The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

Iron-based metal-organic framework co-loaded with buthionine sulfoximine and oxaliplatin for enhanced cancer chemo-ferrotherapy via sustainable glutathione elimination.

BACKGROUND: Emerging ferroptosis-driven therapies based on nanotechnology function either by increasing intracellular iron level or suppressing glutathione peroxidase 4 (GPX4) activity. Nevertheless, the therapeutic strategy of simultaneous iron delivery and GPX4 inhibition remains challenging and has significant scope for improvement. Moreover, current nanomedicine studies mainly use disulfide-thiol exchange to deplete glutathione (GSH) for GPX4 inactivation, which is unsatisfactory because of the compensatory effect of continuous GSH synthesis. METHODS: In this study, we design a two-in-one ferroptosis-inducing nanoplatform using iron-based metal-organic framework (MOF) that combines iron supply and GPX4 deactivation by loading the small molecule buthionine sulfoxide amine (BSO) to block de novo GSH biosynthesis, which can achieve sustainable GSH elimination and dual ferroptosis amplification. A coated lipid bilayer (L) can increase the stability of the nanoparticles and a modified tumor-homing peptide comprising arginine-glycine-aspartic acid (RGD/R) can achieve tumor-specific therapies. Moreover, as a decrease in GSH can alleviate resistance of cancer cells to chemotherapy drugs, oxaliplatin (OXA) was also loaded to obtain BSO&OXA@MOF-LR for enhanced cancer chemo-ferrotherapy in vivo. RESULTS: BSO&OXA@MOF-LR shows a robust tumor suppression effect and significantly improved the survival rate in 4T1 tumor xenograft mice, indicating a combined effect of dual amplified ferroptosis and GSH elimination sensitized apoptosis. CONCLUSION: BSO&OXA@MOF-LR is proven to be an efficient ferroptosis/apoptosis hybrid anti-cancer agent. This study is of great significance for the clinical development of novel drugs based on ferroptosis and apoptosis for enhanced cancer chemo-ferrotherapy.

Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes.

BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences-a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5' end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3' end. RESULTS: Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. CONCLUSIONS: The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening.

Simultaneously Enhanced Singlet Oxygen and Fluorescence Production of Nanoplatform by Surface Plasmon Resonance Coupling for Biomedical Applications.

Photodynamic therapy (PDT) and fluorescence imaging offer the possibility of precise and personalized treatment of cancer, but low singlet oxygen production of a commercial photosensitizer and the quenching effect of fluorescent dyes limit the further application of PDT treatment and fluorescence imaging. In addition, the single nanoplatform that simultaneously achieved singlet oxygen and fluorescence enhancement is rare. In this paper, a novel simultaneously enhanced singlet oxygen and fluorescence production nanoplatform of AuNR@mSiO2-Ce6-Cy5.5 has been successfully designed and synthesized by surface plasmon resonance coupling. The as-synthesized nanoplatform achieved a 1.8-fold enhancement of the singlet oxygen production of Ce6 and a 5.0-fold enhancement of the fluorescence production of Cy5.5 by surface plasmon resonance coupling. The as-synthesized nanoplatform simultaneously enhances the photodynamic therapy and fluorescence imaging of cancer, which will have great potential in biomedical applications.

Near-infrared fluorescent probe for selective detection of H2S and its application in living animals.

In this study, a novel near-infrared fluorescent off-on probe for H2S based on seminaphthorhodafluor fluorophore is designed and constructed, which could be used in detection with 121-fold (23-fold) fluorescent (absorbance) enhancement at 630 nm (572 nm), fast responsiveness (completed within 5 min), high sensitivity, and lower cellular autofluorescence interference. Based on these excellent optical properties, the probe was employed to monitor H2S in red wine samples with satisfactory results. Moreover, the probe was successfully applied for monitoring and imaging H2S quantitatively in Hela cells and live athymic nude mice, indicating its potential application in biological science.

Nonplanar Monocyanines: Meso-Substituted Thiazole Orange with High Photostability and Their Synthetic Strategy as well as a Cell Association Study.

