Dissection of 3D chromosome organization in Streptomyces coelicolor A3(2) leads to biosynthetic gene cluster overexpression.

The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, Streptomyces coelicolor, during the different growth phases. The chromosome undergoes a dramatic global structural change from primary to secondary metabolism, while some biosynthetic gene clusters (BGCs) form special local structures when highly expressed. Strikingly, transcription levels of endogenous genes are found to be highly correlated to the local chromosomal interaction frequency as defined by the value of the frequently interacting regions (FIREs). Following the criterion, an exogenous single reporter gene and even complex BGC can achieve a higher expression after being integrated into the chosen loci, which may represent a unique strategy to activate or enhance the production of natural products based on the local chromosomal 3D organization.

Disrupted Tuzzerella abundance and impaired L-glutamine levels induce Treg accumulation in ovarian endometriosis: a comprehensive multi-omics analysis.

INTRODUCTION: The microbial community plays a crucial role in the pathological microenvironment. However, the structure of the microbial community within endometriotic lesions and its impact on the microenvironment is still limited. METHODS: All 55 tissue samples, including ovarian ectopic (OEMs) and normal (NE) endometrium, were subjected to 16S rRNA sequencing, metabolomic and proteomic analysis. RESULTS: We found the abundance of Tuzzerella is significantly lower in OEMs compared to NE tissue (p < 0.01). We selected samples from these two groups that exhibited the most pronounced difference in Tuzzerella abundance for further metabolomic and proteomic analysis. Our findings indicated that endometriotic lesions were associated with a decrease in L-Glutamine levels. However, proteomic analysis revealed a significant upregulation of proteins related to the complement pathway, including C3, C7, C1S, CLU, and A2M. Subsequent metabolic and protein correlation predictions demonstrated a negative regulation between L-Glutamine and C7. In vitro experiments further confirmed that high concentrations of Glutamine significantly inhibit C7 protein expression. Additionally, immune cell infiltration analysis, multiplex immunofluorescence, and multifactorial testing demonstrated a positive correlation between C7 expression and the infiltration of regulatory T cells (Tregs) in ectopic lesions, while L-Glutamine was found to negatively regulate the expression of chemotactic factors for Tregs. CONCLUSION: In this study, we found a clear multi-omics pathway alteration, "Tuzzerella (microbe)-L-Glutamine (metabolite)-C7 (protein)," which affects the infiltration of Tregs in endometriotic lesions. Our findings provide insights into endometriosis classification and personalized treatment strategies based on microbial structures.

Comparison of microbial abundance and diversity in uterine and peritoneal fluid in infertile patients with or without endometriosis.

INTRODUCTION: Endometriosis (EM) is a multifactorial disease that affects 10 - 15% of women of reproductive age. Additionally, 30-50% of women with EM suffer from infertility. The mechanism of infertility caused by EM has not yet been consistently explained. In recent years, studies have shown a link between infertility associated with EM and changes in the reproductive tract microbiota. METHODS: In this study, we involved 26 EM patients (8 cases of stage I-II and 18 cases of stage III-IV) and 31 control subjects who were tubal obstruction-related infertility (TORI). The samples from peritoneal fluid (PF) and uterine fluid (UF) were collected and sequenced by 16 S rRNA amplicon. RESULTS: In the comparison of microbial diversity, we found no significant differences in the microbial diversity of PF and UF between patients with stage I-II EM and those with TORI. However, there was a significant difference in microbial diversity among patients with stage III-IV EM compared to the previous two groups. Lactobacillus decreased in PF of EM compared to the control group, while it increased in UF. In PF, the abundance of Pseudomonas, Enterococcus, Dubosiella and Klebsiella was significantly higher in patients with stage III-IV compared to TORI patients. And in UF, the main differences existed between stage I-II EM compared to the other two groups. The abundance of pontibacter, aquabacterium, Rikenellaceae and so on at the genus level was significantly enriched in the EM patients with stage I-II. In the analysis based on KEGG database, EM may affect the receptivity related pathways of the endometrium by influencing changes in the uterine microbiota. CONCLUSION: Our results indicated that as EM progresses, the microorganisms in UF and PF keep changing. These changes in the microbiota, as well as the resulting alternations in gene functional classification, may play an important role in the infertility associated with EM.

