Recent advances in enzyme-free and enzyme-mediated single-nucleotide variation assay in vitro.

Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro, particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles.

Accelerated Activity-Based Sensing by Fluorogenic Reporter Engineering Enables to Rapidly Determine Unstable Analyte.

Accurate detection of labile analytes through activity based fluorogenic sensing is meaningful but remains a challenge because of nonrapid reaction kinetic. Herein, we present a signaling reporter engineering strategy to accelerate azoreduction reaction by positively charged fluorophore promoted unstable anion recognition for rapidly sensing sodium dithionite (Na2S2O4), a kind of widespread used but harmful inorganic reducing agent. Its quick decomposition often impedes application reliability of traditional fluorogenic probes in real samples because of their slow responses. In this work, four azo-based probes with different charged fluorophores (positive, zwitterionic, neutral, and negative) were synthesized and compared. Among of them, with sequestration effect of positively charged anthocyanin fluorophore for dithionite anion via electrostatic attraction, the cationic probe Azo-Pos displayed ultrafast fluorogenic response (∼2 s) with the fastest response kinetic (kpos' = 0.373 s-1) that is better than other charged ones (kzwi' = 0.031 s-1, kneu' = 0.013 s-1, kneg' = 0.003 s-1). Azo-Pos was demonstrated to be capable to directly detect labile Na2S2O4 in food samples and visualize the presence of Na2S2O4 in living systems in a timely fashion. This new probe has potential as a robust tool to fluorescently monitor excessive food additives and biological invasion of harmful Na2S2O4. Moreover, our proposed accelerating strategy would be versatile to develop more activity-based sensing probes for quickly detecting other unstable analytes of interest.

A novel prognostic signature based on cancer stemness and metabolism-related genes for cervical squamous cell carcinoma and endocervical adenocarcinoma.

BACKGROUND: CESC is the second most commonly diagnosed gynecological malignancy. Given the pivotal involvement of metabolism-related genes (MRGs) in the etiology of multiple tumors, our investigation aims to devise a prognostic risk signature rooted in cancer stemness and metabolism. METHODS: The stemness index based on mRNA expression (mRNAsi) of samples from the TCGA dataset was computed using the One-class logistic regression (OCLR) algorithm. Furthermore, potential metabolism-related genes related to mRNAsi were identified through weighted gene co-expression network analysis (WGCNA). We construct a stemness-related metabolic gene signature through shrinkage estimation and univariate analysis, thereby calculating the corresponding risk scores. Moreover, we selected corresponding DEGs between groups with high- and low-risk score and conducted routine bioinformatic analyses. Furthermore, we validated the expression of four hub genes at the protein level through immunohistochemistry (IHC) in samples obtained from our patient cohort. RESULTS: According to the findings, it was found that six genes-AKR1B10, GNA15, ALDH1B1, PLOD2, LPCAT1, and GPX8- were differentially expressed in both TCGA-CSEC and GEO datasets among 23 differentially expressed metabolism-related genes (DEMRGs). mRNAsi exhibited a notable association with the extent of key oncogene mutation. The results showed that the AUC values for forecasting survival at 1, 3, and 5 years are 0.715, 0.689, and 0.748, individually. We observed a notable association between the risk score and different immune cell populations, along with enrichment in crucial signaling pathways in CESC. Four genes differentially expressed between different risk score groups were validated by IHC to be highly expressed in the CESC samples at the protein level. CONCLUSION: The current investigation indicated that a 3-gene signature based on stemness-related metabolic and 4 hub genes with differential expression between high and low-risk score subgroups may serve as valuable prognostic markers and potential therapeutic targets in CESC.

Universal crRNA Acylation Strategy for Robust Photo-Initiated One-Pot CRISPR-Cas12a Nucleic Acid Diagnostics.

Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile and efficient strategies to control CRISPR system are still desirable. Here, we proposed a universal and accessible acylation strategy to regulate the CRISPR-Cas12a system by efficient acylation of 2'-hydroxyls (2'-OH) on crRNA strand with photolabile agents (PLGs). The introduction of PLGs confers efficient suppression of crRNA function and rapid restoration of CRISPR-Cas12a reaction upon short light exposure regardless of crRNA sequences. Based on this strategy, we constructed a universal PhotO-Initiated CRISPR-Cas12a system for Robust One-pot Testing (POIROT) platform integrated with recombinase polymerase amplification (RPA), which showed two orders of magnitude more sensitive than the conventional one-step assay and comparable to the two-step assay. For clinical sample testing, POIROT achieved high-efficiency detection performance comparable to the gold-standard quantitative PCR (qPCR) in sensitivity and specificity, but faster than the qPCR method. Overall, we believe the proposed strategy will promote the development of many other universal photo-controlled CRISPR technologies for one-pot assay, and even expand applications in the fields of controllable CRISPR-based genomic editing, disease therapy, and cell imaging.

Inhibition of phosphatidylinositol 3-kinase catalytic subunit alpha by miR-203a-3p reduces hypertrophic scar formation via phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway.

Background: Hypertrophic scar (HS) is a common fibroproliferative skin disease that currently has no truly effective therapy. Given the importance of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) in hypertrophic scar formation, the development of therapeutic strategies for endogenous inhibitors against PIK3CA is of great interest. Here, we explored the molecular mechanisms underlying the protective effects of miR-203a-3p (PIK3CA inhibitor) against excessive scar. Methods: Bioinformatic analysis, immunohistochemistry, immunofluorescence, miRNA screening and fluorescence in situ hybridization assays were used to identify the possible pathways and target molecules mediating HS formation. A series of in vitro and in vivo experiments were used to clarify the role of PIK3CA and miR-203a-3p in HS. Mechanistically, transcriptomic sequencing, immunoblotting, dual-luciferase assay and rescue experiments were executed. Results: Herein, we found that PIK3CA and the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway were upregulated in scar tissues and positively correlated with fibrosis. We then identified miR-203a-3p as the most suitable endogenous inhibitor of PIK3CA. miR-203a-3p suppressed the proliferation, migration, collagen synthesis and contractility as well as the transdifferentiation of fibroblasts into myofibroblasts in vitro, and improved the morphology and histology of scars in vivo. Mechanistically, miR-203a-3p attenuated fibrosis by inactivating the PI3K/AKT/mTOR pathway by directly targeting PIK3CA. Conclusions: PIK3CA and the PI3K/AKT/mTOR pathway are actively involved in scar fibrosis and miR-203a-3p might serve as a potential strategy for hypertrophic scar therapy through targeting PIK3CA and inactivating the PI3K/AKT/mTOR pathway.

Exosomal miR-4645-5p from hypoxic bone marrow mesenchymal stem cells facilitates diabetic wound healing by restoring keratinocyte autophagy.

Background: Refractory diabetic wounds are a common occurrence in patients with diabetes and epidermis-specific macroautophagy/autophagy impairment has been implicated in their pathogenesis. Therefore, identifying and developing treatment strategies capable of normalizing epidermis-specific macroautophagy/autophagy could facilitate diabetic wound healing. The study aims to investigate the potential of bone marrow mesenchymal stem cell-derived exosomes (BMSC-exos) from hypoxic conditions as a treatment to normalize epidermis-specific autophagy for diabetic wound healing. Methods: We compared the effects of bone marrow mesenchymal stem cell (BMSC)-sourced exosomes (BMSC-Exos) from hypoxic conditions to those of BMSC in normoxic conditions (noBMSC-Exos). Our studies involved morphometric assessment of the exosomes, identification of the microRNA (miRNA) responsible for the effects, evaluation of keratinocyte functions and examination of effects of the exosomes on several molecules involved in the autophagy pathway such as microtubule-associated protein 1 light chain 3 beta, beclin 1, sequestosome 1, autophagy-related 5 and autophagy-related 5. The experiments used human BMSCs from the American Type Culture Collection, an in vivo mouse model of diabetes (db/db) to assess wound healing, as well as the human keratinocyte HaCaT cell line. In the methodology, the authors utilized an array of approaches that included electron microscopy, small interfering RNA (siRNA) studies, RNA in situ hybridization, quantitative real-time reverse transcription PCR (qRT-PCR), the isolation, sequencing and differential expression of miRNAs, as well as the use of miR-4645-5p-specific knockdown with an inhibitor. Results: Hypoxia affected the release of exosomes from hypoxic BMSCs (hy-BMSCs) and influenced the size and morphology of the exosomes. Moreover, hyBMSC-Exo treatment markedly improved keratinocyte function, including keratinocyte autophagy, proliferation and migration. miRNA microarray and bioinformatics analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in 'autophagy' and 'process utilizing autophagic mechanism' in the 'biological process' category and miR-4645-5p as a major contributor to the pro-autophagy effect of hyBMSC-Exos. Moreover, mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) was identified as a potential target of exosomal miR-4645-5p; this was confirmed using a dual luciferase assay. Exosomal miR-4645-5p mediates the inactivation of the MAPKAPK2-induced AKT kinase group (comprising AKT1, AKT2, and AKT3), which in turn suppresses AKT-mTORC1 signaling, thereby facilitating miR-4645-5p-mediated autophagy. Conclusions: Overall, the results of this study showed that hyBMSC-Exo-mediated transfer of miR-4645-5p inactivated MAPKAPK2-induced AKT-mTORC1 signaling in keratinocytes, which activated keratinocyte autophagy, proliferation and migration, resulting in diabetic wound healing in mice. Collectively, the findings could aid in the development of a novel therapeutic strategy for diabetic wounds.