A convenient approach for the direct synthesis of meso-substituted thiazole orange (meso-TO) analogues has been unprecedentedly developed through the AlCl3-catalyzed reaction of parent TO with benzyl alcohol derivatives. Single-crystal X-ray structures show that the prepared new meso-TO analogues are nonplanar, forming a sharp contrast to planar TO. The spectral properties show that nonplanar meso-TO analogues do not aggregate, existing in monomer form (M) in PBS buffer, and have little effect of solvatochromism in different solvents. In comparison with the parents, meso-TO analogues exhibit a large Stokes shift, excellent light fastness, and inertness to singlet oxygen. A cellular association study demonstrates that incorporation of a benzyl group at the meso position methine of parent TO can decrease the cytotoxicity, change staining area in cells, and emit long-wavelength fluorescence for an extended time, which are useful for the development of smarter TOs for imaging in biological science.

Genetically engineered macrophages as living cell drug carriers for targeted cancer therapy.

Precise targeting is a major prerequisite for effective cancer therapy because it ensures a sufficient therapeutic dosage in tumors while minimizing off-target side effects. Herein, we report a live-macrophage-based therapeutic system for high-efficiency tumor therapy. As a proof of concept, anti-human epidermal growth factor receptor-2 (HER2) affibodies were genetically engineered onto the extracellular membrane of macrophages (AE-Mφ), which further internalized doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) nanoparticles (NPs) to produce a macrophage-based therapeutic system armed with anti-HER2 affibodies. NPs(DOX)@AE-Mφ were able to target HER2+ cancer cells and specifically elicit affibody-mediated cell therapy. Most importantly, the superior HER2 + -targeting capability of NPs(DOX)@AE-Mφ greatly guaranteed high accumulation at the tumor site for improved chemotherapy, which acted synergistically with cell therapy to significantly enhance anti-tumor efficacy. This study suggests that NPs(DOX)@AE-Mφ could be utilized as an innovative 'living targeted drug' platform for combining both macrophage-mediated cell therapy and targeted chemotherapy for the individualized treatment of solid tumors.

Filamentous-Actin-Mimicking Nanoplatform for Enhanced Cytosolic Protein Delivery.

Despite the potential of protein therapeutics, the cytosolic delivery of proteins with high efficiency and bioactivity remains a significant challenge owing to exocytosis and lysosomal degradation after endocytosis. Therefore, it is important to develop a safe and efficient strategy to bypass endocytosis. Inspired by the extraordinary capability of filamentous-actin (F-actin) to promote cell membrane fusion, a cyanine dye assembly-containing nanoplatform mimicking the structure of natural F-actin is developed. The nanoplatform exhibits fast membrane fusion to cell membrane mimics and thus enters live cells through membrane fusion and bypasses endocytosis. Moreover, it is found to efficiently deliver protein cargos into live cells and quickly release them into the cytosol, leading to high protein cargo transfection efficiency and bioactivity. The nanoplatform also results in the superior inhibition of tumor cells when loaded with anti-tumor proteins. These results demonstrate that this fusogenic nanoplatform can be valuable for cytosolic protein delivery and tumor treatment.

In vivo gastric cancer targeting and imaging using novel symmetric cyanine dye-conjugated GX1 peptide probes.

To facilitate the translation of cancer fluorescence imaging into clinical practice, the development of stable and highly specific and sensitive targeted fluorescence probes with low toxicity is desirable. GX1, a gastric tumor angiogenesis marker candidate, holds promise in the target-specific delivery of molecular imaging probes for early gastric cancer detection in vivo. In this study, we describe the design and synthesis of a series of novel penta-methine cyanine dyes using the symmetric synthesis method and further conjugated the dyes with GX1, allowing specific binding to the vasculature of gastric cancer. This efficient synthetic route can decrease the undesired byproducts, while increasing yield. Furthermore, in vivo fluorescence imaging revealed that this novel targeted probe accumulates selectively in the tumor site of SGC-7901 subcutaneous xenograft models. The combination of such novel vasculature-targeted molecular probes with fluorescence imaging technology may improve early detection, metastasis detection, and antitumor angiogenesis therapy for gastric cancer.

Near-infrared-dye labeled tumor vascular-targeted dimer GEBP11 peptide for image-guided surgery in gastric cancer.