Teadenol B as a Component of Microorganism-Fermented Tea Extract Inhibited Breast Cancers by Promoting Autophagy.

Breast cancer is a significant threat to life and health, which needs more safe and effective drugs to be explored. Teadenol B is a characteristic chemical component of microbial fermented tea. This study discovered that teadenol B could exhibit obvious inhibitory effects on all four different clinical subtype characteristics of breast cancer cells. Proteomic studies show that deoxycytidine triphosphate deaminase (DCTD), which could block DNA synthesis and repair DNA damage, had the most significant and consistent reduction in all four types of breast cancer cells with the treatment of teadenol B. Considering MDA-MB-231 cells exhibit poor clinical prognosis and displayed substantial statistical differences in KEGG pathway enrichment analysis results, we investigated its impact on the size and growth of MDA-MB-231 triple-negative breast tumors transplanted into nude mice and demonstrated that teadenol B significantly suppressed tumor growth without affecting body weight significantly. Finally, we found that the conversion of LC3-I to LC3-II in MDA-MB-231 increased significantly with teadenol B treatment. This proved that teadenol B could be a strong autophagy promotor, which explained the down-regulation of DCTD to some extent and may be the potential mechanism underlying teadenol B's anti-breast cancer effects. This finding provides new evidence for drinking fermented tea to prevent breast cancer and highlights the potential of teadenol B as a novel therapeutic option for breast cancer prevention and treatment, necessitating further investigations to clarify its exact target and the details involved.

CYSTEINE-RICH RECEPTOR-LIKE KINASE5 (CRK5) and CRK22 regulate the response to Verticillium dahliae toxins.

Cysteine-rich receptor-like kinases (CRKs) play critical roles in responses to biotic and abiotic stresses. However, the molecular mechanisms of CRKs in plant defense responses remain unknown. Here, we demonstrated that two CRKs, CRK5 and CRK22, are involved in regulating defense responses to Verticillium dahliae toxins (Vd-toxins) in Arabidopsis (Arabidopsis thaliana). Biochemical and genetic analyses showed that CRK5 and CRK22 may act upstream of MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 to regulate the salicylic acid (SA)-signaling pathway in response to Vd-toxins. In addition, MPK3 and MPK6 interact with the transcription factor WRKY70 to modulate defense responses to Vd-toxins. WRKY70 directly binds the promoter domains of the SA-signaling-related transcription factor genes TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA2) and TGA6 to regulate their expression in response to Vd-toxins. Thus, our study reveals a mechanism by which CRK5 and CRK22 regulate SA signaling through the MPK3/6-WRKY70-TGA2/6 pathway in response to Vd-toxins.

The bioceramic sealer iRoot SP promotes osteogenic differentiation of human stem cells from apical papilla via miR-141-3p/SPAG9/MAPK signalling pathway.