Relieving Macrophage Dysfunction by Inhibiting SREBP2 Activity: A Hypoxic Mesenchymal Stem Cells-Derived Exosomes Loaded Multifunctional Hydrogel for Accelerated Diabetic Wound Healing.

Macrophage dysfunction is one of the primary factors leading to the delayed healing of diabetic wounds. Hypoxic bone marrow mesenchymal stem cells-derived exosomes (hyBMSC-Exos) have been shown to play an active role in regulating cellular function through the carried microRNAs. However, the administration of hyBMSC-Exos alone in diabetic wounds usually brings little effect, because the exosomes are inherently unstable and have a short retention time at the wounds. In this study, a multifunctional hydrogel based on gallic acid (GA) conjugated chitosan (Chi-GA) and partially oxidized hyaluronic acid (OHA) is prepared for sustained release of hyBMSC-Exos. The hydrogel not only exhibits needs-satisfying physicochemical properties, but also displays outstanding biological performances such as low hemolysis rate, strong antibacterial capacity, great antioxidant ability, and excellent biocompatibility. It has the ability to boost the stability of hyBMSC-Exos, leading to a continuous and gradual release of the exosomes at wound locations, ultimately enhancing the exosomes' uptake efficiency by target cells. Most importantly, hyBMSC-Exos loaded hydrogel shows an excellent ability to promote diabetic wound healing by regulating macrophage polarization toward M2 phenotype. This may be because exosomal miR-4645-5p and antioxidant property of the hydrogel synergistically inhibit SREBP2 activity in macrophages. This study presents a productive approach for managing diabetic wounds.

Pyroptosis-related gene signature elicits immune response in rosacea.

Rosacea is a complex chronic inflammatory skin disorder with high morbidity. Pyroptosis is known as a regulated inflammatory cell death. While its association with immune response to various inflammatory disorders is well established, little is known about its functional relevance of rosacea. So, we aimed to explore and enrich the pathogenesis involved in pyroptosis-related rosacea aggravations. In this study, we evaluated the pyroptosis-related patterns of rosacea by consensus clustering analysis of 45 ferroptosis-related genes (FRGs), with multiple immune cell infiltration analysis to identify the pyroptosis-mediated immune response in rosacea using GSE65914 dataset. The co-co-work between PRGs and WGCNA-revealed hub genes has established using PPI network. FRG signature was highlighted in rosacea using multi-transcriptomic and experiment analysis. Based on this, three distinct pyroptosis-related rosacea patterns (non/moderate/high) were identified, and the notably enriched pathways have revealed through GO, KEGG and GSEA analysis, especially immune-related pathways. Also, the XCell/MCPcount/ssGSEA/Cibersort underlined the immune-related signalling (NK cells, Monocyte, Neutrophil, Th2 cells, Macrophage), whose hub genes were identified through WGCNA (NOD2, MYD88, STAT1, HSPA4, CXCL8). Finally, we established a pyroptosis-immune co-work during the rosacea aggravations. FRGs may affect the progression of rosacea by regulating the immune cell infiltrations. In all, pyroptosis with its mediated immune cell infiltration is a critical factor during the development of rosacea.

