Proteome Fishing for CRISPR/Cas12a-Based Orthogonal Multiplex Aptamer Sensing.

Detection of serum protein biomarkers is extremely challenging owing to the superior complexity of serum. Here, we report a method of proteome fishing from the serum. It uses a magnetic nanoparticle-protein corona and a multiplexed aptamer panel, which we incubated with the nanoparticle-protein corona for biomarker recognition. To transfer protein biomarker detection to aptamer detection, we established a CRISPR/Cas12a-based orthogonal multiplex aptamer sensing (COMPASS) platform by profiling the aptamers of protein corona with clinical nonsmall cell lung cancer (NSCLC) serum samples. Furthermore, we determined the four out of nine (FOON) panel (including HE4, NSE, AFP, and VEGF165) to be the most cost-effective and accurate panel for COMPASS in NSCLC diagnosis. The diagnostic accuracy of NSCLC by the FOON panel with internal and external cohorts was 95.56% (ROC-AUC = 99.40%) and 89.58% (ROC-AUC = 95.41%), respectively. Our developed COMPASS technology circumvents the otherwise challenging multiplexed serum protein amplification problem and avoids aptamer degradation in serum. Therefore, this novel COMPASS could lead to the development of a facile, cost-effective, intelligent, and high-throughput diagnostic platform for large-cohort cancer screening.

Immune response induced by oral administration with a Saccharomyces cerevisiae-based SARS-CoV-2 vaccine in mice.

BACKGROUND: The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need to develop safe and effective vaccines with a top priority. Multiple vaccine candidates are under development, and several vaccines are currently available. Efforts need to be undertaken to counter the threat of the global COVID-19 pandemic. RESULTS: We generated a Saccharomyces cerevisiae (S. cerevisiae)-based SARS-CoV-2 vaccine, EBY100/pYD1-RBD, in which the full-length receptor binding domain (RBD) of the spike protein of SARS-CoV-2 was expressed on the surface of yeast. Mice vaccinated orally with unadjuvanted EBY100/pYD1-RBD could produce significant humoral and mucosal responses as well as robust cellular immune responses. Notably, EBY100/pYD1-RBD elicited a mixed Th1/Th2-type cellular immune response with a Th1-biased immune response in a mouse model. CONCLUSIONS: Our findings highlight the importance of the RBD as a key target to design and develop vaccines against SARS-CoV-2 and provide evidence of oral administration of a S. cerevisiae-based SARS-CoV-2 vaccine eliciting significant immune responses. Most importantly, the S. cerevisiae surface display system can serve as a universal technology platform and be applied to develop other oral viral or bacterial vaccines.

A feasible self-assembled near-infrared fluorescence sensor for acid phosphatase detection and cell imaging.

The single signal amplification strategy is significant for detecting various disease biomarkers but is restricted by its limited accuracy. The multi-signal and multi-mode methods have overcome this deficiency. Acid phosphatase (ACP) is an important intracellular enzyme but one-step cell imaging material-based probes are scarce for ACP. Herein, we designed a one-step self-assembled polymer probe using neutral red (NR), modified-(pyridoxal-5'-phosphate (PLP)) and Eu3+. The polymer exhibited non-emission and excellent stability. Upon the catalytic hydrolysis reaction of ACP, the polymer exhibited two strong fluorescence signals at 373 nm and 613 nm and an appreciable decline of absorbance at 395 nm. The probe has excellent selectivity and higher sensitivity with a limit of detection as low as 0.02 mU mL-1. It possesses favorable biocompatibility and has been successfully used to detect and image intracellular ACP in several living cells.

A dual-signal fluorescent probe for detection of acid phosphatase.

Acid phosphatase has become a significant indicator of prognostic and medical diagnosis, and its dysfunction may lead to a series of diseases. A novel dual-signal fluorescence method for acid phosphatase detection based on europium polymer (europium-pyridine dicarboxylicacid-adenine) and pyridoxal phosphate (PLP) was proposed. PLP coordinated with europium polymer via Eu3+ and P-O bonds, and the fluorescence of europium polymer was quenched due to the photoinduced electron transfer (PET) effect between aldehyde and europium polymer. Upon addition of acid phosphatase, the PLP was transformed to phosphate (Pi) and pyridoxal (PL). The PL was released from the surface of europium polymer, and the blue emission was enhanced due to the formation of internal hemiacetal, while the fluorescence of europium polymer recovered. The blue (PL) and red emission (Eu3+) were positively correlated with acid phosphatase activity; thus the sensitive assay of acid phosphatase was effectively achieved. The two signals were applied to determine the acid phosphatase with limits of detection (LOD) of 0.04 mU/mL and 0.38 mU/mL, and the linear ranges were 0.13-5.00 mU/mL and 1.25-20.00 mU/mL, respectively. The probe can be used to trace the acid phosphatase in biological systems and holds promise for use in clinical diagnosis and early prevention.

A peptide with a cysteine terminus: probe for label-free fluorescent detection of thrombin activity.

