Discovery of a novel small-molecule activator of SIRT3 that inhibits cell proliferation and migration by apoptosis and autophagy-dependent cell death pathways in colorectal cancer.

Colorectal cancer (CRC) is well known as a prevalent malignancy affecting the digestive tract, yet its precise etiological determinants remain to be elusive. Accordingly, identifying specific molecular targets for colorectal cancer and predicting potential malignant tumor behavior are potential strategies for therapeutic interventions. Of note, apoptosis (type I programmed cell death) has been widely reported to play a pivotal role in tumorigenesis by exerting a suppressive effect on cancer development. Moreover, autophagy-dependent cell death (type II programmed cell death) has been implicated in different types of human cancers. Thus, investigating the molecular mechanisms underlying apoptosis and autophagy-dependent cell death is paramount in treatment modalities of colorectal cancer. In this study, we uncovered that a new small-molecule activator of SIRT3, named MY-13, triggered both autophagy-dependent cell death and apoptosis by modulating the SIRT3/Hsp90/AKT signaling pathway. Consequently, this compound inhibited tumor cell proliferation and migration in RKO and HCT-116 cell lines. Moreover, we further demonstrated that the small-molecule activator significantly suppressed tumor growth in vivo. In conclusion, these findings demonstrate that the novel small-molecule activator of SIRT3 may hold a therapeutic potential as a drug candidate in colorectal cancer.

Aptamer-bivalent-cholesterol-mediated proximity entropy-driven exosomal protein reporter for tumor diagnosis.

Exosomal proteins from the parental cells are considered to be promising biomarker sets for precise tumor diagnostics and monitoring. However, the accurate quantitative analysis of low-abundance exosomal proteins remains challenging due to the heterogeneity of clinical samples. Here, we standardized the exosomal concentration with a fluorogenic membrane probe and developed an aptamer-bivalent-cholesterol-mediated Proximity Entropy-driven Exosomal Protein Reporter (PEEPR). The proposed PEEPR enables the in-situ analysis of multiple exosomal proteins by integrating bivalent cholesterol anchor (exosomal lipid bilayer) and aptamer (exosomal proteins) with a proximity entropy-driven circuit. Based on this strategy, we successfully achieved detection limits of 3.9 pg/mL exosomal GPC-3 and 3.4 pg/mL exosomal PD-L1. Notably, the standardization of exosome concentrations is designed to avoid errors due to biological heterogeneity. The results showed that evaluating the levels of exosomal GPC-3 and PD-L1 in clinical samples via this strategy could accurately differentiate healthy individuals, hepatitis B patients, and hepatocellular carcinoma patients. In summary, PEEPR is a promising clinical diagnostic strategy for the quantitative analysis of a variety of tumor-associated exosomal proteins for the precise diagnosis and personalized treatment monitoring of tumors.

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs.

This protocol aims to establish a method for identifying small molecular antagonists of ß2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. ß2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling ß2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of ß2 integrins, is utilized to quantify ß2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil ß2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on ß2 integrin inhibition are assessed within 3 h. Molecules that directly target ß2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

Design, synthesis, and pharmacological evaluation of novel benzothiazole derivatives targeting LCK in acute lymphoblastic leukemia.

Lymphocyte-specific protein tyrosine kinase (LCK), a member of the Src family of tyrosine kinases, is implicated in the pathogenesis of almost all types of leukemia via T cells activation and signal transduction. LCK is highly expressed in acute lymphoblastic leukemia (ALL), and knockdown of the LCK gene can significantly inhibit the proliferation of leukemia cell lines. Here, we designed and synthesized a series of benzothiazole derivatives as novel LCK inhibitors using both docking-based virtual screening and activity assays for structural optimization. Among these compounds, 7 m showed a strong inhibitory activity in the proliferation of leukemia cell lines and LCK kinase activity. Moreover, we found that compound 7 m could induce apoptosis while simultaneously blocking cell cycle via decreasing its phosphorylation at Tyr394 of the LCK. Collectively, these findings shed new light on compound 7 m that would be utilized as a promising drug candidate with apoptosis-triggered and cell cycle arrest activities for the future ALL therapy.

Multi-omics approaches identify novel prognostic biomarkers of autophagy in uveal melanoma.

