DNA nanomachines reveal an adaptive energy mode in confinement-induced amoeboid migration powered by polarized mitochondrial distribution.

Energy metabolism is highly interdependent with adaptive cell migration in vivo. Mechanical confinement is a critical physical cue that induces switchable migration modes of the mesenchymal-to-amoeboid transition (MAT). However, the energy states in distinct migration modes, especially amoeboid-like stable bleb (A2) movement, remain unclear. In this report, we developed multivalent DNA framework-based nanomachines to explore strategical mitochondrial trafficking and differential ATP levels during cell migration in mechanically heterogeneous microenvironments. Through single-particle tracking and metabolomic analysis, we revealed that fast A2-moving cells driven by biomimetic confinement recruited back-end positioning of mitochondria for powering highly polarized cytoskeletal networks, preferentially adopting an energy-saving mode compared with a mesenchymal mode of cell migration. We present a versatile DNA nanotool for cellular energy exploration and highlight that adaptive energy strategies coordinately support switchable migration modes for facilitating efficient metastatic escape, offering a unique perspective for therapeutic interventions in cancer metastasis.

Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

Single Multifunctional Nanocabinets-Based Target-Activated Feedback for Simultaneously Precise Monitoring and Therapy.

Stimulus-responsive mode is highly desirable for improving the precise monitoring and physiological efficacy of endogenous biomarkers (EB). However, its integrated application for visual detection and therapy is limited by inappropriate use of responsive triggers and poor delivery of EB signal-transducing agents, which remain challenging in simultaneous monitoring and noninvasive therapy of EB and EB-mediated pathological events. Target microRNA (miRNA) as controllable reaction triggers and DNAzyme as signal-transducing agent are proposed to develop target-stimulated multifunctional nanocabinets (MFNCs) for the visual tracking of both miRNA and miRNA-mediated anticancer events. The MFNCs, equipped with a target-discriminating sequence-incorporated DNAzyme motif, can specifically release therapeutic molecules through target-triggered conformational switches, accompanied by transduction signal output. Target detection and molecule release performance are recorded in parallel via reverse dual-signal feedback at the single-molecule level. In addition, the intrinsic thermal-replenishing of the MFNCs leads to tumor ablation without invasive exogenous aids. The system achieves visual target quantification, anticancer molecule real-time tracking, and tumor suppression in vivo and in vitro. This work proposes a new paradigm for precise visual exploration of EB or EB-mediated bio-events and provides a demonstration of efficacious all-in-one detection and therapy based on the target-triggered multifunctional nanosystem.

Trends in confinement-induced cell migration and multi-omics analysis.

Cell migration is an essential manner of different cell lines that are involved in embryological development, immune responses, tumorigenesis, and metastasis in vivo. Physical confinement derived from crowded tissue microenvironments has pivotal effects on migratory behaviors. Distinct migration modes under a heterogeneous extracellular matrix (ECM) have been extensively studied, uncovering potential molecular mechanisms involving a series of biological processes. Significantly, multi-omics strategies have been launched to provide multi-angle views of complex biological phenomena, facilitating comprehensive insights into molecular regulatory networks during cell migration. In this review, we describe biomimetic devices developed to explore the migratory behaviors of cells induced by different types of confined microenvironments in vitro. We also discuss the results of multi-omics analysis of intrinsic molecular alterations and critical pathway dysregulations of cell migration under heterogeneous microenvironments, highlighting the significance of physical confinement-triggered intracellular signal transduction in order to regulate cellular behaviors. Finally, we discuss both the challenges and promise of mechanistic analysis in confinement-induced cell migration, promoting the development of early diagnosis and precision therapeutics.

Engineering of Multivalent Membrane-Anchored DNA Frameworks for Precise Profiling of Variable Membrane Permeability During Reversible Electroporation.