PANoptosis signaling enables broad immune response in psoriasis: From pathogenesis to new therapeutic strategies.

Background: Accumulating evidence suggests that regulated cell death, such as pyroptosis, apoptosis, and necroptosis, is deeply involved in the pathogenesis of psoriasis. As a newly recognized form of systematic cell death, PANoptosis is involved in a variety of inflammatory disorders through amplifying inflammatory and immune cascades, but its role in psoriasis remains elusive. Objectives: To reveal the role of PANoptosis in psoriasis for a potential therapeutic strategy. Methods: Multitranscriptomic analysis and experimental validation were used to identify PANoptosis signaling in psoriasis. RNA-seq and scRNA-seq analyses were performed to establish a PANoptosis-mediated immune response in psoriasis, which revealed hub genes through WGCNA and predicted disulfiram as a potential drug. The effect and mechanism of disulfiram were verified in imiquimod (IMQ)-induced psoriasis. Results: Here, we found a highlighted PANoptosis signature in psoriasis patients through multitranscriptomic analysis and experimental validation. Based on this, two distinct PANoptosis patterns (non/high) were identified, which were the options for clinical classification. The high-PANoptosis-related group had a higher response rate to immune cell infiltration (such as M1 macrophages and keratinocytes). Subsequently, WGCNA showed the hub genes (e.g., S100A12, CYCS, NOD2, STAT1, HSPA4, AIM2, MAPK7), which were significantly associated with clinical phenotype, PANoptosis signature, and identified immune response in psoriasis. Finally, we explored disulfiram (DSF) as a candidate drug for psoriasis through network pharmacology, which ameliorated IMQ-mediated psoriatic symptoms through antipyroptosis-mediated inflammation and enhanced apoptotic progression. By analyzing the specific ligand-receptor interaction pairs within and between cell lineages, we speculated that DSF might exert its effects by targeting keratinocytes directly or targeting M1 macrophages to downregulate the proliferation of keratinocytes. Conclusions: PANoptosis with its mediated immune cell infiltration provides a roadmap for research on the pathogenesis and therapeutic strategies of psoriasis.

The functions and clinical application potential of exosomes derived from mesenchymal stem cells on wound repair: a review of recent research advances.

Wound repair is a complex problem for both clinical practitioners and scientific investigators. Conventional approaches to wound repair have been associated with several limitations, including prolonged treatment duration, high treatment expenses, and significant economic and psychological strain on patients. Consequently, there is a pressing demand for more efficacious and secure treatment modalities to enhance the existing treatment landscapes. In the field of wound repair, cell-free therapy, particularly the use of mesenchymal stem cell-derived exosomes (MSC-Exos), has made notable advancements in recent years. Exosomes, which are small lipid bilayer vesicles discharged by MSCs, harbor bioactive constituents such as proteins, lipids, microRNA (miRNA), and messenger RNA (mRNA). These constituents facilitate material transfer and information exchange between the cells, thereby regulating their biological functions. This article presents a comprehensive survey of the function and mechanisms of MSC-Exos in the context of wound healing, emphasizing their beneficial impact on each phase of the process, including the regulation of the immune response, inhibition of inflammation, promotion of angiogenesis, advancement of cell proliferation and migration, and reduction of scar formation.

Endogenous Enzyme-Activatable Spherical Nucleic Acids for Spatiotemporally Controlled Signal Amplification Molecular Imaging and Combinational Tumor Therapy.

Due to the adjustable hybridization activity, antinuclease digestion stability, and superior endocytosis, spherical nucleic acids (SNAs) have been actively developed as probes for molecular imaging and the development of noninvasive diagnosis and image-guided surgery. However, since highly expressed biomarkers in tumors are not negligible in normal tissues, an inevitable background signal and the inability to precisely release probes at the chosen region remain a challenge for SNAs. Herein, we proposed a rationally designed, endogenous enzyme-activatable functional SNA (Ep-SNA) for spatiotemporally controlled signal amplification molecular imaging and combinational tumor therapy. The self-assembled amphiphilic polymer micelles (SM-ASO), which were obtained by a simple and rapid copper-free strain-promoted azide-alkyne cycloaddition click reaction between dibenzocyclooctyne-modified antisense oligonucleotide and azide-containing aliphatic polymer polylactic acid, were introduced as the core elements of Ep-SNA. This Ep-SNA was then constructed by connecting two apurinic/apyrimidinic (AP) site-containing trailing DNA hairpins, which could occur via a hybridization chain reaction in the presence of low-abundance survivin mRNA to SM-ASO through complementary base pairing. Notably, the AP site-containing trailing DNA hairpins also empowered the SNA with the feasibility of drug delivery. Once this constructed intelligent Ep-SNA nanoprobe was specifically cleaved by the highly expressed cytoplasmic human apurinic/apyrimidinic endonuclease 1 in tumor cells, three key elements (trailing DNA hairpins, antisense oligonucleotide, and doxorubicin) could be released to enable subsequent high-sensitivity survivin mRNA imaging and combinational cancer therapy (gene silencing and chemotherapy). This strategy shows great application prospects of SNAs as a precise platform for the integration of disease diagnosis and treatment and can contribute to basic biomedical research.

