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

Pinpoint Diagnostic Kit for Heat Stroke by Monitoring Lysosomal pH

Heat stroke is one of the most serious causes of mortality. To prevent the situation, it is fundamental to research the mechanism of heat cytotoxicity. The preliminary results revealed that heat stroke and the change of lysosome acidity had some certain correlation. To further clarify their relationship, herein, we report a highly selective and sensitive fluorescence probe (<b>NT1</b>) for turn-on sensing of the pH value. <b>NT2</b> was synthesized as control compound. Compared to <b>NT2</b>, <b>NT1</b> showed accurate lysosome target ability. In addition, the suitable p<i>K</i>_a value (5.67) allows <b>NT1</b> to response to the changes of lysosomal pH values. Most importantly, <b>NT1</b> could be used to study the correlation between the change of lysosomal pH and heat stroke. It was shown that the lysosomal pH value increasing with temperature during heat stroke. Thus, <b>NT1</b> was an excellent candidate for research of the complex biological mechanism of heat stroke

A lanthanide metal-organic framework based on a custom-designed macrocyclic ligand

<p>The lanthanum-based metal macrocyclic framework, MMCF-3, where the ligand is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetra-<i>p</i>-methylbenzoic acid, has been synthesized and characterized by powder X-ray diffraction, single crystal X-ray diffraction, and thermogravimetric analysis. MMCF-3 forms in closely packed 2-D sheets. In MMCF-3, the azamacrocyclic-based ligand coordinates tetradentate to four separate lanthanum ions via its carboxylate groups, leaving the macrocycle site unoccupied. The lanthanum ions are 10-coordinate with a distorted bi-capped square anti-prism coordination environment. The vacancy of the macrocycle site within the framework encourages the utilization of the framework as a cation receptor. The availability of these sites allows for the possibility to coordinate to newly introduced metals to produce heterometallic frameworks, which could exhibit intriguing properties.</p

Data_Sheet_2_Production and immunogenicity of a deoxyribonucleic acid Alphavirus vaccine expressing classical swine fever virus E2-Erns protein and porcine Circovirus Cap-Rep protein.pdf

Classical swine fever virus (CSFV) and porcine Circovirus type 2 (PCV2) are economically pivotal infectious disease viruses of swine. Alphaviral RNA replicon plasmids have been used as an important vector for constructing nucleic acid vaccines. Here, we aimed to construct a recombinant alphaviral plasmid vaccine pSCA1-E2-Erns-Cap-Rep for the prevention and control of CSFV and PCV2. Our results showed that the recombinant alphaviral plasmid vaccine pSCA1-E2-Erns-Cap-Rep was successfully constructed. The vaccine encoding E2 and Erns of CSFV, Cap, and Rep of PCV2 can induce E2, Erns, Cap, and Rep protein expression. ELISA analysis showed that mice-immunized pSCA1-E2-Erns-Cap-Rep plasmid vaccine produced higher anti–CSFV- and anti–PCV2-specific antibodies with dose- and time-dependent manners. Furthermore, neutralizing assays were measured using IF and ELISA methods. The results showed the production of neutralizing antibodies could neutralize CSFV (up to 210.13) and PCV2 (28.6) effectively, which exhibited the immune efficacy of the pSCA1-E2-Erns-Cap-Rep plasmid vaccine. Taken together, this pSCA1-E2-Erns-Cp-Rep plasmid vaccine could be considered a novel candidate vaccine against CSFV and PCV2.</p

Data_Sheet_1_Production and immunogenicity of a deoxyribonucleic acid Alphavirus vaccine expressing classical swine fever virus E2-Erns protein and porcine Circovirus Cap-Rep protein.pdf

Classical swine fever virus (CSFV) and porcine Circovirus type 2 (PCV2) are economically pivotal infectious disease viruses of swine. Alphaviral RNA replicon plasmids have been used as an important vector for constructing nucleic acid vaccines. Here, we aimed to construct a recombinant alphaviral plasmid vaccine pSCA1-E2-Erns-Cap-Rep for the prevention and control of CSFV and PCV2. Our results showed that the recombinant alphaviral plasmid vaccine pSCA1-E2-Erns-Cap-Rep was successfully constructed. The vaccine encoding E2 and Erns of CSFV, Cap, and Rep of PCV2 can induce E2, Erns, Cap, and Rep protein expression. ELISA analysis showed that mice-immunized pSCA1-E2-Erns-Cap-Rep plasmid vaccine produced higher anti–CSFV- and anti–PCV2-specific antibodies with dose- and time-dependent manners. Furthermore, neutralizing assays were measured using IF and ELISA methods. The results showed the production of neutralizing antibodies could neutralize CSFV (up to 210.13) and PCV2 (28.6) effectively, which exhibited the immune efficacy of the pSCA1-E2-Erns-Cap-Rep plasmid vaccine. Taken together, this pSCA1-E2-Erns-Cp-Rep plasmid vaccine could be considered a novel candidate vaccine against CSFV and PCV2.</p

Reversible Ratiometric Fluorescent Probe for Sensing Bisulfate/H₂O₂ and Its Application in Zebrafish

Herein, a novel near-infrared fluorescent probe for ratiometric detection of bisulfate was designed and developed based on a conjugation of naphthopyran-benzothiazolium system. The sensor showed excellent selectivity, high sensitivity and a rapid response toward bisulfite in aqueous solution. Upon the addition of HSO₃^–, the sensor displayed 37-fold (<i>I</i>₅₂₀/<i>I</i>₆₃₀) fluorescence intensity enhancement, accompanied by an apparent color change from violet to colorless, suggesting that the sensor can be used to detect HSO₃^– with “naked-eye”. Notably, the addition product can be applied to the design of regenerative chemodosimeters based on the H₂O₂ promoted elimination of bisulfite and recovery of probe <b>1</b>. Further cell and zebrafish imaging experiment demonstrated that the sensor could image the bisulfite/H₂O₂ redox cycle in biological system with ratiometric manners

Table6_Enhanced protein–protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.XLSX

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein–protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.</p

DataSheet1_Enhanced protein–protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.PDF

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein–protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.</p

Table5_Enhanced protein–protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.XLSX

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein–protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.</p

Table1_Enhanced protein–protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment.XLSX

Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein–protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.</p