Electroporation techniques have emerged as attractive tools for intracellular delivery, rendering promising prospects towards clinical therapies. Transient disruption of membrane permeability is the critical process for efficient electroporation-based cargo delivery. However, smart nanotools for precise characterization of transient membrane changes induced by strong electric pulses are extremely limited. Herein, multivalent membrane-anchored fluorescent nanoprobes (MMFNPs) that take advantages of flexible functionalization and spatial arrangement of DNA frameworks are developed for in situ evaluation of electric field-induced membrane permeability during reversible electroporation . Single-molecule fluorescence imaging techniques are adopted to precisely  verify the excellent analytical performance of the engineered MMFNPs. Benefited from tight membrane anchoring and sensitive adenosine triphosphate (ATP) profiling, varying degrees of membrane disturbances are visually exhibited under different intensities of the microsecond pulse electric field (µsPEF). Significantly, the dynamic process of membrane repair during reversible electroporation is well demonstrated via ATP fluctuations monitored by the designed MMFNPs. Furthermore, molecular dynamics (MD) simulations are performed for accurate verification of electroporation-driven dynamic cargo entry via membrane nanopores. This work provides an avenue for effectively capturing transient fluctuations of membrane permeability under external stimuli, offering valuable guidance for developing efficient and safe electroporation-driven delivery strategies for clinical diagnosis and therapeutics.

Transformable DNA Nanorobots Reversibly Regulating Cell Membrane Receptors for Modulation of Cellular Migrations.

Oligomerization of cellular membrane receptors plays crucial roles in activating intracellular downstream signaling cascades for controlling cellular behaviors in physiological and pathological processes. However, the reversible and controllable regulation of receptors in a user-defined manner remains challenging. Herein, we developed a versatile DNA nanorobot (nR) with installed aptamers and hairpin structures to reversibly and controllably regulate cell migration. This was achieved by dimerization and de-dimerization of mesenchymal-epithelial transition (Met) receptors through DNA strand displacement reactions. The functionalized DNA nR not only plays similar roles as hepatocyte growth factor (HGF) in inducing cell migration but also allows a downgrade to the original state of cell migration. The advanced DNA nanomachines can be flexibly designed to target other receptors for manipulating cellular behaviors and thus represent a powerful tool for the future of biological and medical engineering.

Nanoarray Enabled Size-Dependent Isolation and Proteomics Profiling of Small Extracellular Vesicle Subpopulations toward Accurate Cancer Diagnosis and Prognosis.

Small extracellular vesicles (sEVs) have emerged as noninvasive biomarkers in liquid biopsy due to their significant function in pathology and physiology. However, the phenotypic heterogeneity of sEVs presents a significant challenge to their study and has significant implications for their applications in liquid biopsies. In this study, anodic aluminum oxide films with different pore sizes (AAO nanoarray) were introduced to enable size-based isolation and downstream proteomics profiling of sEV subpopulations. The adjustable pore size and abundant Al3+ on the framework of AAOs allowed size-dependent isolation of sEV subpopulations through nanoconfined effects and Lewis acid-base interaction between AAOs and sEVs. Benefiting from the strong concerted effect, the simple AAO nanoarray enabled specific isolation of three sEV subpopulations, termed "50", "90", and "150 nm" groups, from 10 µL of complex biological samples within 10 min with high capture efficiencies and purities. Moreover, the nanopores of AAOs also acted as nanoreactors for comprehensive proteomic profiling of the captured sEV subpopulations to reveal their heterogeneity. The AAO nanoarray was first investigated on sEVs from a cell culture medium, where sEV subpopulations could be clearly distinguished, and three traditional sEV-specific proteins (CD81, CD9, and FLOT1) could be identified by proteomic analysis. A total of 3946, 3951, and 3940 proteins were identified from 50, 90, and 150 nm sEV subpopulations, respectively, which is almost twice the number compared to those obtained from the conventional approach. The concept was further applied to complex real-case sample analysis from prostate cancer patients. Machine learning and gene ontology (GO) information analysis of the identified proteins indicate that different-sized sEV subpopulations contain unique protein cargos and have distinct cellular components and molecular functions. Further receiver operating characteristic curve (ROC) analysis of the top five differential proteins from the three sEV subpopulations demonstrated the high accuracy of the proposed approach toward prostate cancer diagnosis (AUC > 0.99). More importantly, several proteins involved in focal adhesion and antigen processing and presentation pathways were found to be upregulated in prostate cancer patients, which may serve as potential biomarkers of prostate cancer. These results suggest that the sEV subpopulation-based AAO nanoarray is of great value in facilitating the early diagnosis and prognosis of cancer and opens a new avenue for sEVs in liquid biopsy.

Natural products modulate NLRP3 in ulcerative colitis.