Reaction-Activated Disassembly of the NIR-II Probe Enables Fast Detection and Ratiometric Photoacoustic Imaging of Glutathione In Vivo.

Glutathione (GSH), the most abundant nonprotein biothiol, is a significant endogenous molecule that plays a key role in redox equilibrium in vivo and is regarded as a critical biomarker of cancer. Currently, various fluorescent probes have been designed and synthesized for imaging GSH at the cellular level in the visible range and the first near-infrared window (NIR-I, 750-900 nm). However, the application of these fluorescent probes for bioimaging and biosensing in vivo has been extremely hindered by the high biobackground and low tissue penetration. Herein, based on the self-assembly and disassembly of J-aggregation, we designed and synthesized a GSH-activatable probe MC-PSE for second near-infrared window (NIR-II) fluorescence and ratiometric photoacoustic imaging of GSH in vivo. The anionic cyanine-based MC-PSE tends to form stable J-aggregates in an aqueous solution. Upon the reaction with GSH, the J-aggregates of MC-PSE disassembled, the emission peak intensity of MC-PSE at 940 nm significantly increased by about 20 times, and the PA900/PA980 ratio increased by 4 times within 15 min in vitro. Notably, we used MC-PSE to visualize GSH in tumor-bearing mice and to distinguish normal and tumor areas successfully by virtue of NIR-II FL and PA dual-modal imaging. The design strategy of MC-PSE provides a novel method for ratiometric photoacoustic imaging, and MC-PSE is expected to be a powerful tool for the accurate detection of GSH in cancer diagnosis.

A pyroptosis-related gene signature that predicts immune infiltration and prognosis in colon cancer.

Background: Colon cancer (CC) is a highly heterogeneous malignancy associated with high morbidity and mortality. Pyroptosis is a type of programmed cell death characterized by an inflammatory response that can affect the tumor immune microenvironment and has potential prognostic and therapeutic value. The aim of this study was to evaluate the association between pyroptosis-related gene (PRG) expression and CC. Methods: Based on the expression profiles of PRGs, we classified CC samples from The Cancer Gene Atlas and Gene Expression Omnibus databases into different clusters by unsupervised clustering analysis. The best prognostic signature was screened and established using least absolute shrinkage and selection operator (LASSO) and multivariate COX regression analyses. Subsequently, a nomogram was established based on multivariate COX regression analysis. Next, gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were performed to explore the potential molecular mechanisms between the high- and low-risk groups and to explore the differences in clinicopathological characteristics, gene mutation characteristics, abundance of infiltrating immune cells, and immune microenvironment between the two groups. We also evaluated the association between common immune checkpoints and drug sensitivity using risk scores. The immunohistochemistry staining was utilized to confirm the expression of the selected genes in the prognostic model in CC. Results: The 1163 CC samples were divided into two clusters (clusters A and B) based on the expression profiles of the 33 PRGs. Genes with prognostic value were screened from the DEGs between the two clusters, and an eight PRGs prognostic model was constructed. GSEA and GSVA of the high- and low-risk groups revealed that they were mainly enriched in inflammatory response-related pathways. Compared to those in the low-risk group, patients in the high-risk group had worse overall survival, an immunosuppressive microenvironment, and worse sensitivity to immunotherapy and drug treatment. Conclusion: Our findings provide a foundation for future research targeting pyroptosis and new insights into prognosis and immunotherapy from the perspective of pyroptosis in CC.