Introduction: Positive resection margins occur in about 2.8%-8.2% gastric cancer surgeries and is associated with poor prognosis. Intraoperative guidance using Nearinfrared (NIR) fluorescence imaging is a promising technique for tumor detection and margin assessment. The goal of this study was to develop a tumor-specific probe for real-time intraoperative NIR fluorescence imaging guidance. Methods: The tumor vascular homing peptide specific for gastric cancer, GEBP11, was conjugated with a near-infrared fluorophore, Cy5.5. The binding specificity of the GEBP11 probes to tumor vascular endothelial cells were confirmed by immunofluorescent staining. The ability of the probe to detect tumor lesions was evaluated in two xenograft models. An orthotopic gastric cancer xenograft model was used to evaluate the efficacy of the GEBP11 NIR probes in real-time surgical guidance. Results: In vitro assay suggested that both mono and dimeric GEBP11 NIR probes could bind specifically to tumor vascular epithelial cells, with dimeric peptides showed better affinity. In tumor xenograft mice, live imaging suggested that comparing with free Cy5.5 probe, significantly stronger NIR signals could be detected at the tumor site at 24-48h after injection of mono or dimeric GEBP11 probes. Dimeric GEBP11 probe showed prolonged and stronger NIR signals than mono GEBP11 probe. Biodistribution assay suggested that GEBP11 NIR probes were enriched in gastric cancer xenografts. Using dimeric GEBP11 NIR probes in real-time surgery, the tumor margins and peritoneal metastases could be clearly visualized. Histological examination confirmed the complete resection of the tumor. Conclusion: (GEBP11)2-ACP-Cy5.5 could be a potential useful probe for intraoperative florescence guidance in gastric cancer surgery.

Liposome-templated gold nanoparticles for precisely temperature-controlled photothermal therapy based on heat shock protein expression.

Mild temperature photothermal therapy is gaining more and more attention due to high safety, high specificity and moderate efficacy. However, the therapeutical outcome of mild photothermal therapy is limited due to the overexpression of heat shock proteins (HSPs). Therefore, the precise management of HSP expression is the key to improvement of mild temperature photothermal therapy. However, the correlation between HSP expression and photothermal temperature in vivo is still unclear. To precisely control the photothermal temperature by managing the HSP expression, we quantified the HSP expression at different photothermal temperatures after irradiation on liposome-templated gold nanoparticles, which have high photostability, high photothermal conversion efficiency and low temperature fluctuation (smaller than 1 â„ƒ). We found that the expression of HSP70 was least at 47 â„ƒ, which was the optimal temperature for HSP management. We chose to co-administrate HSP70 inhibitor during 47 â„ƒ photothermal therapy, leading to greatly enhanced tumor inhibition. Our precise temperature-controlled photothermal therapy based on HSP expression offers a new strategy for clinical tumor photothermal therapy.

Liposomal Glucose Oxidase for Enhanced Photothermal Therapy and Photodynamic Therapy against Breast Tumors.

Organic near-infrared fluorescent dye mediated photothermal therapy (PTT) and photodynamic therapy (PDT) suffer from heat shock response, since, heat shock proteins (HSPs) are overexpressed and can repair the proteins damaged by PTT and PDT. Starvation therapy by glucose oxide (GOx) can inhibit the heat shock response by limiting the energy supply. However, the delivery of sufficient and active GOx remains a challenge. To solve this problem, we utilize liposomes as drug carriers and prepare GOx loaded liposome (GOx@Lipo) with a high drug loading content (12.0%) and high enzymatic activity. The successful delivery of GOx shows excellent inhibition of HSPs and enhances PTT and PDT. Additionally, we apply the same liposome formulation to load near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbo cyanine iodide (DiR) and prepare DiR contained liposomes (DiR@Lipo) for PTT and PDT. The liposomal formulation substantially enhances the PTT and PDT properties of DiR as well as the cellular uptake and tumor accumulation. Finally, the combination therapy shows excellent tumor inhibition on 4T1 tumor-bearing mice. Interestingly, we also find that the starvation therapy can efficiently inhibit tumor metastasis, which is probably due to the immunogenic effect. Our work presents a biocompatible and effective carrier for the combination of starvation therapy and phototherapy, emphasizing the importance of auxiliary starvation therapy against tumor metastasis and offering important guidance for clinical PTT and PDT.

Insight into the spatial interaction of D-π-A bridge derived cyanines and nitroreductase for fluorescent cancer hypoxia detection.