Ulcerative colitis (UC) is a clinically common, progressive, devastating, chronic inflammatory disease of the intestine that is recurrent and difficult to treat. Nod-like receptor protein 3 (NLRP3) is a protein complex composed of multiple proteins whose formation activates cysteine aspartate protease-1 (caspase-1) to induce the maturation and secretion of inflammatory mediators such as interleukin (IL)-1ß and IL-18, promoting the development of inflammatory responses. Recent studies have shown that NLRP3 is associated with UC susceptibility, and that it maintains a stable intestinal environment by responding to a wide range of pathogenic microorganisms. The mainstay of treatment for UC is to control inflammation and relieve symptoms. Despite a certain curative effect, there are problems such as easy recurrence after drug withdrawal and many side effects associated with long-term medication. NLRP3 serves as a core link in the inflammatory response. If the relationship between NLRP3 and gut microbes and inflammation-associated factors can be analyzed concerning its related inflammatory signaling pathways, its expression status as well as specific mechanism in the course of IBD can be elucidated and further considered for clinical diagnosis and treatment of IBD, it is expected that the development of lead compounds targeting the NLRP3 inflammasome can be developed for the treatment of IBD. Research into the prevention and treatment of UC, which has become a hotbed of research in recent years, has shown that natural products are rich in therapeutic means, and multi-targets, with fewer adverse effects. Natural products have shown promise in treating UC in numerous basic and clinical trials over the past few years. This paper describes the regulatory role of the NLRP3 inflammasome in UC and the mechanism of recent natural products targeting NLRP3 against UC, which provides a reference for the clinical treatment of this disease.

Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

Lamin A/C and Vimentin as a Coordinated Regulator during Amoeboid Migration in Microscale Confined Microenvironments.

Cell migration occurs in confined microenvironments, which plays a vital role in the process of tumor metastasis. However, it is challenging to study their behaviors in vivo. Here we developed a cell squeeze system that can be scaled down to micrometers to mimic native physical confined microenvironments, wherein degrees of surface adhesion and mechanical constraints could be manipulated in order to investigate cell-migrating behaviors. Based on the microscale cell squeeze system, we found the synergistic role of lamin A/C and vimentin in cell transition and migration under strong confinement. The dynamic variations in lamin A/C and vimentin expression establish a positive feedback loop in response to confinement, effectively promoting amoeboid migration by modulating nuclear deformability while ensuring cell viability. This work shed light on modulating cell response to microenvironments by altering the expression of lamin A/C and/or vimentin, which may be a more efficient way of inhibiting cancer metastasis.

Host-Guest Self-Assembled Interfacial Nanoarrays for Precise Metabolic Profiling.

Accurate and rapid metabolic profiling of cerebrospinal fluid (CSF) is urgently needed but remains challenging for clinical diagnosis of central nervous system diseases and biomarker discovery. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) holds promise for metabolic analysis. Its low signal reproducibility, however, severely restricts acquisition of quantitative MS data in clinical practice. Herein, a multifunctional self-assembled AuNPs array (MSANA)-based LDI-MS platform for direct amino acids analysis and metabolic profiling in patient CSF samples is developed. MSANA featuring a highly ordered and closely packed two-dimensional nanostructure permits capture and direct analysis of aromatic amino acids by LDI-MS with high selectivity and micromolar sensitivity. Meanwhile, the MSANA-based LDI-MS platform exhibits excellent reproducibility (RSD < 10%), largely outperforming the direct matrix spotting approach widely used now (RSD < 44%). The platform is successfully used in metabolic profiling of CSF (1 µL) within minutes for discrimination of medulloblastoma patients from non-tumor controls. Taken together, the MSANA-based LDI-MS platform shows potential clinical values toward large-scale metabolic diagnostics and pathogenic mechanism study.

Activity-Based Self-Enriched SERS Sensor for Blood Metabolite Monitoring.