PURPOSE: Uveal melanoma (UVM) is a rare yet malignant ocular tumor that metastases in approximately half of all patients, with the majority of those developing metastasis typically succumbing to the disease within a year. Hitherto, no effective treatment for UVM has been identified. Autophagy is a cellular mechanism that has been suggested as an emerging regulatory process for cancer-targeted therapy. Thus, identifying novel prognostic biomarkers of autophagy may help improve future treatment. METHODS: Consensus clustering and similarity network fusion approaches were performed for classifying UVM patient subgroups. Weighted correlation network analysis was performed for gene module screening and network construction. Gene set variation analysis was used to evaluate the autophagy activity of the UVM subgroups. Kaplan-Meier survival curves (Log-rank test) were performed to analyze patient prognosis. Gene set cancer analysis was used to estimate the level of immune cell infiltration. RESULTS: In this study, we employed multi-omics approaches to classify UVM patient subgroups by molecular and clinical characteristics, ultimately identifying HTR2B, EEF1A2, FEZ1, GRID1, HAP1, and SPHK1 as potential prognostic biomarkers of autophagy in UVM. High expression levels of these markers were associated with poorer patient prognosis and led to reshaping the tumor microenvironment (TME) that promotes tumor progression. CONCLUSION: We identified six novel potential prognostic biomarkers in UVM, all of which are associated with autophagy and TME. These findings will shed new light on UVM therapy with inhibitors targeting these biomarkers expected to regulate autophagy and reshape the TME, significantly improving UVM treatment outcomes.

Targeting autophagy with small-molecule activators for potential therapeutic purposes.

Autophagy is well-known to be a lysosome-mediated catabolic process for maintaining cellular and organismal homeostasis, which has been established with many links to a variety of human diseases. Compared with the therapeutic strategy for inhibiting autophagy, activating autophagy seems to be another promising therapeutic strategy in several contexts. Hitherto, mounting efforts have been made to discover potent and selective small-molecule activators of autophagy to potentially treat human diseases. Thus, in this perspective, we focus on summarizing the complicated relationships between defective autophagy and human diseases, and further discuss the updated progress of a series of small-molecule activators targeting autophagy in human diseases. Taken together, these inspiring findings would provide a clue on discovering more small-molecule activators of autophagy as targeted candidate drugs for potential therapeutic purposes.

IL-2 can signal via chemokine receptors to promote regulatory T cells' suppressive function.

Canonical interleukin-2 (IL-2) signaling via the high-affinity CD25-containing IL-2 receptor-Janus kinase (JAK)1,3-signal transducer and activator of transcription 5 (STAT5) pathway is essential for development and maintenance of CD4+CD25HiFoxp3+ regulatory T cells (Tregs) that support immune homeostasis. Here, we report that IL-2 signaling via an alternative CD25-chemokine receptor pathway promotes the suppressive function of Tregs. Using an antibody against CD25 that biases IL-2 signaling toward this alternative pathway, we establish that this pathway increases the suppressive activity of Tregs and ameliorates murine experimental autoimmune encephalomyelitis (EAE). Furthermore, heparan sulfate, an IL-2-binding element of cell surfaces and extracellular matrix, or an engineered IL-2 immunocytokine can also direct IL-2 signaling toward this alternative pathway. Overall, these data reveal a non-canonical mechanism for IL-2 signaling that promotes suppressive functions of Tregs, further elucidates how IL-2 supports immune homeostasis, and suggests approaches to promote or suppress Treg functions.

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

Programmable Nanoscale Crack Lithography for Multiscale PMMA Patterns.

Crack lithography is important for preparing microstructured materials. This strategic use of cracking breaks with the traditional idea that cracks are unwanted and has great potential for high-resolution and high-throughput production. However, the ability to control nanoscale crack patterning is still insufficient. Here, we present a nanoscale, programmable angle-dependent technique to control crack generation that relies on standard electron-beam lithography. Multiscale patterns of poly(methyl methacrylate) of arbitrary shape, geometric size, and large area were obtained, greatly expanding the processing capacity of electron-beam lithography. In addition, we observed the interaction between adjacent structures and cracks, which resulted in crack suppression or second-order cracks. We also demonstrated that angle-dependent nanoscale cracks can be used in physical unclonable functions and have great application prospects in the field of information security. We believe that our strategy for programmable nanoscale crack patterning provides new opportunities and perspectives for nanofabrication.

Opportunities and challenges of natural killer cell-derived extracellular vesicles.

Extracellular vesicles (EVs) are increasingly recognized as important intermediaries of intercellular communication. They have significant roles in many physiological and pathological processes and show great promise as novel biomarkers of disease, therapeutic agents, and drug delivery tools. Existing studies have shown that natural killer cell-derived EVs (NEVs) can directly kill tumor cells and participate in the crosstalk of immune cells in the tumor microenvironment. NEVs own identical cytotoxic proteins, cytotoxic receptors, and cytokines as NK cells, which is the biological basis for their application in antitumor therapy. The nanoscale size and natural targeting property of NEVs enable precisely killing tumor cells. Moreover, endowing NEVs with a variety of fascinating capabilities via common engineering strategies has become a crucial direction for future research. Thus, here we provide a brief overview of the characteristics and physiological functions of the various types of NEVs, focusing on their production, isolation, functional characterization, and engineering strategies for their promising application as a cell-free modality for tumor immunotherapy.