Nitroreductase (NTR) detection in tumor is critical because NTR level is correlated with hypoxia degree and cancer prognosis. With the feature of high sensitivity and selectivity, fluorescence organic probes for NTR detection exhibited a promising future for tumor hypoxia detection. However, the discovery and design of such probes have been impeded due to the lack of the understanding of spatial match and mismatch of these probes with NTR. Here, we have developed two new nitrophenyl-functionalized trimethincyanine (Cy3) probes with para- or meta- positions of nitro-group in phenyl ring. Para-nitrophenyl substituted Cy3 (pNP-Cy3) exhibited a remarkable response to NTR (20-fold fluorescence enhancement) with good selectivity and sensitivity. Experimental and theoretical analysis verified that the substituent position of nitro group on phenyl ring of dyes altered the spatial arrangement of nitro-substituent group, thereby modulated the spatial match and mismatch between Cy3 dyes and binding domain of NTR, and consequently led to a different fluorescent turn-on response. In tumor-bearing mice model, hypoxia status of A549 xenografted tumor of mice was successfully delineated by using pNP-Cy3. These results may provide a clue for designing new cyanine-derived NTR probe to monitor NTR-overexpressed hypoxia cancer cells.

Bioorthogonally activatable cyanine dye with torsion-induced disaggregation for in vivo tumor imaging.

Advancement of bioorthogonal chemistry in molecular optical imaging lies in expanding the repertoire of fluorophores that can undergo fluorescence signal changes upon bioorthogonal ligation. However, most available bioorthogonally activatable fluorophores only emit shallow tissue-penetrating visible light via an intramolecular charge transfer mechanism. Herein, we report a serendipitous "torsion-induced disaggregation (TIDA)" phenomenon in the design of near-infrared (NIR) tetrazine (Tz)-based cyanine probe. The TIDA of the cyanine is triggered upon Tz-transcyclooctene ligation, converting its heptamethine chain from S-trans to S-cis conformation. Thus, after bioorthogonal reaction, the tendency of the resulting cyanine towards aggregation is reduced, leading to TIDA-induced fluorescence enhancement response. This Tz-cyanine probe sensitively delineates the tumor in living mice as early as 5 min post intravenous injection. As such, this work discovers a design mechanism for the construction of bioorthogonally activatable NIR fluorophores and opens up opportunities to further exploit bioorthogonal chemistry in in vivo imaging.

Albumin-based fluorescence resonance energy transfer nanoprobes for multileveled tumor tissue imaging and dye release imaging.

Tumor tissue imaging and drug release imaging are both crucial for tumor imaging and image-guided drug delivery. It is urgent to develop a multileveled tumor imaging platform to realize the multiple imaging applications. In this work, we synthesized an albumin-based fluorescence resonance energy transfer (FRET) probe Cy5/7@HSA NPs containing two near-infrared cyanine dyes (CyBI5 and CyBI7) with high FRET efficiency (97 %). Excellent brightness and efficient FRET inside Cy5/7@HSA NPs enabled high-sensitive cell imaging and tumor imaging. This unique nanoprobe imaged 4T1 tumor-bearing mice with high sensitivity (TBR = 5.2) at 24 h post-injection and the dyes penetrated the tumor interior around 4 h post-injection. The release of dyes from nanoprobes was also tracked. This result shows the strong potential of this albumin-based FRET nanoprobe as multileveled tumor imaging platform for in vivo tumor imaging, drug delivery and image-guided surgery.

An activatable liposomal fluorescence probe based on fluorescence resonance energy transfer and aggregation induced emission effect for sensitive tumor imaging.

Liposomes are of great interest and importance in tumor imaging, since they can greatly improve the imaging sensitivity and specificity by increasing the accumulation of contrast agents. Still, most liposome-based probes have high background signals during blood circulation, which limits enhancement of S/B ratio and tumor imaging sensitivity. To enhance the S/B ratio of tumor imaging, we construct a fluorescence resonance energy transfer (FRET) and aggregation induced emission (AIE) based liposomal fluorescence probe TPE/BHQ-lipo with excellent FRET effect (99 %) and great fluorescence enhancement upon liposome rupture (120-fold) as well as efficient fluorescence recovery in tumor cell imaging. Finally, we used the TPE/BHQ-lipo to image 4T1 tumor upon intravenous injection of liposomes and the group showed enhanced signal to background ratio of 4.1, compared to 1.8 from control AIE-based liposomal group (TPE-lipo). Our work offers an excellent FRET and AIE-based liposomal probe for high-sensitive tumor imaging.