The monitoring of metabolites in biofluids provides critical clues for disease diagnosis and evaluation. Yet, the quantitative detection of metabolites remains challenging for surface-enhanced Raman spectroscopy (SERS) due to poor reproducibility in preparation and manipulation of SERS nanoprobes. Herein, we develop an activity-based, slippery liquid-infused porous surface SERS (abSLIPSERS) sensor for facile quantification of metabolites with unmodified naked metal nanoparticles (NPs) by integrating biocatalysis-boronate oxidation cascades with SLIPS-driven self-concentration and delivering. Upon mixing the target metabolite with a specific oxidase, a H2O2-sensitive phenylboronate probe, and the naked Au NPs, H2O2 produced from the biocatalytic reaction oxidizes the phenylboronate probe to phenol, resulting in a ratiometric SERS response. Meanwhile, the SLIPS enables the complete enrichment of molecules and NPs within an evaporating liquid droplet, delivering the probes to the SERS-active sites for Raman amplification. Compared with conventional SERS biosensors, abSLIPSERS avoids multistep synthesis and biofunctionalization of nanoprobes, which significantly simplifies the detection workflow and improves the reproducibility. The abSLIPSERS sensor also shows tunable dynamic range beyond 4 orders of magnitude and allows quantifying any other metabolites with specific enzymes. We demonstrate abSLIPSERS sensing of lactate, glucose, and choline in human serum for exploring energy metabolism in lung cancer. This study opens up a new opportunity for future point-of-care testing of circulating metabolites by SERS and will help to facilitate the translation of SERS bioanalysis to clinical settings.

Spatially resolved single-molecule profiling of microRNAs in migrating cells driven by microconfinement.

Cancer cells utilize a range of migration modes to navigate through a confined tissue microenvironment in vivo, while regulatory roles of key microRNAs (miRNAs) remain unclear. Precisely engineered microconfinement and the high spatial-resolution imaging strategy offer a promising avenue for deciphering the molecular mechanisms that drive cell migration. Here, enzyme-free signal-amplification nanoprobes as an effective tool are developed for three-dimensional (3D) high-resolution profiling of key miRNA molecules in single migrating cells, where distinct migration modes are precisely driven by microconfinement-engineered microchips. The constructed nanoprobes exhibit intuitive and ultrasensitive miRNA characterization in vitro by virtue of a single-molecule imaging microscope, and the differential expression and intracellular locations in different cell lines are successfully monitored. Furthermore, 3D spatial distribution of miR-141 at high resolution in flexible phenotypes of migrating cells is reconstructed in the engineered biomimetic microenvironment. The results indicate that miR-141 may be involved in the metastatic transition from a slow to a fast migration state. This work offers a new opportunity for investigating regulatory mechanisms of intracellular key biomolecules during cell migration in biomimetic microenvironments, which may advance in-depth understanding of cancer metastasis in vivo.

Integrated microfluidic-SERS for exosome biomarker profiling and osteosarcoma diagnosis.

Osteosarcoma is one of the most frequent primary sarcoma of bone among adolescents. Early diagnosis of osteosarcoma is the key factor to achieve high survival rate of patients. Nevertheless, traditional histological biopsy is highly invasive and associated with the risk of arousing tumor spread. Herein, we develop a method integrating microfluidics and surface-enhanced Raman spectroscopy (SERS) to isolate plasma-derived exosomes and profile multiple exosomal biomarkers for the diagnosis of osteosarcoma. The method showed highly efficient isolation of exosomes directly from human plasma and can profile exosomes based on protein biomarkers, with the detection limit down to 2 exosomes per µL. The whole assay can be performed in 5 h and only consumed 50 µL of plasma for one analysis. With the method, we analyzed the level of three protein biomarkers, i.e., CD63, vimentin (VIM) and epithelial cell adhesion molecule (EpCAM), on plasma-derived exosomes from 20 osteosarcoma patients and 20 heathy controls. Significantly higher levels of CD63, VIM and EpCAM were observed on plasma exosomes from the osteosarcoma patients compared to the healthy controls. Based on the level of the exosomal biomarkers, a classification model was built for the rapid diagnosis of osteosarcoma, with the sensitivity, specificity and accuracy of 100%, 90% and 95%, respectively. The proposed method does not require complex operations nor expensive equipment, and has great promise in clinical diagnosis of cancer as a liquid biopsy technique.

Nanomaterials assisted exosomes isolation and analysis towards liquid biopsy.