Self-assembled super-small AIEgen nanoprobe for highly sensitive and selective detection of protamine and trypsin.

Amphiphilic aggregation-induced emission (AIE) molecules show superior potential for fabricating novel ultrasmall nanoprobes. Here, an anionic dipyridyl tetraphenylethene (TPE) derivative is rationally designed and a super-small self-assembled AIEgen nanoprobe (TPE-2Py-SO3NaNPs, ca. 2.48 nm) is thus conveniently constructed for the supersensitive detection of protamine and trypsin. In HEPES/DMSO solution (8 : 2, v/v, pH = 7.4), negatively charged TPE-2Py-SO3NaNPs exhibited an AIE effect in the presence of positively charged protamine, presenting a fluorescence enhancement at 498 nm together with a large Stokes shift of 150 nm and a low detection limit of 8.0 ng mL-1. In addition, the in situ formed TPE-2Py-SO3Na/protamine nanocomposite can be dissociated by trypsin due to the highly selective degradation of protamine via enzymatic hydrolysis, achieving a detection limit for trypsin as low as 5.0 ng mL-1.

Identification and verification of ferroptosis-related genes in diabetic foot using bioinformatics analysis.

Ferroptosis is a novel form of cell death that plays a key role in several diseases, including inflammation and tumours; however, the role of ferroptosis-related genes in diabetic foot remains unclear. Herein, diabetic foot-related genes were downloaded from the Gene Expression Omnibus and the ferroptosis database (FerrDb). The least absolute shrinkage and selection operator regression algorithm was used to construct a related risk model, and differentially expressed genes were analysed through immune infiltration. Finally, we identified relevant core genes through a protein-protein interaction network, subsequently verified using immunohistochemistry. Comprehensive analysis showed 198 genes that were differentially expressed during ferroptosis. Based on functional enrichment analysis, these genes were primarily involved in cell response, chemical stimulation, and autophagy. Using the CIBERSORT algorithm, we calculated the immune infiltration of 22 different types of immune cells in diabetic foot and normal tissues. The protein-protein interaction network identified the hub gene TP53, and according to immunohistochemistry, the expression of TP53 was high in diabetic foot tissues but low in normal tissues. Accordingly, we identified the ferroptosis-related gene TP53 in the diabetic foot, which may play a key role in the pathogenesis of diabetic foot and could be used as a potential biomarker.

Rigidity-Dependent Emission: Inspired Selection of an ATP-Specific Polyvalent Hydrogen Binding-Lighted Fluorophore for Intracellular Amplified Imaging.

ATP, a small molecule with high intracellular concentration (mM level), provides a fuel to power signal amplification, which is meaningful for biosensing. However, traditional ATP-powered amplification is based on ATP/aptamer recognition, which is susceptible to the complex biological microenvironment (e.g., nuclease). In this work, we communicate a signaling manner termed as ATP-specific polyvalent hydrogen binding (APHB), which is mimetic to ATP/aptamer binding but can avoid interference from biomolecules. The key in APHB is a functional fluorophore that can selectively bind with ATP via polyvalent hydrogen, and the fluorescence was lighted with the changes of the molecular structure from flexibility to rigidity. By designing, synthesizing, and screening a series of compounds, we successfully obtained an ATP-specific binding-lighted fluorophore (ABF). Experimental verification and a complex analogue demonstrated that two melamine brackets in the ABF dominate the polyvalent hydrogen binding between the ABF and ATP. Then, to achieve amplification biosensing, fibroblast activation protein (FAP) in activated hepatic stellate cells was taken as a model target, and a nanobeacon consisting of an ABF, a quencher, and an FAP-activated polymer shell was constructed. Benefiting from the ATP-powered amplification, the FAP was sensitively detected and imaged, and the potential relationship between differentiation of hepatocytes and FAP concentration was first revealed, highlighting the great potential of APHB-mediated signaling for intracellular sensing.

Collagen scaffolds derived from bovine skin loaded with MSC optimized M1 macrophages remodeling and chronic diabetic wounds healing.