AIM: The premixed bioceramic sealer iRoot SP that is widely used clinically has been reported to kill bacterial biofilms and promote osteogenic differentiation of human stem cells from the apical papilla (hSCAPs). Although miR-141-3p has been substantiated to be involved in the osteogenic process, the underlying mechanisms remain unclear. The aim of this study was to investigate the role of miR-141-3p in osteogenic differentiation and underlying mechanisms of iRoot SP-treated hSCAPs. METHODOLOGY: hSCAPs were extracted from tissue blocks with enzyme digestion and identified by using immunofluorescence, flow cytometry and alizarin red staining. The mRNA expression level of miR-141-3p in hSCPAs after culture with iRoot SP was examined by quantitative real-time PCR (qRT-PCR) assay. SPAG9 was identified as a downstream target gene of miR-141-3p by dual-luciferase report assay. Alkaline phosphatase (ALP) staining and activity detection, alizarin red staining, calcium concentration assay, qRT-PCR and western blot were used to estimate osteogenic differentiation ability and involved protein expression levels of the osteogenic makers and signalling pathway-related factors in iRoot SP-treated hSCAPs. Data were analysed by one-way anova and post hoc Tukey's test to determine any statistical differences between the experimental groups and the control group. p < .05 was considered statistically significant. RESULTS: Expression of miR-141-3p was reduced in iRoot SP-treated hSCAPs with the increased exposure time up to 7 days, and the western blot and qRT-PCR results revealed that the osteogenic markers osteocalcin (OCN), osterix (OSX), runt-related transcription factor 2 (RUNX2) and dentin sialophosphoprotein (DSPP) were inversely correlated with miR-141-3p. The negative regulatory relationship between miR-141-3p and SPAG9/ mitogen-activated protein kinases (MAPK) signalling axis was validated in this in vitro experiments. CONCLUSIONS: The bioceramic sealer iRoot SP promoted osteogenic differentiation of hSCAPs by inhibiting miR-141-3p following down-regulated SPAG9 expression, and activated MAPK pathway. These findings proposed a novel therapeutic impact of bioceramic sealer iRoot SP inducing bone regeneration in refractory periapical periodontitis.

Differential expression of circular RNAs in interleukin 6-promoted osteogenic differentiation of human stem cells from apical papilla.

INTRODUCTION: Studies have shown that interleukin 6 (IL-6) can regulate stem cell osteogenic differentiation; however, the exact mechanism is not clear. Circular RNAs (circRNAs) are closed circular non-coding RNAs that are involved in the process of stem cell osteogenic differentiation. Therefore, the purpose of this present study was to investigate the effect of IL-6 treatment on osteogenic differentiation of human apical tooth papillae stem cells (hSCAPs), and to detect the difference in circRNA expression using gene microarray technology. METHODS: After extraction and identification of hSCAPs, alkaline phosphatase (ALP) activity, alizarin red staining, and calcium ion quantitative assay were used to determine the changes of ALP enzyme, mineralized nodules, and matrix calcium levels before and after IL-6 treatment of hSCAPs gene microarray technology was used to analyze the changes in circRNA expression levels before and after IL-6 induction of mineralization. The four selected circRNAs were validated by qRT-PCR. Moreover, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to predict the potential functions and biological signaling pathways of circRNAs. Finally, these data are integrated and analyzed to construct circRNA-microRNA-mRNA networks. RESULTS: Alp and Alizarin red staining confirmed that IL-6 promoted the osteogenic differentiation of hSCAPs. The gene microarray results identified 132 differentially expressed circRNAs, of which 117 were upregulated and 15 were downregulated. Bioinformatic analysis predicted that the circRNA-406620/miR-103a-3p/FAT atypical cadherin 4 (FAT4) pathway might be involved in regulating IL-6 to promote osteogenic differentiation of hSCAPs. CONCLUSION: Differentially expressed circRNAs might be closely involved in regulating IL-6 to promote osteogenic differentiation of hSCAPs.

Circular RNA differential expression profiles and bioinformatics analysis of hsa_circRNA_079422 in human endometrial carcinoma.

