Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

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.

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.

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.

Orthogonal Radical and Cationic Single-Unit Monomer Insertions for Engineering Polymer Architectures.

The single-unit monomer insertion (SUMI), derived from living/controlled polymerization, can be directly functionalized at the end or within the chain of polymers prepared by living/controlled polymerization, offering potential applications in the preparation of polymers with complex architectures. Many scenarios demand the simultaneous incorporation of monomers suitable for different polymerization methods into complex polymers. Therefore, it becomes imperative to utilize SUMI technologies with diverse mechanisms, especially those that are compatible with each other. Here, we reported the orthogonal SUMI technique, seamlessly combining radical and cationic SUMI approaches. Through the careful optimization of monomer and chain transfer agent pairs and adjustments to reaction conditions, we can efficiently execute both radical and cationic SUMI processes in one pot without mutual interference. The utilization of orthogonal SUMI pairs facilitates the integration of radical and cationic reversible addition-fragmentation chain transfer (RAFT) polymerization in various configurations. This flexibility enables the synthesis of diblock, triblock, and star polymers that incorporate both cationically and radically polymerizable monomers. Moreover, we have successfully implemented a mixing mechanism of free radicals and cations in RAFT step-growth polymerization, resulting in the creation of a side-chain sequence-controlled polymer brushes.

Cationic Single-Unit Monomer Insertion (cSUMI): From Discrete Oligomers to the α-/ω-End and In-Chain Sequence-Regulated Polymers.

Single-unit monomer insertion (SUMI) has become an important strategy for the synthesis of sequence-controlled vinyl polymers due to its strong versatility and high efficiency. However, all reported SUMI processes are based on a free-radical mechanism, resulting in a limited number of monomer types being applicable to SUMI or a limited number of sequences of structural units that SUMI can synthesize. Herein, we developed a novel SUMI based on a cationic mechanism (cSUMI), which operates through a degenerative (similar to radical SUMI) but cationic chain transfer process. By optimizing the chain transfer agent (CTA) and monomer pairs, a high-efficiency cSUMI was achieved for vinyl ether and styrene monomers. Based on this reaction, a range of discrete oligomers containing vinyl ether and styrene moieties, and even α-/ω-end and in-chain sequence-regulated polymers were synthesized, most of which cannot be achieved by radical SUMI. In addition, we explored the application of these sequence-regulated polymers in the preparation of miktoarm star polymers, delivery of photosensitizers, and solubilization of fluorescence probes. The development of SUMI with a new mechanism will certainly broaden the scope of structures and sequences in precise vinyl-based polymers.

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.

Ultrasensitive CRISPR/Cas13a-Mediated Photoelectrochemical Biosensors for Specific and Direct Assay of miRNA-21.

Sensitive and specific assay of microRNAs (miRNAs) is beneficial to early disease screening. Herein, we for the first time proposed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated photoelectrochemical biosensors for the direct assay of miRNA-21. In this study, compared with traditional nucleic acid-based signal amplification strategies, the CRISPR/Cas13a system can greatly improve the specificity and sensitivity of target determination due to its accurate recognition and high-efficient trans-cleavage capability without complex nucleic acid sequence design. Moreover, compared with the CRISPR/Cas12a-based biosensing platform, the developed CRISPR/Cas13a-mediated biosensor can directly detect RNA targets without signal transduction from RNA to DNA, thereby avoiding signal leakage and distortion. Generally, the proposed biosensor reveals excellent analysis capability with a wider linear range from 1 fM to 5 nM and a lower detection limit of 1 fM. Additionally, it also shows satisfactory stability in the detection of human serum samples and cell lysates, manifesting that it has great application prospects in the areas of early disease diagnosis and biomedical research.

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.

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.

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.

Bioinformatics analysis and verification of m6A related genes based on the construction of keloid diagnostic model.

Keloids are formed due to abnormal hyperplasia of the skin connective tissue. We explored the relationship between N6-methyladenosine (m6A)-related genes and keloids. The transcriptomic datasets (GSE44270 and GSE185309) of keloid and normal skin tissues samples were obtained from the Gene Expression Omnibus database. We constructed the m6A landscape and verified the corresponding genes using immunohistochemistry. We extracted hub genes for unsupervised clustering analysis using protein-protein interaction (PPI) network; gene ontology enrichment analysis was performed to determine the biological processes or functions affected by the differentially expressed genes (DEGs). We performed immune infiltration analysis to determine the relationship between keloids and the immune microenvironment using single-sample gene set enrichment analysis and CIBERSORT. Differential expression of several m6A genes was observed between the two groups; insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was significantly upregulated in keloid patients. PPI analysis elucidated six genes with significant differences between the two keloid sample groups. Enrichment analysis revealed that the DEGs were mainly enriched in cell division, proliferation, and metabolism. Moreover, significant differences in immunity-related pathways were observed. Therefore, the results of this study will provide a reference for the elucidation of the pathogenesis and therapeutic targets of keloids.

