Targeted Extracellular Protein Degradation by Dendronized DNA Chimeras.

Extracellular soluble proteins are key agents in the development of various diseases. However, strategies to remove therapeutically relevant extracellular targets are still scarce. Here, we establish dendronized DNA chimera (DENTAC) as an efficient approach for targeted degradation of the extracellular protein of interest (ePOI). DENTAC consists of a DNA dendron against cell-surface scavenger receptors (SRs), a protein ligand, and a connecting linker, which harnesses SRs as a lysosome-trafficking receptor to mediate the lysosomal degradation of the ePOI. We interrogate and optimize structure-activity relationships of DENTAC. Using neutravidin as a model ePOI, we show that both branch number and DNA length in the DNA dendron are important determinants for efficient lysosomal delivery and degradation of the protein. We demonstrate three branches and 10 nucleotide-length polythymidine as the optimal DNA dendron components to construct DENTAC. We further exemplify the anticancer application of DENTAC by targeting matrix metalloproteinase-9 (MMP-9), where we find linker property as another factor important for DENTAC performance. We reveal that MMP-9-targeting DENTAC effectively restrain cancer cell proliferation, migration, and invasion. This study thus provides a potent strategy to delete extracellular proteins that are commonly difficult to target.

Age-related secretion of grancalcin by macrophages induces skeletal stem/progenitor cell senescence during fracture healing.

Skeletal stem/progenitor cell (SSPC) senescence is a major cause of decreased bone regenerative potential with aging, but the causes of SSPC senescence remain unclear. In this study, we revealed that macrophages in calluses secrete prosenescent factors, including grancalcin (GCA), during aging, which triggers SSPC senescence and impairs fracture healing. Local injection of human rGCA in young mice induced SSPC senescence and delayed fracture repair. Genetic deletion of Gca in monocytes/macrophages was sufficient to rejuvenate fracture repair in aged mice and alleviate SSPC senescence. Mechanistically, GCA binds to the plexin-B2 receptor and activates Arg2-mediated mitochondrial dysfunction, resulting in cellular senescence. Depletion of Plxnb2 in SSPCs impaired fracture healing. Administration of GCA-neutralizing antibody enhanced fracture healing in aged mice. Thus, our study revealed that senescent macrophages within calluses secrete GCA to trigger SSPC secondary senescence, and GCA neutralization represents a promising therapy for nonunion or delayed union in elderly individuals.

Building a highly efficient Streptomyces super-chassis for secondary metabolite production by reprogramming naturally-evolved multifaceted shifts.

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae. We initially identified and characterized a set of multiplex targets (afsQ1, bldD, bldA, and miaA) that contribute to SM (jadomycin) production when subjected to ethanol shock. Subsequently, we developed an ethanol-induced orthogonal amplification system (EOAS), enabling dynamic and precise control over targets. Ultimately, we integrated these multiplex targets into functional units governed by the EOAS, generating a universal and plug-and-play Streptomyces super-chassis. In addition to achieving the unprecedented titer and yield of jadomycin B, we also evidenced the potential of this super-chassis for production of diverse heterologous SMs, including antibiotic oxytetracycline, anticancer drug doxorubicins, agricultural herbicide thaxtomin A, and plant growth regulator guvermectin, all with the yields of >10 mg/g glucose in a simple mineral medium. Given that the production of SMs all required complexed medium and the cognate yields were usually much lower, our achievement of using a universal super-chassis and engineering pipeline in a simple mineral medium is promising for convenient heterologous production of SMs.

Self-Assembled Nano-PROTAC Enables Near-Infrared Photodynamic Proteolysis for Cancer Therapy.

Confining the protein degradation activity of proteolysis-targeting chimera (PROTAC) to cancer lesions ensures precision treatment. However, it still remains challenging to precisely control PROTAC function in tumor regions in vivo. We herein describe a near-infrared (NIR) photoactivatable nano-PROTAC (NAP) for remote-controllable proteolysis in tumor-bearing mice. NAP is formed by molecular self-assembly from an amphiphilic conjugate of PROTAC linked with an NIR photosensitizer through a singlet oxygen (1O2)-cleavable linker. The activity of PROTAC is initially silenced but can be remotely switched on upon NIR photoirradiation to generate 1O2 by the photosensitizer. We demonstrated that NAP enabled tumor-specific degradation of bromodomain-containing protein 4 (BRD4) in an NIR light-instructed manner. This in combination with photodynamic therapy (PDT) elicited an effective suppression of tumor growth. This work thus presents a novel approach for spatiotemporal control over targeted protein degradation by PROTAC.

α-Mangostin Exhibits a Therapeutic Effect on Spinal Cystic Echinococcosis by Affecting Glutamine Metabolism.

