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Abstract Tumor‐associatedmacrophages (TAMs) exhibit remarkable heterogeneity in glioblastoma. Spatially resolved single‐cell transcriptomic studies identified a monocyte‐derived TAM subset localized in the peri‐necrotic niche, driven by hypoxic cues to acquire ahypoxia response signature. These hypoxia‐TAMs destabilize endothelial adherens junctions through adrenomedullin paracrine signaling, promoting the formation of hyperpermeable neovasculature that impedes drug delivery. Blocking adrenomedullin produced by hypoxia‐TAMs restores vascular integrity, increases drug deliveryinto tumors, and provides combinatorial therapeutic benefits. Here we discuss the heterogeneity of TAMs regarding functional states and locations in glioblastomas, and propose future directions for studying the temporospatial dynamics of multifaceted TAM. Highlights Single‐cell omics reveal a functionally and spatially distinct hypoxia‐TAM subset in glioblastoma. Adrenomedullin secreted by hypoxia‐TAM destabilizes tumor vasculature and its blockade enhances vessel integrity and drug delivery.

Abstract Dysregulation of the transforming growth factor‐β (TGF‐β) signaling pathway regulates cancer stem cells (CSCs) and drug sensitivity, whereas it remains largely unknown how feedback regulatory mechanisms are hijacked to fuel drug‐resistant CSCs. Through a genome‐wide CRISPR activation screen utilizing stem‐like drug‐resistant properties as a readout, the TGF‐β receptor‐associated binding protein 1 (TGFBRAP1) is identified as a TGF‐β‐inducible positive feedback regulator that governs sensitivity to tyrosine kinase inhibitors (TKIs) and promotes liver cancer stemness. By interacting with and stabilizing the TGF‐β receptor type 1 (TGFBR1), TGFBRAP1 plays an important role in potentiating TGF‐β signaling. Mechanistically, TGFBRAP1 competes with E3 ubiquitin ligases Smurf1/2 for binding to TGFΒR1, leading to impaired receptor poly‐ubiquitination and proteasomal degradation. Moreover, hyperactive TGF‐β signaling in turn up‐regulates TGFBRAP1 expression in drug‐resistant CSC‐like cells, thereby constituting a previously uncharacterized feedback mechanism to amplify TGF‐β signaling. As such, TGFBRAP1 expression is correlated with TGFΒR1 levels and TGF‐β signaling activity in hepatocellular carcinoma (HCC) tissues, as well as overall survival and disease recurrence in multiple HCC cohorts. Therapeutically, blocking TGFBRAP1‐mediated stabilization of TGFBR1 by selective inhibitors alleviates Regorafenib resistance via reducing CSCs. Collectively, targeting feedback machinery of TGF‐β signaling pathway may be an actionable approach to mitigate drug resistance and liver cancer stemness.

Abstract The drug response phenotype is determined by a combination of genetic and environmental factors. The high clinical conversion failure rate of gene‐targeted drugs might be attributed to the lack of emphasis on environmental factors and the inherent individual variability in drug response (IVDR). Current evidence suggests that environmental variables, rather than the disease itself, are the primary determinants of both gut microbiota composition and drug metabolism. Additionally, individual differences in gut microbiota create a unique metabolic environment that influences the in vivo processes underlying drug absorption, distribution, metabolism, and excretion (ADME). Here, we discuss how gut microbiota, shaped by both genetic and environmental factors, affects the host's ADME microenvironment within a new evaluation system for drug–microbiota interactions. Furthermore, we propose a new top‐down research approach to investigate the intricate nature of drug–microbiota interactions in vivo. This approach utilizes germ‐free animal models, providing foundation for the development of a new evaluation system for drug–microbiota interactions.

Abstract The peptidyl-prolyl cis-trans isomerase Pin1 is a pivotal therapeutic target in cancers, but the regulation of Pin1 protein stability is largely unknown. High Pin1 expression is associated with SUMO1-modified protein hypersumoylation in glioma stem cells (GSCs), but the underlying mechanisms remain elusive. Here we demonstrate that Pin1 is deubiquitinated and stabilized by USP34, which promotes isomerization of the sole SUMO E2 enzyme Ubc9, leading to SUMO1-modified hypersumoylation to support GSC maintenance. Pin1 interacts with USP34, a deubiquitinase with preferential expression and oncogenic function in GSCs. Such interaction is facilitated by Plk1-mediated phosphorylation of Pin1. Disruption of USP34 or inhibition of Plk1 promotes poly-ubiquitination and degradation of Pin1. Furthermore, Pin1 isomerizes Ubc9 to upregulate Ubc9 thioester formation with SUMO1, which requires CDK1-mediated phosphorylation of Ubc9. Combined inhibition of Pin1 and CDK1 with sulfopin and RO3306 most effectively suppresses orthotopic tumor growth. Our findings provide multiple molecular targets to induce Pin1 degradation and suppress hypersumoylation for cancer treatment.