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.

CENPF as an independent prognostic and metastasis biomarker corresponding to CD4+ memory T cells in cutaneous melanoma.

Owing to recent advances in immunotherapies, the overall survival of patients with skin cutaneous melanoma (SKCM) has increased; however, the 5-year survival rate of metastatic patients remains poor. Skin cutaneous melanoma-upregulated genes were screened via analysis of differentially expressed genes (GSE3189 and GSE46517), and metastasis-related oncogenes were identified via weighted gene coexpression network analysis of the GSE46517 dataset. As confirmed by the Tumor Immune Estimation Resource, we found highly expressed centromere protein F (CENPF) in SKCM and its metastases. Immunostaining of human melanoma tissues demonstrated high CENPF expression. According to the Kaplan-Meier survival curve log-rank test, receiver-operating characteristic curve, and univariate and multivariate analyses, the Cancer Genome Atlas (TCGA) database suggested CENPF be a typical independent predictor of SKCM. The CIBERSORT algorithm classified the types of the immune cells from GSE46517 and showed higher proportion of CD4+ memory-activated T cells in metastatic melanoma. Single-sample gene set enrichment analysis of TCGA data confirmed the correlation between CENPF and activated CD4+ T cells. Centromere protein F was positively correlated with tumor mutational burden and CD4+ memory T cell markers (interleukin [IL]-23A, CD28, and CD62L), negatively associated with memory T cell maintenance factors (IL-7 and IL-15) by correlation analysis. Moreover, immunofluorescence showed high coexpression of CENPF and IL23A, CD4 in melanoma. Upregulated CENPF might lead to premature depletion of CD4+ memory T cells and immunosuppression. Nomogram indicated CENPF clinical predictive value for 1-, 3-, 5-, and 7-year melanoma overall survival. Therefore, CENPF plays a vital role in the progression and metastasis of melanoma and can be an effective therapeutic target.

Brain-targeted Near-Infrared Nanobeacon for In Situ Monitoring H2S Fluctuation during Epileptic Seizures.

Epilepsy is a neurological brain disease, and its recurrent seizures are related to the reductive substance-powered antioxidant defense system (ADS). However, until now, there has been no report on the study of in situ antioxidant fluctuation during epilepsy of varying severity. In this work, hydrogen sulfide (H2S) was selected as the model target, a H2S-responsive near-infrared fluorophore was designed and synthesized, and an amphiphilic molecule was synthesized and modified with angiopep-2 peptide at its hydrophilic terminus. A nanobeacon termed as BFPP was prepared by the formation of micelles with the package of the fluorophore. The nanobeacon was sensitive to H2S, with a low detection limit of 17 nM. The H2S fluctuation in cells can be monitored by fluorescence imaging. In addition, angiopep-2 peptide at the surface of BFPP helps it cross the blood-brain barrier, and near-infrared fluorescence improves in vivo imaging. BFPP revealed that H2S was at a moderate level in the normal brain, but its level was obviously elevated during mild epilepsy because of the activation of the ADS while significantly suppressed during severe epilepsy due to neuronal damage. This approach is generally accessible for other targets by altering the responsive fluorophore, with significance for in situ analysis of brain pathology.

[Diagnosis and treatment of rectal injury during laparoscopic radical prostatectomy: Analysis of 7 cases].

OBJECTIVE: To explore the risk factors and management principles of rectal injury during laparoscopic radical prostatectomy (LRP). METHODS: We retrospectively analyzed the clinical data on 7 cases of LRP complicated with rectal injury and treated in Huzhou Central Hospital from January 2010 to June 2021. Four of the 7 PCa patients were found with complete rectal rupture during LRP, of whom 2 were treated by laparoscopic rectal repair (LRR) and the other 2 by LRR + colostomy during surgery. Another case of rectal muscle injury also underwent LRR. Two cases of delayed rectal rupture were observed postoperatively and treated by colostomy + transrectal repair in the second-stage operation. RESULTS: The rectal injuries were found in the apex of the prostate in all the 7 cases, pathologically staged as pT2b��pT3b and with Gleason scores of 7��10. Postoperative follow-up lasted 2 to 18 months, during which the 5 cases of intraoperative rectal repair recovered well without complications, and of the 2 cases of postoperative rectal repair, 1 made a good recovery and the other 1 developed rectourethral fistula. CONCLUSION: Rectal injury during radical prostatectomy tends to occur in the apex of the prostate and can be effectively managed by laparoscopic repair. Meanwhile, attention should be paid to the postoperative complication of rectourethral fistula.