The aim of our study was to explore circular RNA (circRNA) expression profiles associated with human endometrial carcinoma (EC) and to analyse the molecular mechanisms involved in cancer development and their potential clinical importance. Differential expression profiles were revealed by Arraystar human circRNA microarray analysis. The results of the circRNA microarray were confirmed by quantitative real-time PCR. Interactions between circRNAs and microRNAs (miRNAs) were predicted using Arraystar's miRNA target prediction software. The functions of the circRNA-miRNA coexpression network were identified by KEGG pathway analysis and GO analysis. Compared with para-tumorous tissues, 14 genes were significantly upregulated and 12 genes were significantly downregulated in EC tissues (P < 0.05). The quantitative real-time PCR data demonstrated consistency with the results of the microarray profile analysis. We generated a circRNA-miRNA coexpression network. Hsa_circRNA_079422 expression was significantly lower and miR-136-5p expression was higher in EC tissues than in normal endometrial tissues. KEGG pathway analysis and GO analysis indicated that hsa_circRNA_079422 might play roles in different signalling pathways and biological functions. We confirmed the presence of different circRNA expression profiles and predicted the circRNA-miRNA coexpression network in human EC tissues. Hsa_circRNA_079422 might be involved in the pathogenesis and biological process of EC via interactions with miRNAs.IMPACT STATEMENTWhat is already known on this subject? EC is a common malignancy of the female reproductive system. CircRNAs were demonstrated to exert critical roles in cancers, including EC.What do the results of this study add? The results of this study add circRNAs expression profiles, the circRNA-miRNA coexpression network and cancer-related circRNA-miRNA target genes in EC. It was first found that hsa_circRNA_079422 was downregulated, while miR-136-5p was upregulated in EC tissues.What are the implications of these findings for clinical practice and/or further research? In clinical practice, early EC diagnosis lacks specific biomarkers, so most EC patients are diagnosed at an advanced stage. In the management of EC patients, we also lack personalised adjuvant treatment that combines the clinical pathological characteristics. For the existing literature, we identified a new EC differential expression biomarker, hsa_circ_079422. It can be used to verify the correlation with EC clinical severity or poor prognosis. Its targeting can also be used to stratify EC patients with different molecular types, including to guide adjuvant therapy. In addition, we can verify and analyse regulatory pathways associated with it for the design of regulating engineering circRNA.

Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly.

Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

Moisture-resistant MXene-sodium alginate sponges with sustained superhydrophobicity for monitoring human activities.

Wearable mechanical sensors are easily influenced by moisture resulting in inaccuracy for monitoring human health and body motions. Though the superhydrophobic barrier has been extensively explored as passive water repel strategy on the sensor surface, the dense superhydrophobic surface not only limits the sensor working under large deformations but also inevitable degradation in high humidity or saturation water vapor environments. This work reports a superhydrophobic MXene-sodium alginate sponge (SMSS) pressure sensor with a low voltage Joule heating effect to provide sustain moisture-insensitive property for both sensing performance and superhydrophobicity by heating-driven water molecules away. Because of the positive temperature coefficient under pressure applied, the Joule heating can provides a stable temperature to the moisture-insensitivity property during the whole dynamic pressure cycled. Therefore, the pressure sensor with a simple spray-coating superhydrophobic coating on the outer layer demonstrates key capabilities even in extreme use scenarios with high humidity or water vapor and also provides stable and reliable bio-signal monitoring.

Synthesis of tryptophan-dehydrobutyrine diketopiperazine and biological activity of hangtaimycin and its co-metabolites.

An improved synthesis for tryptophan-dehydrobutyrine diketopiperazine (TDD), a co-metabolite of the hybrid polyketide/non-ribosomal peptide hangtaimycin, starting from ʟ-tryptophan is presented. Comparison to TDD isolated from the hangtaimycin producer Streptomyces spectabilis confirmed its S configuration. The X-ray structure of the racemate shows an interesting dimerisation through hydrogen bridges. The results from bioactivity testings of hangtaimycin, TDD and the hangtaimycin degradation product HTM222 are given.

H2Bub1 Regulates RbohD-Dependent Hydrogen Peroxide Signal Pathway in the Defense Responses to Verticillium dahliae Toxins.