Spinal cystic echinococcosis, a severely neglected, rare disease, is characterized by high morbidity, disability, and mortality in prevalent regions. Due to the high-risk nature of surgical treatment and the ineffectiveness of conventional drugs, there is an unmet need for novel safe and effective drugs for the treatment of this disease. In this study, we examined the therapeutic effects of α-mangostin for spinal cystic echinococcosis, and explored its potential pharmacological mechanism. The repurposed drug exhibited a potent in vitro protoscolicidal effect and significantly inhibited the evolution of larval encystation. Moreover, it demonstrated a remarkable anti-spinal cystic echinococcosis effect in gerbil models. Mechanistically, we found that α-mangostin intervention led to intracellular depolarization of mitochondrial membrane potential and reactive oxygen species generation. In addition, we observed elevated expression of autophagic proteins, aggregation of autophagic lysosomes, activated autophagic flux, and disrupted larval microstructure in protoscoleces. Further metabolite profiling showed that glutamine was imperative for autophagic activation and anti-echinococcal effects mediated by α-mangostin. These results suggest that α-mangostin is a potentially valuable therapeutic option against spinal cystic echinococcosis through its effect on glutamine metabolism.

Bone marrow mesenchymal stem cells derived exosomal miRNAs can modulate diabetic bone-fat imbalance.

Background: Diabetes mellitus is a chronic metabolic disease with systemic complications. Patient with diabetes have increased risks of bone fracture. Previous studies report that diabetes could affect bone metabolism, however, the underlying mechanism is still unclear. Methods: We isolated exosomes secreted by bone marrow mesenchymal stem cells of normal and diabetic mice and test their effects on osteogenesis and adipogenesis. Then we screened the differential microRNAs by high-throughput sequencing and explored the function of key microRNA in vitro and in vivo. Results: We find that lower bone mass and higher marrow fat accumulation, also called bone-fat imbalance, exists in diabetic mouse model. Exosomes secreted by normal bone marrow mesenchymal stem cells (BMSCs-Exos) enhanced osteogenesis and suppressed adipogenesis, while these effects were diminished in diabetic BMSCs-Exos. miR-221, as one of the highly expressed miRNAs within diabetic BMSCs-Exos, showed abilities of suppressing osteogenesis and promoting adipogenesis both in vitro and in vivo. Elevation of miR-221 level in normal BMSCs-Exos impairs the ability of regulating osteogenesis and adipogenesis. Intriguingly, using the aptamer delivery system, delivery normal BMSCs-Exos specifically to BMSCs increased bone mass, reduced marrow fat accumulation, and promoted bone regeneration in diabetic mice. Conclusion: We demonstrate that BMSCs derived exosomal miR-221 is a key regulator of diabetic osteoporosis, which may represent a potential therapeutic target for diabetes-related skeletal disorders.

Inhibition of SMAD3 effectively reduces ADAMTS-5 expression in the early stages of osteoarthritis.

OBJECTIVE: As one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-ß signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown. METHODS: Sprague-Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3. RESULTS: In vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete. CONCLUSION: The results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.

Dendronized DNA Chimeras Harness Scavenger Receptors To Degrade Cell Membrane Proteins.

Bispecific chimeras bridging cell membrane proteins with lysosome-trafficking receptors (LTRs) provide an effective therapeutic approach through lysosomal degradation of disease-relevant targets. Here, we report a novel dendronized DNA chimera (DENTAC) strategy that uses a dendritic DNA to engage cell surface scavenger receptors (SRs) as LTR. Using bioorthogonal strain-promoted alkyne-azide cycloaddition to conjugate the dendritic DNA with protein binder, the resulting DENTAC is able to traffic the protein target into the lysosome for elimination. We demonstrated the utility of DENTAC by degrading oncogenic membrane nucleolin (NCL) and epidermal growth factor receptor (EGFR). The anti-cancer application of NCL-targeting DENTAC was validated in a mouse xenograft model of lung cancer. This work thus presents a new avenue for rapid development of potent degraders against membrane proteins, with also broad research and therapeutic prospects.

A Small-Molecule Probe with a Dual Function of miRNA Inhibition and Target identification.

By virtue of their key roles in pathologies, miRNAs represent a promising class of therapeutic targets. While high-fidelity small-molecule modulators of miRNAs can be identified via high-throughput screening using cellular reporter systems, their modes of action are elusive due to the lack of proper tools. Here, we report a small-molecule probe, 1 a, that is capable of elucidating its biological target along miRNA inhibition. Derived from norathyriol, a nature product, 1 a possessed a bioorthogonal alkyne moiety for subsequent labeling via copper-catalyzed azide-alkyne cycloaddition chemistry. We demonstrated that 1 a inhibited a panel of different miRNAs by blocking their loading onto argonaute 2 (AGO2), which is the key protein responsible for miRNA function. With the alkyne handle, we successfully identified AGO2 as an intracellular target of 1 a. Therefore, this work presents a novel small-molecule tool for suppressing and probing miRNA regulatory pathways.

Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach.

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.

Status and prospect of novel treatment options toward alveolar and cystic echinococcosis.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are the two most important global parasitic infectious diseases caused by species of Echinococcus granulosus and E. multilocularis, respectively. Although numerous trials have been performed in search of novel therapeutic options to curb the neglected zoonosis, no other nonsurgical options are currently available to replace the licensed anti echinococcal drugs albendazole (ABZ) and mebendazole (MBZ). A safer and more effective treatment plan for echinococcosis is therefore urgently needed to compensate for this therapeutic shortfall. Here, we present a review of the literature for state-of-the-art valuable anti-parasitic compounds and novel strategies that have proved effective against CE and AE, which includes details about the pharmaceutical type, practical approach, experimental plan, model application and protoscolecidal effects in vivo and in vitro. The content includes the current application of traditional clinical chemicals, the preparation of new compounds with various drug loadings, repurposing findings, combined programs, the prospects for Chinese herbal medicines, non-drug administrations and the exploration of target inhibitors based on open-source information for parasitic genes. Next the conventional experimental projects and pharmacodynamic evaluation methods are systematically summarized and evaluated. The demands to optimize the construction of the echinococcosis model and improve the dynamic monitoring method in vivo are also discussed given the shortcomings of in vivo models and monitoring methods.

In Vitro and In Vivo Efficacy of Albendazole Chitosan Microspheres with Intensity-Modulated Radiation Therapy in the Treatment of Spinal Echinococcosis.

Currently, there is a lack of clinically safe and effective treatment for spinal cystic echinococcosis (CE). Recent studies have shown that albendazole chitosan microspheres (ABZ-CS-MPs) and irradiation have certain anti-abdominal echinococcosis ability, so this study aims to compare the in vitro and in vivo therapeutic effects of ABZ-CS-MPs, intensity-modulated radiation therapy (IMRT), and combination therapy on spinal echinococcosis. First, protoscoleces were processed by different treatments to evaluate their respective antiechinococcosis effects by monitoring the viability change of protoscoleces. Then, the apoptotic status of protoscoleces was evaluated by detecting the changes of mitochondrial membrane potential, the expression of apoptosis proteins, and the ultrastructural alterations of protoscoleces. After that, we constructed a gerbil model of spinal CE and further applied B-ultrasound and magnetic resonance imaging (MRI) technology to assess the size of hydatid in vivo. Finally, the cysts were obtained and weighed to compare the inhibition rate in different groups. The combined therapy increased protoscoleces mortality to over 90% after 18 days, which showed the highest scolicidal effect. Moreover, confocal imaging, expression of apoptotic proteins, and ultrastructural changes of protoscoleces showed the highest apoptotic rate in this group. In vivo, the combination treatment also exhibited the highest cyst inhibition rate (61.4%). In conclusion, our results showed that ABZ-CS-MPs combined with IMRT could be a new treatment option for spinal CE. We also provided a method to evaluate the growth and metastasis of hydatid in animals with B-ultrasound and MRI technologies.

Investigation of the Behavior and Mechanism of Action of Ether-Based Polycarboxylate Superplasticizers Absorption on Large Bibulous Stone Powder.

The aim of this paper is to study the adsorption behavior of polycarboxylate superplasticizers (PCE) on coarse aggregates with a property of high water consumption (above 2%). The coarse aggregates were ground into a powder to create large bibulous stone powder, and it was observed that significant amounts of the ether-based PCE were absorbed onto large bibulous stone powder. The adsorption rate immediately reached a maximum after 5 min and then gradually decreased until an equilibrium absorption was established after 30 min. Zeta potential, infrared spectroscopy, and thermogravimetric analysis (TGA) measurements confirmed that the polycarboxylate superplasticizer adsorbed on the surface of the stone powder. Hydrodynamic diameter measurements showed that the polycarboxylate superplasticizer molecules were smaller than pore size, and the surface area and pore volume were reduced by the polymer incorporation in the pores.

Polyketide pesticides from actinomycetes.

Natural product derived pesticides have increased in popularity worldwide because of their high efficacy, eco-friendly nature and favorable safety profile. The development of polyketide pesticides from actinomycetes reflects this increase in popularity in the past decades. These pesticides, which include avermectins, spinosyns, polynactins, tetramycin and their analogues, have been successfully applied in crop protection. Moreover, the advance of biotechnology has led to continuous improvement in the discovery and production processes. In this review, we summarize these polyketide pesticides, their activities and provide insight into their development. We also discuss engineering strategies and the current status of industrial production for these pesticides. Given that actinomycetes are known to produce a wide range of bioactive secondary metabolites, the description of pesticide development and high yield strain improvement presented herein will facilitate further development of these valuable polyketide pesticides from actinomycetes.