A Polymeric Nanobeacon for Monitoring the Fluctuation of Hydrogen Polysulfides during Fertilization and Embryonic Development.

Fertilization and early embryonic development as the beginning of a new life are key biological events. Hydrogen polysulfide (H2 Sn ) plays important roles during physiological regulation, such as antioxidation-protection. However, no report has studied in situ H2 Sn fluctuation during early embryonic development because of the low abundance of H2 Sn and inadequate sensitivity of probes. We herein construct a polymeric nanobeacon from a H2 Sn -responsive polymer and fluorophores, which is capable of detecting H2 Sn selectively and of signal amplification. Taking the zebrafish as a model, the polymeric nanobeacon revealed that the H2 Sn level was significantly elevated after fertilization due to the activation of cell multiplication, suppressed partially during embryonic development, and finally kept steady up to zebrafish emergence. This strategy is generally accessible for biomarkers by altering the responsive unit and significant for facilitating biological analysis during life development.

Identification of a novel tumour microenvironment-based prognostic biomarker in skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is one of the most destructive skin malignancies and has attracted worldwide attention. However, there is a lack of prognostic biomarkers, especially tumour microenvironment (TME)-based prognostic biomarkers. Therefore, there is an urgent need to investigate the TME in SKCM, as well as to identify efficient biomarkers for the diagnosis and treatment of SKCM patients. A comprehensive analysis was performed using SKCM samples from The Cancer Genome Atlas and normal samples from Genotype-Tissue Expression. TME scores were calculated using the ESTIMATE algorithm, and differential TME scores and differentially expressed prognostic genes were successively identified. We further identified more reliable prognostic genes via least absolute shrinkage and selection operator regression analysis and constructed a prognostic prediction model to predict overall survival. Receiver operating characteristic analysis was used to evaluate the diagnostic efficacy, and Cox regression analysis was applied to explore the relationship with clinicopathological characteristics. Finally, we identified a novel prognostic biomarker and conducted a functional enrichment analysis. After considering ESTIMATEScore and tumour purity as differential TME scores, we identified 34 differentially expressed prognostic genes. Using least absolute shrinkage and selection operator regression, we identified seven potential prognostic biomarkers (SLC13A5, RBM24, IGHV3OR16-15, PRSS35, SLC7A10, IGHV1-69D and IGHV2-26). Combined with receiver operating characteristic and regression analyses, we determined PRSS35 as a novel TME-based prognostic biomarker in SKCM, and functional analysis enriched immune-related cells, functions and signalling pathways. Our study indicated that PRSS35 could act as a potential prognostic biomarker in SKCM by investigating the TME, so as to provide new ideas and insights for the clinical diagnosis and treatment of SKCM.

Long-Lasting Bioluminescence Imaging of the Fibroblast Activation Protein by an Amphiphilic Block Copolymer-Based Probe.

Long-term specific tracing of the fibroblast activation protein (FAP) has been of great importance because it is heavily expressed by stromal fibroblasts of multiple diseases, and several disorders associated with FAP are chronical. Bioluminescence (BL) imaging has its advantages to detect FAP in vivo since no external excitation is required, but the current FAP-responsive BL probe was constructed by covalently masking the firefly luciferase substrate and easily secreted out from the animal, resulting in transient BL imaging of FAP. To circumvent this problem, a peptide-linked amphiphilic block copolymer-based probe (PABC) was developed and applied to the long-lasting BL image of FAP in vivo. For this purpose, an amphiphilic block copolymer containing an FAP-responsive peptide was fabricated to self-assemble into micelles, which act as a depot to load amounts of d-luciferin for constructing the BL probe. Upon reaction with FAP, the micelle would be destroyed to release the internal d-luciferin for BL emission by a luciferase-catalyzed reaction. By virtue of the high loading capability of micelles, the FAP was determined from 0.5 to 10 ng/mL with a detection limit of 0.105 ng/mL, and the high sensitivity makes the PABC capable of distinguishing cancer cells from normal ones. Importantly, compared with free d-luciferin, PABC can be used to persistently image the FAP in living cells and in vivo. This characteristic of long-lasting specific tracing of the FAP makes us envision that this BL probe could be used for screening of FAP inhibitors and diagnosing various FAP-related diseases in future.