Histone H2B monoubiquitination (H2Bub1) plays critical roles in regulating growth and development as well as stress responses in Arabidopsis (Arabidopsis thaliana). In this study, we used wild-type and HUB1 and HUB2 loss-of-function Arabidopsis plants to elucidate the mechanisms involved in the regulation of the plant's defense responses to Verticillium dahliae toxins (Vd-toxins). We demonstrated that HUB-mediated H2Bub1 regulates the expression of the NADPH oxidase RbohD by enhancing the enrichment of histone H3 trimethylated on Lys-4 in response to Vd-toxins. RbohD-dependent hydrogen peroxide (H2O2) signaling is a critical modulator in the defense response against Vd-toxins. Moreover, H2Bub1 also affects posttranscriptional mitogen-activated protein kinase (or MPK) signaling. H2Bub1 was required for the activation of MPK3 and MPK6. MPK3 and MPK6 are involved in regulating RbohD-mediated H2O2 production. MPK3 and MPK6 are associated with protein tyrosine phosphatases (PTPs), such as Tyr-specific phosphatase1 and mitogen-activated protein kinases phosphatase1, which negatively regulated H2O2 production. In addition, H2Bub1 is involved in regulating the expression of WRKY33 WRKY33 directly binds to RbohD promoter and functions as a transcription factor to regulate the expression of RbohD Collectively, our results indicate that H2Bub1 regulates the NADPH oxidase RbohD-dependent H2O2 production and that the PTP-MPK3/6-WRKY pathway plays an important role in the regulation of RbohD-dependent H2O2 signaling in defense responses to Vd-toxins in Arabidopsis.

Methyltransferases of gentamicin biosynthesis.

Gentamicin C complex from Micromonospora echinospora remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific gene deletion and chemical complementation, that the gentamicin pathway up to the branch point is defined by the selectivity of the methyltransferases GenN, GenD1, and GenK. Unexpectedly, they comprise a methylation network in which early intermediates are ectopically modified. Using whole-genome sequence, we have also discovered the terminal 6'-N-methyltransfer required to produce gentamicin C2b from C1a or gentamicin C1 from C2, an example of an essential biosynthetic enzyme being located not in the biosynthetic gene cluster but far removed on the chromosome. These findings fully account for the methylation pattern in gentamicins and open the way to production of individual gentamicins by fermentation, as starting materials for semisynthesis.

Production of 3-desmethyl protostreptovaricin I from the genetically engineered Streptomyces spectabilis CCTCC M2017417.

A new streptovaricin analogue, namely 3-desmethyl protostreptovaricin I (1), was isolated from the culture of the genetically engineered strain ΔstvM2 derived from Streptomyces spectabilis CCTCC M2017417. Its structure was elucidated on the basis of extensive spectroscopic analyses, including 1D and 2D NMR tests, and high resolution mass spectrometry analysis. Compound 1 is the first example of 3-desmethyl streptovaricin analogues reported so far, however, it showed no antibacterial activities against Staphylococcus aureus ATCC 29213.

The Mechanism of Dehydrating Bimodules in trans-Acyltransferase Polyketide Biosynthesis: A Showcase Study on Hepatoprotective Hangtaimycin.

A bioassay-guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthesis. The biosynthetic gene cluster was identified and resembles a hybrid trans-AT PKS/NRPS biosynthetic machinery whose first PKS enzyme contains an internal dehydrating bimodule, which is usually found split in other trans-AT PKSs. The mechanisms of such dehydrating bimodules have often been proposed, but have never been deeply investigated. Here we present in vivo mutations and in vitro enzymatic experiments that give first and detailed mechanistic insights into catalysis by dehydrating bimodules.

NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells.

Little is known about the molecules mediating the cross-talk between post-traumatic axons and scar-forming cells after spinal cord injury. We found that a sustained NB-3 induction was simultaneously present in the terminations of post-traumatic corticospinal axons and scar-forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced in vivo with NB-3 deficiency or interruption of NB-3 trans-homophilic interactions. Biochemical, in vitro and in vivo evidence demonstrated that NB-3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar-forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB-3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB-3 trans-homophilic interactions mediate the cross-talk between post-traumatic axons and scar-forming cells and impair the intrinsic growth ability of injured axons.

E2 conjugases UBC1 and UBC2 regulate MYB42-mediated SOS pathway in response to salt stress in Arabidopsis.

