized by osteoblasts and thought to regulate energy metabolism, testosterone synthesis and brain development. However, its function in bone is not fully understood. Mice have three Ocn genes: Bglap, Bglap2 and Bglap3. Due to the long span of these genes in the mouse genome and the low expression of Bglap3 in bone, researchers commonly use Bglap and Bglap2 knockout mice to investigate the function of Ocn. However, it is unclear whether Bglap3 has any compensatory mechanisms when Bglap and Bglap2 are knocked out. Considering the controversy surrounding the role of Ocn in bone, we constructed an Ocn-deficient mouse model by knocking out all three genes (Ocn-/-) and analyzed bone quality by Raman spectroscopy (RS), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and MicroCT (µCT). The RS test showed that the alignment of hydroxyapatite crystals and collagen fibers was significantly poorer in Ocn-/- mice than in wild-type (WT) mice. Ocn deficiency resulted in a looser surface structure of bone particles and a larger gap area proportion. FTIR analysis showed few differences in bone mineral index between WT and Ocn-/- mice, while µCT analysis showed no significant difference in cortical and trabecular regions. However, under tail-suspension simulating bone loss condition, the disorder of hydroxyapatite and collagen fiber alignment in Ocn-/- mice led to more obvious changes in bone mineral composition. Collectively, our results revealed that Ocn is necessary for regulating the alignment of minerals parallel to collagen fibrils.

Sustainable management of hazardous asbestos-containing materials: Containment, stabilization and inertization.

Asbestos is a group of six major silicate minerals that belong to the serpentine and amphibole families, and include chrysotile, amosite, crocidolite, anthophyllite, tremolite and actinolite. Weathering and human disturbance of asbestos-containing materials (ACMs) can lead to the emission of asbestos dust, and the inhalation of respirable asbestos fibrous dust can lead to 'mesothelioma' cancer and other diseases, including the progressive lung disease called 'asbestosis'. There is a considerable legacy of in-situ ACMs in the built environment, and it is not practically or economically possible to safely remove ACMs from the built environment. The aim of the review is to examine the three approaches used for the sustainable management of hazardous ACMs in the built environment: containment, stabilization, and inertization or destruction. Most of the asbestos remaining in the built environment can be contained in a physically secured form so that it does not present a significant health risk of emitting toxic airborne fibres. In settings where safe removal is not practically feasible, stabilization and encapsulation can provide a promising solution, especially in areas where ACMs are exposed to weathering or disturbance. Complete destruction and inertization of asbestos can be achieved by thermal decomposition using plasma and microwave radiation. Bioremediation and chemical treatment (e.g., ultrasound with oxalic acid) have been found to be effective in the inertization of ACMs. Technologies that achieve complete destruction of ACMs are found to be attractive because the treated products can be recycled or safely disposed of in landfills.

Molecular Imaging Reveals Two Distinct Mixing States of PM2.5 Particles Sampled in a Typical Beijing Winter Pollution Case.

Mixing states of aerosol particles are crucial for understanding the role of aerosols in influencing air quality and climate. However, a fundamental understanding of the complex mixing states is still lacking because most traditional analysis techniques only reveal bulk chemical and physical properties with limited surface and 3-D information. In this research, 3-D molecular imaging enabled by ToF-SIMS was used to elucidate the mixing states of PM2.5 samples obtained from a typical Beijing winter haze event. In light pollution cases, a thin organic layer covers separated inorganic particles; while in serious pollution cases, ion exchange and an organic-inorganic mixing surface on large-area particles were observed. The new results provide key 3-D molecular information of mixing states, which is highly potential for reducing uncertainty and bias in representing aerosol-cloud interactions in current Earth System Models and improving the understanding of aerosols on air quality and human health.

Applications, impacts, and management of biochar persistent free radicals: A review.

Biochar is a promising environmental contaminant remediation agent because of its adsorptive and catalytic properties. However, the environmental effects of persistent free radicals (PFRs) produced by biomass pyrolysis (biochar production) are still poorly understood, though they have received increasing research attention in recent years. Although PFRs both directly and indirectly mediate biochar's removal of environmental pollutants, they also have the potential to cause ecological damage. In order to support and sustain biochar applications, effective strategies are needed to control the negative effects of biochar PFRs. Yet, there has been no systematic evaluation of the environmental behavior, risks, or management techniques of biochar PFRs. Thus, this review: 1) outlines the formation mechanisms and types of biochar PFRs, 2) evaluates their environmental applications and potential risks, 3) summarizes their environmental migration and transformation, and 4) explores effective management strategies for biochar PFRs during both production and application phases. Finally, future research directions are recommended.

Artificial intelligence-based comprehensive analysis of immune-stemness-tumor budding profile to predict survival of patients with pancreatic adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy. CD8+ T cells, cancer stem cells (CSCs), and tumor budding (TB) have been significantly correlated with the outcome of patients with PDAC, but the correlations have been independently reported. In addition, no integrated immune-CSC-TB profile for predicting survival in patients with PDAC has been established. METHODS: Multiplexed immunofluorescence and artificial intelligence (AI)-based comprehensive analyses were used for quantification and spatial distribution analysis of CD8+ T cells, CD133+ CSCs, and TB. In vivo humanized patient-derived xenograft (PDX) models were established. Nomogram analysis, calibration curve, time-dependent receiver operating characteristic curve, and decision curve analyses were performed using R software. RESULTS: The established 'anti-/pro-tumor' models showed that the CD8+ T cell/TB, CD8+ T cell/CD133+ CSC, TB-adjacent CD8+ T cell, and CD133+ CSC-adjacent CD8+ T cell indices were positively associated with survival of patients with PDAC. These findings were validated using PDX-transplanted humanized mouse models. An integrated nomogram-based immune-CSC-TB profile that included the CD8+ T cell/TB and CD8+ T cell/CD133+ CSC indices was established and shown to be superior to the tumor-node-metastasis stage model in predicting survival of patients with PDAC. CONCLUSIONS: 'Anti-/pro-tumor' models and the spatial relationship among CD8+ T cells, CSCs, and TB within the tumor microenvironment were investigated. Novel strategies to predict the prognosis of patients with PDAC were established using AI-based comprehensive analysis and machine learning workflow. The nomogram-based immune-CSC-TB profile can provide accurate prognosis prediction for patients with PDAC.