Exosomes has attracted tremendous research interests as they are emerging as a new paradigm of liquid biopsy. Although the concentration of exosomes in blood is relatively abundant, there still exists various vesicle-like nanoparticles, such as microvesicles, apoptotic bodies. It's an urgent need to isolate and enrich exosomes from the complex contaminants in biofluid samples. Moreover, the expressing level of exosomal biomarkers varies a lot, which make the sensitive molecular detection of exosomes in high demand. Unfortunately, the efficient isolation and sensitive molecular quantification of exosomes is still a major obstacle hindering the further development and clinical application of exosome-based liquid biopsy. Nanomaterials, with unique physiochemical properties, have been widely used in biosensing and analysis aspects, thus they are thought as powerful tools for effective purification and molecular analysis of exosomes. In this review, we summarized the most recent progresses in nanomaterials assisted exosome isolation and analysis towards liquid biopsy. On the one hand, nanomaterials can be used as capture substrates to afford large binding area and specific affinity to exosomes. Meanwhile, nanomaterials can also be served as promising signal transducers and amplifiers for molecular detection of exosomes. Furthermore, we also pointed out several potential and promising research directions in nanomaterials assisted exosome analysis. It's envisioned that this review will give the audience a complete outline of nanomaterials in exosome study, and further promote the intersection of nanotechnology and bio-analysis.

Nonenzymatic Multiamplified Electrochemical Detection of Medulloblastoma-Relevant MicroRNAs from Cerebrospinal Fluid.

The sensitive analysis of microRNAs (miRNAs) in cerebrospinal fluid (CSF) holds promise for the minimally invasive early diagnosis of brain cancers such as pediatric medulloblastoma but remains challenging due partially to a lack of facile yet sensitive sensing methods. Herein, an enzyme-free triple-signal amplification electrochemical assay for miRNA was developed by integrating the target-triggered cyclic strand-displacement reaction (TCSDR), hybridization chain reaction (HCR), and methylene blue (MB) intercalation. In this assay, the presence of target miRNA (miR-9) initiated the TCSDR and produced primers that triggered the subsequent HCR amplification to generate copious double-stranded DNAs (dsDNAs) on the electrode surface. Intercalation of a large number of MB reporters into the long nicked double helixes of dsDNAs yielded a more enhanced signal of differential pulse voltammetry. The enzyme-free multiple-amplification approach allowed for highly sensitive (detection limit: 6.5 fM) and sequence-specific (single-base mismatch resolution) detection of miR-9 from tumor cells and human CSF with minimal sample consumption (10 µL). Moreover, the clinical utilization of this method was documented by accurate discrimination of five medulloblastoma patients from the nontumoral controls. In light of its sensitivity, specificity, and convenience of use, this electrochemical method was expected to facilitate the early detection of malignant brain tumors.

All-in-One Strategy for Downstream Molecular Profiling of Tumor-Derived Exosomes.

In light of the significance of exosomes in cancer diagnosis and treatment, it is important to understand the components and functions of exosomes. Herein, an all-in-one strategy has been proposed for comprehensive characterization of exosomal proteins based on nanoporous TiO2 clusters acting as both an extractor for exosome isolation and a nanoreactor for downstream molecular profiling. With the improved hydrophilicity and inherent properties of TiO2, exosomes can be captured by a versatile nanodevice through the specific binding and hydrophilicity interaction synergistically. The strong concerted effect between exosomes and nanodevices ensured high efficiency and specificity of exosome isolation with high recovery and low contaminations. Meanwhile, highly efficient downstream proteomic analysis of the purified exosomes was also enabled by the nanoporous TiO2 clusters. Benefiting from the porous structure of the nanodevice, the lysed exosomal proteins are highly concentrated in the nanopore to achieve high-efficiency in situ proteolytic digestion. Therefore, the unique features of the TiO2 clusters ensured that all the complex steps about isolation and analysis of exosomes were completed efficiently in one simple nanodevice. The concept was first proved with exosomes from cell culture medium, where a high number of identified total proteins and protein groups in exosomes were obtained. Taking advantage of these attractive merits, the first example of the integrated platform has been successfully applied to the analysis of exosomes in complex real-case samples. Not only 196 differential protein biomarker candidates were discovered, but also many more significant cellular components and functions related to gastric cancer were found. These results suggest that the nanoporous TiO2 cluster-based all-in-one strategy can serve as a simple, cost-effective, and integrated platform to facilitate comprehensive analysis of exosomes. Such an approach will provide a valuable tool for the study of exosome markers and their functions.