AIF1 was identified as an up-regulated gene contributing to CSFV Shimen infection in porcine alveolar macrophage 3D4/21 cells.

Classical swine fever (CSF) is a disease that is characterized by diffuse hemorrhaging, high fever, and high mortality rates. The pro-inflammatory characteristics of allograft inflammatory factor 1 (AIF1) have been well documented; however, insufficient attention has been given to porcine AIF1. In the present study, AIF1 was identified as a key player contributing to CSFV Shimen infection in porcine alveolar macrophage (PAM) 3D4/21 cell line. Our evaluation showed that AIF1 mRNA and protein are expressed at a time-dependent high level in CSFV Shimen-infected PAM 3D4/21 cells. The transcription and translation of IL6 were also significantly upregulated in infected PAM 3D4/21 cells. By utilizing overexpression RNAs approach, we showed that the cellular AIF1 induced an increased IL6 in PAM 3D4/21 cells. Furthermore, silencing of AIF1 suppressed CSFV Shimen-induced IL6 production in PAM 3D4/21 cells and also inhibited CSFV replication, whereas overexpression of recombinant AIF1 was beneficial for the replication of CSFV Shimen and promoting IL6 production in CSFV Shimen-infected PAM 3D4/21 cells. It is suggested CSFV Shimen induced IL6 in PAM 3D4/21 cells via AIF1 activation, which help clarify the AIF1-related inflammatory processes that occur on CSFV Shimen infected macrophages.

Rhodol-based fluorescent probes for the detection of fluoride ion and its application in water, tea and live animal imaging.

Herein, we presented two novel turn-on colorimetric and fluorescent probes based on a F- triggered SiO bond cleavage reaction, which displayed several desired properties for the quantitative detection for F-, such as high specificity, rapid response time (within 3 min) and naked-eye visualization. The fluorescence intensity at 574 nm (absorbance at 544 nm) of the solution was found to increase linearly with the concentration of F- (0.00-30.0 µM) with the detection limit was estimated to be 0.47 µM/0.48 µM. Based on these excellent optical properties, the probes were employed to monitor F- in real water samples and tea samples with satisfactory. Furthermore, it was successfully applied for fluorescent imaging of F- in living nude mice, suggesting that it could be used as a powerful tool to predict and explore the biological functions of F- in physiological and pathological processes.

Liposome-based probes for molecular imaging: from basic research to the bedside.

Molecular imaging is very important in disease diagnosis and prognosis. Liposomes are excellent carriers for different types of molecular imaging probes. In this work, we summarize current developments in liposome-based probes used for molecular imaging and their applications in image-guided drug delivery and tumour surgery, including computed tomography (CT), ultrasound imaging (USI), magnetic resonance imaging (MRI), positron emission tomography (PET), fluorescence imaging (FLI) and photoacoustic imaging (PAI). We also summarized liposome-based multimodal imaging probes and new targeting strategies for liposomes. This work will offer guidance for the design of liposome-based imaging probes for future clinical applications.

In Vivo and in Situ Activated Aggregation-Induced Emission Probes for Sensitive Tumor Imaging Using Tetraphenylethene-Functionalized Trimethincyanines-Encapsulated Liposomes.

The design and exploration of fluorescent probes with high-sensitivity and low-background are essential for noninvasive optical molecular imaging. The in vivo and in situ activated aggregation-induced emission (AIE) probes were found to be ideal for achieving higher signal-to-background ratios for tumor detections. We herein developed novel tetraphenylethene-encapsulated liposomes (TPE-LPs) constructed by loading TPE-trimethincyanine into liposomes for the first time, and the probes were applied to tumor bioimaging in vivo. TPE-functionalized trimethincyanines were synthesized with a new and efficient one-pot reaction. In TPE-LPs, TPE-functionalized bicarboxylic acids benzoindole trimethinecyanine (TPE-BICOOH) fluorophores were found to be well dispersed in lipid bilayers (with non-restricted rotation) during the blood circulation, and then aggregated (with restriction of intramolecular rotation) upon liposome rupture in the tumor tissue, achieving a low-background and high-target signal for tumor imaging. The in situ activated AIE probes not only had great accumulation at the tumor site after intravenous injection in 4T1 tumor-bearing mice but also demonstrated excellent signal-to-background ratios, as well as low cytotoxicity and excellent biocompatibility. The proposed strategy is believed to be a simple and powerful tool for the sensitive detection of tumors.