BackgroundPersistent inflammation following vascular injury drives neointimal hyperplasia (NIH). Specialized lipid mediators (SPM) mediate resolution which attenuates inflammation and downstream NIH. We investigated the effects of a synthetic analogue of resolvin D1 (RvD1) on vascular cells and in a model of rat carotid angioplasty.MethodsHuman venous VSMC and endothelial cells (EC) were employed in migration, cell shape, toxicity, proliferation and p65 nuclear translocation assays. Murine RAW 264.7 cells were utilized to test the effect of pro-resolving compounds on phagocytic activity. A model of rat carotid angioplasty was used to evaluate the effects of 17R/S-benzo-RvD1 (benzo-RvD1) and 17R-RvD1 applied to the adventitia via 25% Pluronic gel. Immunostaining was utilized to examine Ki67 expression and leukocyte recruitment. Morphometric analysis was performed on arteries harvested 14 days after injury.ResultsExposure to benzo-RvD1 attenuated PDGF- stimulated VSMC migration across a range of concentrations (0.1-100 nM), similar to that observed with 17R-RvD1. Pre-treatment with either Benzo-RvD1 or 17R-RvD1 (10, 100nM) attenuated PDGF-BB-induced VSMC cytoskeletal changes to nearly baseline dimensions. Benzo-RvD1 demonstrated modest anti-proliferative activity on VSMC and EC at various concentrations, without significant cytotoxicity. Benzo-RvD1 (10nM) inhibited p65 nuclear translocation in cytokine-stimulated EC by 21% (p

Background: Guidance has showed positive effects on promoting inquiry-based learning in science education. While an increasing number of studies focus on the design of guidance in simulation-based inquiry learning due to recent technology developments, how different designs of a same type of guidance affect learning remains a question. Objectives: This study reviews the (quasi-)experimental research on the learning effects of differently designed guidance in simulation-based inquiry learning in the past decade (2011-2020). The investigation is guided by two questions: how differently designed guidance affects inquiry-based learning in simulated environments in terms of learning process and learning outcomes, and what role technology plays in the design of guidance. In particular, we report on studies that compares the effects of a same type (process constraints/prompts/heuristics/scaffolds/metacognitive supports/direct presentation of information) of guidance with different instructional designs, and select a total of 28 peer-reviewed journal articles. The results indicate no equivocal tendency of effectiveness towards a specific design. Instead, each type of guidance has related factors that may influence its effectiveness. Three major factors that related to optimization of inquiry learning guidance in simulation-based environments are identified: the learner factor, the pedagogical factor, and the technological factor.

Abstract Effective treatment for metastasis, a leading cause of cancer‐associated death, is still lacking. To seed on a distal organ, disseminated cancer cells (DCCs) must adapt to the local tissue microenvironment. However, it remains elusive how DCCs respond the pro‐metastatic niche signals. Here, systemic motif‐enrichment identified myocyte enhancer factor 2D (MEF2D) as a critical sensor of niche signals to regulate DCCs adhesion and colonization, leading to intrahepatic metastasis and recurrence of liver cancer. In this context, MEF2D transactivates Itgb1 (coding β1‐integrin) and Itgb4 (coding β4‐integrin) to execute temporally unique functions, where ITGB1 recognizes extracellular matrix for early seeding, and ITGB4 acts as a novel sensor of neutrophil extracellular traps‐DNA (NETs‐DNA) for subsequent chemotaxis and colonization. In turn, an integrin‐FAK circuit promotes a phosphorylation‐dependent USP14‐orchastrated deubiquitination switch to stabilize MEF2D via circumventing degradation by the E3‐ubiquitin‐ligase MDM2. Clinically, the USP14(pS432)‐MEF2D‐ITGB1/4 feedback loop is often hyper‐active and indicative of inferior outcomes in human malignancies, while its blockade abrogated intrahepatic metastasis of DCCs. Together, DCCs exploit a deubiquitination‐dependent switch on MEF2D to integrate niche signals in the liver mesenchyme, thereby amplifying the pro‐metastatic integrin‐FAK signaling. Disruption of this feedback loop is clinically applicable with fast‐track potential to block microenvironmental cues driving metastasis.