Owing to the persistent inflammatory microenvironment and unsubstantial dermal tissues, chronic diabetic wounds do not heal easily and their recurrence rate is high. Therefore, a dermal substitute that can induce rapid tissue regeneration and inhibit scar formation is urgently required to address this concern. In this study, we established biologically active dermal substitutes (BADS) by combining novel animal tissue-derived collagen dermal-replacement scaffolds (CDRS) and bone marrow mesenchymal stem cells (BMSCs) for the healing and recurrence treatments of chronic diabetic wounds. The collagen scaffolds derived from bovine skin (CBS) displayed good physicochemical properties and superior biocompatibility. CBS loaded with BMSCs (CBS-MCSs) could inhibit M1 macrophage polarization in vitro. Decreased MMP-9 and increased Col3 at the protein level were detected in CBS-MSCs-treated M1 macrophages, which may be attributed to the suppression of the TNF-α/NF-κB signaling pathway (downregulating phospho-IKKα/ß/total IKKα/ß, phospho-IκB/total IκB, and phospho-NFκB/total NFκB) in M1 macrophages. Moreover, CBS-MSCs could benefit the transformation of M1 (downregulating iNOS) to M2 (upregulating CD206) macrophages. Wound-healing evaluations demonstrated that CBS-MSCs regulated the polarization of macrophages and the balance of inflammatory factors (pro-inflammatory: IL-1ß, TNF-α, and MMP-9; anti-inflammatory: IL-10 and TGF-ß3) in db/db mice. Furthermore, CBS-MSCs facilitated the noncontractile and re-epithelialized processes, granulation tissue regeneration, and neovascularization of chronic diabetic wounds. Thus, CBS-MSCs have a potential value for clinical application in promoting the healing of chronic diabetic wounds and preventing the recurrence of ulcers.

A New Paradigm in Spinal Cord Injury Therapy: from Cell-free Treatment to Engineering Modifications.

Spinal cord injury (SCI) is an intractable and poorly prognostic neurological disease, and current treatments are still unable to cure it completely and avoid sequelae. Extracellular vesicles (EVs), as important carriers of intercellular communication and pharmacological effects, are considered to be the most promising candidates for SCI therapy because of their low toxicity and immunogenicity, their ability to encapsulate endogenous bioactive molecules (e.g., proteins, lipids, and nucleic acids), and their ability to cross the blood-brain/cerebrospinal barriers. However, poor targeting, low retention rate, and limited therapeutic efficacy of natural EVs have bottlenecked EV-based SCI therapy. A new paradigm for SCI treatment will be provided by engineering modified EVs. Furthermore, our limited understanding of the role of EVs in SCI pathology hinders the rational design of novel EVbased therapeutic approaches. In this study, we review the pathophysiology after SCI, especially the multicellular EVs-mediated crosstalk; briefly describe the shift from cellular to cell-free therapies for SCI treatment; discuss and analyze the issues related to the route and dose of EVs administration; summarize and present the common strategies for EVs drug loading in the treatment of SCI and point out the shortcomings of these drug loading methods; finally, we analyze and highlight the feasibility and advantages of bio-scaffold-encapsulated EVs for SCI treatment, providing scalable insights into cell-free therapy for SCI.

A Quencher-Based Blood-Autofluorescence-Suppression Strategy Enables the Quantification of Trace Analytes in Whole Blood.

Precise quantification of trace components in whole blood via fluorescence is of great significance. However, the applicability of current fluorescent probes in whole blood is largely hindered by the strong blood autofluorescence. Here, we proposed a blood autofluorescence-suppressed sensing strategy to develop an activable fluorescent probe for quantification of trace analyte in whole blood. Based on inner filter effect, by screening fluorophores whose absorption overlapped with the emission of blood, a redshift BODIPY quencher with an absorption wavelength ranging from 600-700 nm was selected for its superior quenching efficiency and high brightness. Two 7-nitrobenzo[c] [1,2,5] oxadiazole ether groups were introduced onto the BODIPY skeleton for quenching its fluorescence and the response of H2 S, a gas signal molecule that can hardly be quantified because of its low concentration in whole blood. Such detection system shows a pretty low background signal and high signal-to-back ratio, the probe thus achieved the accurate quantification of endogenous H2 S in 20-fold dilution of whole blood samples, which is the first attempt of quantifying endogenous H2 S in whole blood. Moreover, this autofluorescence-suppressed sensing strategy could be expanded to other trace analytes detection in whole blood, which may accelerate the application of fluorescent probes in clinical blood test.