Osteosarcoma Cell-Derived Exosomal miR-1307 Promotes Tumorgenesis via Targeting AGAP1.

The occurrence of osteosarcoma (OS) is associated with abnormal expression of many microRNAs (miRNAs). Exosomal miRNAs get much more attentions in intracellular communications. miR-1307 has been studied in many cancers, but its effects in OS have not been studied. We hypothesized that OS-derived exosomal miR-1307 regulates OS tumorigenesis. First, we found OS cell-derived exosomes (Exos) significantly promoted the proliferation, migration, and invasion of OS cells. Secondly, we found miR-1307 was highly expressed in OS cell-derived exosomes (OS-Exos), human OS tissues, and OS cell lines. Then, OS-Exos were extracted after OS cells were cultured and transfected with miR-1307 inhibitor, and the level of miR-1307 in OS-Exos was significantly reduced. When the level of miR-1307 in OS-Exos was significantly reduced, the effects of OS-Exos on migration, invasion, and proliferation of OS cells were also significantly weakened. Furthermore, using TargetScan, miRDB, and mirDIP databases, we identified that AGAP1 was a target gene of miR-1307. Overexpression of miR-1307 could inhibit the expression of AGAP1 gene. We also found AGAP1 was lower expressed in human OS tissues and OS cell lines. Luciferase gene indicated that miR-1307 directly bound the 3'-UTR of AGAP1. miR-1307 was negatively correlated with AGAP1 in clinical study. miR-1307 could significantly promote the proliferation, migration, and invasion of OS cells. In addition, upregulation of AGAP1 could significantly inhibit the role of miR-1307 in OS. In conclusion, our study suggests that OS cell-derived exosomal miR-1307 promotes the proliferation, migration, and invasion of OS cells via targeting AGAP1, and miR-1307-AGAP1 axis may play an important role in the future treatment of OS.

A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes.

Rapid and sensitive detection of various analytes is in high demand. Apart from its application in genome editing, CRISPR-Cas also shows promises in nucleic acid detection applications. To further exploit the potential of CRISPR-Cas for detection of diverse analytes, we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12a. We demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background, i.e., the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 µmol/L, respectively, highlighting the advantages of simplicity, sensitivity, short detection time, and low cost compared with the state-of-the-art biosensing approaches. Altogether, this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes.

Mechanism of the Regulatory Effect of Overexpression of circMTO1 on Proliferation and Apoptosis of Hepatoma Cells via miR-9-5p/NOX4 Axis.

PURPOSE: To investigate the potential role of the circMTO1/miR-9-5p/NOX4 axis in liver cancer. MATERIALS AND METHODS: Human genome-wide circrna microarray V2 was used for analyzing the expression profile of circRNAs in human tissue samples. The TargetScan database was used to predict target genes. Gene overexpression and silencing in hepatoma cell lines were achieved by transfecting the cells with suitable constructs. Quantitative real time PCR and Western blotting were used to analyze gene and protein expression levels. CCK-8 analysis was performed to detect cell proliferation and the transwell assay for analyzing cell migration. Annexin V-FITC/PI staining and immunohistochemistry were respectively used to detect apoptosis and protein expression. RESULTS: CircMTO1 were down-regulated in the liver cancer tissues and cell lines compared to their respective normal controls. TargetScan database screening and dual luciferase assay revealed that circMTO1 was a molecular sponge of miR-9-5p, and NOX4 was the target gene of miR-9-5p. Overexpression of circMTO1 and NOX4 inhibited proliferation and migration of hepatoma cells, while the overexpression of miR-9-5p had the opposite effects. In contrast, overexpression of circMTO1 and NOX4 promoted apoptosis, while that of miR-9-5p decreased the cell apoptosis rates. CONCLUSION: Overexpression of CircMTO1 acts as tumor suppressor in liver cancer by sponging miR-9-5p, which upregulates NOX4.

Discrimination of live and dead cells with two different sets of signals and unique application in vivo imaging.

Discriminating living and dead cells is of great significance for the study of apoptosis. In this work, we have developed a unique fluorescent probe (RPIC) for discriminating live and dead cells with duel-channel fluorescence imaging under double excitation and double emission mode. Dead cells treated with RPIC shows weak fluorescence signals in red channel, however, strong fluorescence signals are appeared in red channel in live cells. Weak and strong green fluorescence signals present at live cells and dead cells, respectively. Moreover, RPIC can detect successfully apoptosis of cancer cells. For in-vivo imaging, RPIC can discriminate successfully live and dead zebrafish with the same method. More interestingly, it is found that RPIC possesses the ability of discriminating normal mice and tumor mice.