Accurate and rapid quantification of PD-L1 positive exosomes by a triple-helix molecular probe.

Programmed death ligand-1 (PD-L1) positive exosomes (P-Exo) have been widely used for tumor diagnosis. However, accurate and rapid quantification of P-Exo remains challenging due to the heterogeneity of clinical individuals and isolation techniques. In this study, the triple-helix molecular probe (THMP) coupled with high-affinity silica-based TiO2 magnetic beads was used to isolate exosomes and to analyze the relative abundance of P-Exo in total exosomes (T-Exo). By employing this strategy, the entire analysis was completed within 70 min and the detection limit for P-Exo was 880 particles µL-1. Additionally, the relative abundance of P-Exo in T-Exo (RAP-Exo/T-Exo) was calculated from their fluorescence ratio, which could avoid errors due to differences in samples and separation methods, and identify 1.5 × 103 P-Exo from 5 × 106 T-Exo per microliter. RAP-Exo/T-Exo values were not only effective in distinguishing healthy volunteers from breast cancer patients, but also highly positively correlated with the stage of breast carcinoma. Overall, this strategy opens a new avenue for rapid and quantitative analysis of P-Exo, providing an opportunity for precise diagnosis and prediction of treatment efficacy in cancer.

Programming 3D curved mesosurfaces using microlattice designs.

Cellular microstructures form naturally in many living organisms (e.g., flowers and leaves) to provide vital functions in synthesis, transport of nutrients, and regulation of growth. Although heterogeneous cellular microstructures are believed to play pivotal roles in their three-dimensional (3D) shape formation, programming 3D curved mesosurfaces with cellular designs remains elusive in man-made systems. We report a rational microlattice design that allows transformation of 2D films into programmable 3D curved mesosurfaces through mechanically guided assembly. Analytical modeling and a machine learning-based computational approach serve as the basis for shape programming and determine the heterogeneous 2D microlattice patterns required for target 3D curved surfaces. About 30 geometries are presented, including both regular and biological mesosurfaces. Demonstrations include a conformable cardiac electronic device, a stingray-like dual mode actuator, and a 3D electronic cell scaffold.

Ultrasonic Phased Array Imaging Approach Using Omni-Directional Velocity Correction for Quantitative Evaluation of Delamination in Composite Structure.

The ultrasonic detectability of buried defects within composite materials is dependent on the anisotropy of the composite material by which the propagation property of acoustic wave in each direction is variably affected. In this study, the characteristics of acoustic waves propagating in different directions for composite materials are explored based on the full matrix capture (FMC) data using an ultrasonic phased array. The elastic constant of multidirectional carbon fiber reinforced plastic (CFRP) laminate is first derived based on the genetic algorithm. The characteristics of transmitted and reflected waves in higher angles are predicted by implementing the Christoffel equation, and the focal law used in post-processing of FMC data can be optimized accordingly. The imaging results of the total focusing method (TFM) using the improved focal law are compared with the results of the conventional TFM. The results suggest that the optimized TFM can effectively characterize the defect by reducing the background noise. Furthermore, since it is impractical to theoretically correct angle-dependent velocity for in situ inspection, a linear extrapolation method based on the experimentally measurable velocity at low angles is proposed to estimate the velocity profile at higher angles. The imaging results using the fast extrapolated velocity profile is then compared with the theoretical, and it has been demonstrated that while the difference between the images using the theoretical focal law and the linearly extrapolated one is barely visible, the later one is overwhelmingly advantageous to be realiszd for engineering practices.

Protein-free, ultrasensitive miRNA analysis based on an entropy-driven catalytic reaction switched on a smart-responsive DNAzyme dual-walker amplification strategy.

MicroRNAs (miRNAs), useful biomarkers for cancer diagnosis, play an important role in tumorigenesis and progression, but many of the current analysis methods can suffer from excessive protease dependence, being time-consuming and unsatisfactory performance. Therefore, a reliable sensing strategy for the protein-free, ultrasensitive analysis of tumor-associated miRNAs is desired. The proposed dual-walker biosensing strategy based on an entropy-driven catalytic (EDC) walker coupled with a smart-responsive DNAzyme walker was demonstrated for the dual-amplification detection of miRNA-21. Namely, the target miRNA-21 initiates the three-stranded substrate complex of the traditional EDC circuit to release the input trigger of the Dz walker, which recognizes the circular binding domain to restore the cleavage activity of the DzS-AuNP walker. The fluorescence signal continuously released from the AuNPs was recorded by a fluorescence reader for miRNA-21 sensing. The optimized dual-walker exhibited appreciable sensitivity with a detection limit of 70 fM, satisfactory flexibility, fine specificity and ideal stability for clinical serum sample assays. The proposed strategy may open a new avenue for the development of powerful DNA molecular tools for cancer diagnosis.