Eimeria falciformis secretes extracellular vesicles to modulate proinflammatory response during interaction with mouse intestinal epithelial cells.

BACKGROUND: Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs). METHODS: Eimeria falciformis sporozoites were allowed to interact with inactivated MIECs. Parasite secretions were separated into EV and vesicle-free (VF) fractions by discontinuous centrifugation and ultracentrifugation. Secreted EVs were purified in an iodixanol density gradient medium and the protein composition of both EV and VF fractions were analyzed by liquid chromatoraphy-tandem mass spectroscopy. The inflammatory activities of E. falciformis sporozoite EV on MIECs were then investigated. RESULTS: During the interaction of E. falciformis sporozoites with inactivated MIECs, the parasite secreted VF and vesicle-bound molecules. Eimeria falciformis vesicles are typical pathogenic protozoan EVs with a mean diameter of 264 ± 2 nm, and enclosed heat shock protein (Hsp) 70 as classical EV marker. Refractile body-associated aspartyl proteinase (or eimepsin), GAP45 and aminopeptidase were the main components of E. falciformis sporozoite EVs, while VF proteins include Hsp90, actin, Vps54 and kinases, among others. Proteomic data revealed that E. falciformis EV and VF proteins are aggregates of bioactive, antigenic and immunogenic molecules which act in concert for E. falciformis sporozoite motility, pathogenesis and survival. Moreover, in MIECs, E. falciformis EVs induced upregulation of gene expression and secretion of IL-1ß, IL-6, IL-17, IL-18, MCP1 as well as pyroptosis-dependent caspase 11 and NLRP6 inflammasomes with the concomitant secretion of lactate dehydrogenase. CONCLUSIONS: Eimeria falciformis sporozoite interaction with MIECs triggered the secretion of immunogenic and antigenic proteins. In addition, E. falciformis sporozoite EVs constitute parasite-associated molecular pattern that induced inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs.

In situ ratiometric SERS imaging of intracellular protease activity for subtype discrimination of human breast cancer.

Accurate discrimination between different cells at the molecular level is of fundamental importance for disease diagnosis. Endogenous proteases are such molecular candidates for cancer cell subtype study. But in situ probing their activity in live cells remains challenging for surface-enhanced Raman scattering (SERS). Here, we present a sensitive ratio-type SERS nanoprobe for imaging of matrix metalloproteinase-2 (MMP-2) in different cancer cells subtypes. The nanoprobe contained three components: a plasmon-active gold nanoparticle as the SERS enhancing matrix, Raman dye rhodamine B (Rh B)-labelled substrate peptides as the specific MMP-2 recognizer, and 2-naphthalenethiol (2-NT) as the internal standard. MMP-2-responsive cleavage of peptides from the nanoprobe surface results in decrease or even disappearance of SERS emission of Rh B, which was ratioed over the emission of 2-NT for the quantification of MMP-2 activity. Both in-tube assay and in-cell imaging results show that the MMP-responsive nanoprobe can work and serve to differentiate the normal breast cells from the tumorous ones, to differentiate two breast cancer cell subtypes with a different degree of malignancy. We believe that this SERS nanoprobe could find a wide application in the fields of tumor biology and accurate disease diagnosis.

In Situ Single-Molecule Imaging of MicroRNAs in Switchable Migrating Cells under Biomimetic Confinement.

Spatial imaging of RNAs in single cells is extremely charming for deciphering of regulatory mechanisms in multiple migration modes during tumor metastasis. Herein, enzyme-free-mediated cascade amplified nanoprobes were designed for in situ single-molecule imaging of dual-microRNAs (miRNAs) in switchable migrating cells. Differential expression and localization of dual-miRNAs were clearly exhibited in multiple cell lines attributed to enhanced sensitivity via the cascade signal amplification strategy. Significantly, in situ three-dimensional (3D) imaging of dual-miRNAs in transition of cell migration phenotypes was successfully reconstructed in both non-confined and confined microenvironments in vitro, of which differential spatial distribution was observed in a single cell. This is very promising for exploring key roles of spatial RNA distribution in migrating cells at the single-molecule level, which will advance revealing the molecular mechanism and physical principle in 3D cell migration in vivo.