Thrombin has been implicated in atherosclerotic disease development. However, thrombin activity detection is currently limited because of the lack of convenient fluorescent probes. We developed a label-free fluorescent method to assay thrombin activity on the basis of a designed peptide probe with a thrombin-cleavable peptide sequence and a cysteine terminus. The peptide probe can be conjugated to DNA-templated silver nanoclusters (DNA-AgNCs) through Ag-S bonding; as a result, the fluorescence of DNA-AgNCs was enhanced. As the DNA-AgNCs-peptide conjugate was adsorbed to graphene oxide (GO), the enhanced fluorescence of DNA-AgNCs was quenched. Once the peptide probe was cleaved by thrombin, the resulting release of the DNA-AgNCs from the surface of GO restored the enhanced fluorescence. Thrombin can be determined with a linear range of 0.0-50.0 nM with a detection limit of 1 nM. The thrombin-sensitive probe with a cysteine terminus may be developed into probes to detect other proteases.

TiO2nanotubes promote osteogenic differentiation of human bone marrow stem cells via epigenetic regulation ofRMRP/DLEU2/EZH2 pathway.

TiO2nanotubes (TNTs) significantly promote osteogenic differentiation and bone regeneration of cells. Nevertheless, the biological processes by which they promote osteogenesis are currently poorly understood. Long non-coding RNAs (lncRNAs) are essential for controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Epigenetic chromatin modification is one of the pathways in which lncRNAs regulate osteogenic differentiation. Here, we reported that TNTs could upregulate lncRNARMRP, and inhibition of lncRNARMRPin human BMSCs (hBMSCs) grown on TNTs could decrease runt-related transcription factor 2 (RUNX2), alkaline phosphatase, osteopontin, and osteocalcin (OCN) expression. Furthermore, we discovered that inhibiting lncRNARMRPelevated the expression of lncRNADLEU2, and lncRNADLEU2knockdown promoted osteogenic differentiation in hBMSCs. RNA immunoprecipitation experiments showed that lncRNADLEU2could interact with EZH2 to induce H3K27 methylation in the promoter regions of RUNX2 and OCN, suppressing gene expression epigenetically. According to these results, lncRNARMRPis upregulated by TNTs to promote osteogenic differentiation throughDLEU2/EZH2-mediated epigenetic modifications.

A novel H2O2 activated NIR fluorescent probe for accurately visualizing H2S fluctuation during oxidative stress.

Hydrogen sulfide (H2S) is a typical gas signal molecule that plays a vital role in various pathological courses. Despite having the accumulated knowledge on the physiological functions of H2S in many diseases, the process in the measurement of its level in mitochondria during oxidative stress is still far away from the expectation. In this regard, it is great significant to design a fluorescent probe for accurately monitoring the dynamics of H2S during oxidative stress. In this work, we firstly synthesized an oxidative stress activated fluorescence probe QM-RSH for tracking H2S level in mitochondria. It exhibited high selectivity and sensitivity for detecting H2S with lower limit of detection (LOD = 44.6 nM) in the presence of H2O2. QM-RSH can be successfully applied to accurately monitor the fluctuation of H2S level during oxidative stress without interference from other physiological processes in living cells and zebrafish. Therefore, this multifunctional probe QM-RSH has great potential as an image tool in biological research. It also provides a novel strategy for designing fluorescent probe to investigate biomolecular information and signaling pathways under specific physiological conditions.

One-pot synthesis of a peroxidase-like nanozyme and its application in visual assay for tyrosinase activity.

Both single-atom nanozymes (SAzymes) and protein-template metal nanoparticles have attracted comprehensive attention in several respects owing to their excellent catalytic performance, green facile synthesis process, and robustness. Herein, the peroxidase-like activity of single-atom copper anchored on bovine hemoglobin-template gadolinium nanoparticles (Cu,Gd@BHbFITC NPs) were successfully synthesized and two sensitive turn-on fluorescence strategies for tyrosinase (TYR) activity sensing were proposed for the first time. For strategy Ⅰ, TYR sensing was carried out from 1.00 to 7.80 U/mL with the detection limit (LOD) of 0.20 U/mL based on the fluorescence resonance energy transfer (FRET) between the fluorescein isothiocyanate (FITC) and the in situ generated polydopamine dots (PDA-dots). For strategy Ⅱ, The LOD of TYR was 0.05 U/mL with the linear range of 0.40-19.70 U/mL based on the elimination of inner-filter effect (IEF) between FITC and the reaction product (RC) of phenol and 4-Aminoantipyrine (AAP). The smartphone-assisted sensing platform was applied to construct the on-site detection of TYR with both strategies. The developed probe possessed good selectivity and was successfully utilized to TYR detection in serum samples.

A water-soluble near-infrared fluorescent probe for monitoring change of hydrogen sulfide during cell damage and repair process.