Nexinhib20 Inhibits Neutrophil Adhesion and ß2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting ß2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced ß2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and ß2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Roles of mitochondria in neutrophils.

Neutrophils are the most abundant leukocyte in human blood. They are critical for fighting infections and are involved in inflammatory diseases. Mitochondria are indispensable for eukaryotic cells, as they control the biochemical processes of respiration and energy production. Mitochondria in neutrophils have been underestimated since glycolysis is a major metabolic pathway for fuel production in neutrophils. However, several studies have shown that mitochondria are greatly involved in multiple neutrophil functions as well as neutrophil-related diseases. In this review, we focus on how mitochondrial components, metabolism, and related genes regulate neutrophil functions and relevant diseases.

Integrin Regulators in Neutrophils.

Neutrophils are the most abundant leukocytes in humans and are critical for innate immunity and inflammation. Integrins are critical for neutrophil functions, especially for their recruitment to sites of inflammation or infections. Integrin conformational changes during activation have been heavily investigated but are still not fully understood. Many regulators, such as talin, Rap1-interacting adaptor molecule (RIAM), Rap1, and kindlin, are critical for integrin activation and might be potential targets for integrin-regulating drugs in treating inflammatory diseases. In this review, we outline integrin activation regulators in neutrophils with a focus on the above critical regulators, as well as newly discovered modulators that are involved in integrin activation.

Galectin 3 enhances platelet aggregation and thrombosis via Dectin-1 activation: a translational study.

AIMS: Galectin-3, a ß-galactoside-binding lectin, is abnormally increased in cardiovascular disease. Plasma Galectin-3 receives a Class II recommendation for heart failure management and has been extensively studied for multiple cellular functions. The direct effects of Galectin-3 on platelet activation remain unclear. This study explores the direct effects of Galectin-3 on platelet activation and thrombosis. METHODS AND RESULTS: A strong positive correlation between plasma Galectin-3 concentration and platelet aggregation or whole blood thrombus formation was observed in patients with coronary artery disease (CAD). Multiple platelet function studies demonstrated that Galectin-3 directly potentiated platelet activation and in vivo thrombosis. Mechanistic studies using the Dectin-1 inhibitor, laminarin, and Dectin-1-/- mice revealed that Galectin-3 bound to and activated Dectin-1, a receptor not previously reported in platelets, to phosphorylate spleen tyrosine kinase and thus increased Ca2+ influx, protein kinase C activation, and reactive oxygen species production to regulate platelet hyperreactivity. TD139, a Galectin-3 inhibitor in a Phase II clinical trial, concentration dependently suppressed Galectin-3-potentiated platelet activation and inhibited occlusive thrombosis without exacerbating haemorrhage in ApoE-/- mice, which spontaneously developed increased plasma Galectin-3 levels. TD139 also suppressed microvascular thrombosis to protect the heart from myocardial ischaemia-reperfusion injury in ApoE-/- mice. CONCLUSION: Galectin-3 is a novel positive regulator of platelet hyperreactivity and thrombus formation in CAD. As TD139 has potent antithrombotic effects without bleeding risk, Galectin-3 inhibitors may have therapeutic advantages as potential antiplatelet drugs for patients with high plasma Galectin-3 levels.

Direct Binding of Rap1 to Talin1 and to MRL Proteins Promotes Integrin Activation in CD4+ T Cells.

Agonist-induced Rap1 GTP loading results in integrin activation involved in T cell trafficking and functions. MRL proteins Rap1-interacting adapter molecule (RIAM) and lamellipodin (LPD) are Rap1 effectors that can recruit talin1 to integrins, resulting in integrin activation. Recent work also implicates direct Rap1-talin1 interaction in integrin activation. Here, we analyze in mice the connections between Rap1 and talin1 that support integrin activation in conventional CD4+ T (Tconv) and CD25HiFoxp3+CD4+ regulatory T (Treg) cells. Talin1(R35E, R118E) mutation that disrupts both Rap1 binding sites results in a partial defect in αLß2, α4ß1, and α4ß7 integrin activation in both Tconv and Treg cells with resulting defects in T cell homing. Talin1(R35E,R118E) Tconv manifested reduced capacity to induce colitis in an adoptive transfer mouse model. Loss of RIAM exacerbates the defects in Treg cell function caused by the talin1(R35E,R118E) mutation, and deleting both MRL proteins in combination with talin1(R35E,R118E) phenocopy the complete lack of integrin activation observed in Rap1a/b-null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable αLß2, α4ß1, and α4ß7 integrin activation in CD4+ T cells.