Histone H2B monoubiquitination (H2Bub1) is recognized as a crucial eukaryotic regulatory mechanism that controls a range of cellular processes during both development and adaptation to environmental changes. In Arabidopsis, the E2 conjugated enzymes UBIQUITIN CARRIER PROTEINs (UBCs) -1 and -2 mediate ubiquitination of H2B. Here, we elucidated the functions of UBC1 and -2 in salt-stress responses and revealed their downstream target genes. Real-time quantitative PCR assays showed that UBC1 and -2 positively regulated the salt-induced expression of MYB42 and Mitogen-Activated Protein Kinase 4 (MPK4). Chromatin immunoprecipitation assays revealed that H2Bub1 was enriched weakly on the chromatin of MYB42 and MPK4 in the ubc1,2 mutant. We further determined that UBC1/2-mediated H2Bub1 enhanced the level of histone H3 tri-methylated on K4 (H3K4me3) in the chromatin of MYB42 and MPK4 under salt-stress conditions. MPK4 interacted with and phosphorylated MYB42. The MPK4-mediated MYB42 phosphorylation enhanced the MYB42 protein stability and transcriptional activity under salt-stress conditions. We further showed that MYB42 directly bound to the SALT OVERLY SENSITIVE 2 (SOS2) promoter and mediated the rapid induction of its expression after a salt treatment. Our results indicate that UBC1 and -2 positively regulate salt-stress responses by modulating MYB42-mediated SOS2 expression.

Ansavaricin J, a New Heterocyclic Ring-Fused Streptovaricin from Gene stvP5-Deleted Mutant of Streptomyces spectabilis CCTCC M2017417.

A new ring-fused streptovaricin analogue, named ansavaricin J, was unprecedently isolated from the culture of the genetically modified strains ΔstvP5 which derived from Streptomyces spectabilis CCTCC M2017417. Its structure was elucidated via comprehensive spectroscopic analyses, including 1D- and 2D-NMR tests, and HR-ESI-MS data analysis. Notably, ansavaricin J and E represent the only two reported examples of heterocyclic ring-fused streptovaricins thus far, however, it only showed insignificant antibacterial activities against Staphylococcus aureus.

Cross-Module Enoylreduction in the Azalomycin†F Polyketide Synthase.

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis.

Luminescence-Resonance-Energy-Transfer-Based Luminescence Nanoprobe for In Situ Imaging of CD36 Activation and CD36-oxLDL Binding in Atherogenesis.

Macrophage foam cell formation mediated by CD36 receptor dependent internalization of oxidized low-density lipoprotein (oxLDL) is an important hallmark of early atherosclerosis. Activation of CD36 and its binding to oxLDL are the key points in foam cell formation. Herein, we develop a site-specific luminescence resonance energy transfer (LRET) system for the simultaneous imaging of CD36 activity and CD36-oxLDL binding on the cell surface. The system utilizes CD36-antibody-modified, SiO2-coated upconversion luminescent nanoparticles (UCNPs) as an energy donor to target the plasma membranes of macrophages, and DiI-oxLDL (energy acceptor) binds to CD36 and passes through the membrane during macrophage foam cell formation. Upon excitation at 980 nm, the LRET signal can be obtained because of the short distance between DiI-oxLDL and the nanoprobe. Additionally, the very specific fluorescence can be used to visualize distinct features of CD36. The nanoprobe also exhibits high sensitivity, good stability, simplicity, and low cost for the accurate detection and evaluation of macrophage foam cell formation. Moreover, using this novel nanoprobe, we also investigate the mechanism by which reactive oxygen species (ROS) signaling enhances the binding of oxLDL to CD36. ROS, especially O2·-, alter endothelial permeability and facilitate CD36 clustering, ultimately promoting the entry and internalization of oxLDL. Because of these advantages, this nanoprobe may provide a versatile platform for monitoring the progression of atherogenesis and elucidating atherogenesis signaling at the cellular level.