ObjectiveThe 2020 Global Vascular Guidelines aim at improving decision making in Chronic Limb-Threatening Ischemia (CLTI) by providing a framework for evidence-based revascularization. Herein, the Global Limb Anatomic Staging System (GLASS) serves to estimate the chance of success and patency of arterial pathway revascularization based on the extent and distribution of the atherosclerotic lesions. We report the preliminary feasibility results and observer variability of the GLASS. GLASS is a part of the new global guideline and posed as a promising additional tool for EBR strategies to predict the success of lower extremity arterial revascularization. This study reports on the consistency of GLASS scoring to maximize inter-observer agreement and facilitate its application.MethodsGLASS separately scores the femoropopliteal (FP) and infrapopliteal (IP) segment based on stenosis severity, lesion length and the extent of calcification within the target artery pathway (TAP). In our stepwise approach, we used two angiographic datasets. Each following step was based on the lessons learned from the previous step. The primary outcome was inter-observer agreement measured as Cohen's Kappa, scored by two (step 1 + 2) and four (step 3) blinded and experienced observers, respectively. Steps 1 (n = 139) and 2 (n = 50) were executed within a dataset of a Dutch interventional RCT in CLTI. Step 3 (n = 100) was performed in randomly selected all-comer CLTI patients from two vascular centers in the United States.ResultsIn step 1, kappa values were 0.346 (FP) and 0.180 (IP). In step 2, applied in the same dataset, the use of other experienced observers and a provided TAP, resulted in similar low kappa values 0.406 (FP) and 0.089 (IP). Subsequently, in step 3, the formation of an altered stepwise approach using component scoring, such as separate scoring of calcification and adding a ruler to the images resulted in kappa values increasing to 0.796 (FP) and 0.730 (IP).ConclusionThis retrospective GLASS validation study revealed low inter-observer agreement for unconditioned scoring. A stepwise component scoring provides acceptable agreement and a solid base for further prospective validation studies to investigate how GLASS relates to treatment outcomes.

Abstract Necroptosis plays a double-edged sword role in necroptotic cancer cell death and tumor immune escape. How cancer orchestrates necroptosis with immune escape and tumor progression remains largely unclear. We found that RIP3, the central activator of necroptosis, was methylated by PRMT1 methyltransferase at the amino acid of RIP3 R486 in human and the conserved amino acid R479 in mouse. The methylation of RIP3 by PRMT1 inhibited the interaction of RIP3 with RIP1 to suppress RIP1-RIP3 necrosome complex, thereby blocking RIP3 phosphorylation and necroptosis activation. Moreover, the methylation-deficiency RIP3 mutant promoted necroptosis, immune escape and colon cancer progression due to increasing tumor infiltrated myeloid-derived immune suppressor cells (MDSC), while PRMT1 reverted the immune escape of RIP3 necroptotic colon cancer. Importantly, we generated a RIP3 R486 di-methylation specific antibody (RIP3ADMA). Clinical patient samples analysis revealed that the protein levels of PRMT1 and RIP3ADMA were positively correlated in cancer tissues and both of them predicted the longer patient survival. Our study provides insights into the molecular mechanism of PRMT1-mediated RIP3 methylation in the regulation of necroptosis and colon cancer immunity, as well as reveals PRMT1 and RIP3ADMA as the valuable prognosis markers of colon cancer.

With the aggressive resource conservation necessary to face the coronavirus disease 2019 pandemic, vascular surgeons have faced unique challenges in managing the health of their high-risk patients. An early analysis of patient outcomes after pandemic-related practice changes suggested that patients with chronic limb threatening ischemia have been presenting with more severe foot infections and are more likely to require major limb amputation compared with 6 months previously. As our society and health care system adapt to the new changes required in the post-coronavirus disease 2019 era, it is critical that we pay special attention to the most vulnerable subsets of patients with vascular disease, particularly those with chronic limb threatening ischemia and limited access to care.