As a signal molecule involved in autophagy, hydrogen sulfide (H2S) is considered to be essential in the development and treatment of diseases. In order to clarify the complex role of H2S in organism and the participation of H2S in disease process, it is urgently needed to visualize the dynamics of H2S. In this contribution, a water-soluble near-infrared (695 nm emission) self-immolative fluorescent probe CySO3N3 was constructed for H2S detection. The ability of self-immolative strategy to detect H2S was verified to increase the metabolic capacity and reduce the toxicity of probe. This probe can not only be used to detect H2S in living cell and mice, but also shows great potential in detecting H2S changes to monitor cell self-repair during inflammation and myocardial injury.

Design strategy for an analyte-compensated fluorescent probe to reduce its toxicity.

During biological detection, the toxicity caused by probes to living organisms is neglected. In this study, an analyte-compensated fluorescent probe (NP-SN3) was constructed for the detection of H2S. Through experiments with HepG2 cells and zebrafish embryos and larvae, the NP-SN3 probe showed no significant difference in imaging performance compared with the traditional probe (NP-N3) but exhibited lower detection-induced toxicity in the imaging of liver fibrosis in activated HSC-T6 cells. During the development of zebrafish embryos and continuous administration in rats, NP-SN3 showed a lower death rate, higher hatchability and lower malformation in zebrafish embryos and milder pathological symptoms in stained rat tissues.

Oral administration of Saccharomyces cerevisiae displaying VP28-VP24 confers protection against white spot syndrome virus in shrimp.

White spot syndrome virus (WSSV) is the major pathogen that leads to severe mortalities in cultured shrimp worldwide. The envelope proteins VP28 and VP24 of WSSV are considered potential vaccine candidate antigens. In this study, we utilized a Saccharomyces cerevisiae (S. cerevisiae) surface display system to demonstrate the feasibility of this platform for developing a vaccine candidate against WSSV. EBY100/pYD1-VP28-VP24 was generated, and the fusion protein VP28-VP24 was present on the surface of S. cerevisiae. Penaeus vannamei (P. vannamei) was used as an animal model. Oral administration of EBY100/pYD1-VP28-VP24 could induce significant activities of immune-related enzymes such as superoxide dismutase (SOD) and phenoloxidase (PO). Importantly, WSSV challenge indicated that oral administration of EBY100/pYD1-VP28-VP24 could confer 100% protection with a corresponding decrease in the viral load. The collective results strongly highlight the potential of a S. cerevisiae-based oral vaccine as an efficient control strategy for combating WSSV infection in shrimp aquaculture.

High immune efficacy against different avian influenza H5N1 viruses due to oral administration of a Saccharomyces cerevisiae-based vaccine in chickens.

A safe and effective vaccine is the best way to control large-scale highly pathogenic avian influenza virus (HPAI) A (H5N1) outbreaks. Saccharomyces cerevisiae (S. cerevisiae) is an ideal mucosal delivery vector for vaccine development, and we have previously shown that conventional administration of a S. cerevisiae-based vaccine (EBY100/pYD1-HA) via injection led to protection against the homologous H5N1 virus in a mouse model. Because the diameter of S. cerevisiae is approximately 10 µm, which results in a severe inflammation by injection route, therefore, oral administration is a more suitable approach for EBY100/pYD1-HA conferring protection in poultry. We extended our work by evaluating the immunogenicity and protective efficacy of oral vaccination with EBY100/pYD1-HA in the chicken model. Oral immunization with EBY100/pYD1-HA could induce robust serum IgG, mucosal IgA and cellular immune responses. Importantly, EBY100/pYD1-HA provided protection against challenges with a homologous and a heterologous H5N1 viruses. These findings suggest that EBY100/pYD1-HA, a promising H5N1 oral vaccine candidate, can avoid potential reassortment of other avian influenza viruses in oral administration of live virus vaccines and overcome the limitations of conventional injection routes. Importantly, this platform will be able to provide opportunities for broader applications in poultry during HPAI A (H5N1) outbreaks.

A fluorescent probe for bioimaging of Hexosaminidases activity and exploration of drug-induced kidney injury in living cell.

Hexosaminidases (Hexs) as an exoglycosidase participates in the catalytic hydrolysis of non-reducing end of glycoconjugates in the biological system. The fluctuation of Hexs level could cause many hereditary neurodegenerative diseases such as Tay-Sachs and Sandhoff. The Hexs activity is significantly up-regulated in colorectal cancer and kidney injury tissue so that it is particularly important to construct a fluorescent probe with significant signal change to understand its physiological role. In this work, DyOH was selected as fluorophore scaffolds to synthesize probe Hex-1 for detection of Hexs with good water solubility, high specificity, large stokes shift and quick response. Hex-1 can sensitively detect Hexs with the low detection limit (0.025 mU mL-1) in vitro by "naked eye" due to superior spectral properties of DyOH. Furthermore, Hex-1 was not only employed for imaging Hexs in living cells with low toxicity, but also successfully applied to evaluate the fluctuation of Hexs activity during drug induced kindey injury in living HK-2 cells. These results indicated that Hex-1 could be used as a potential image tool to further explore the pathogenesis of kidney disease and cancer.

Fluorescent RGD-based pro-apoptotic peptide conjugates as mitochondria-targeting probes for enhanced anticancer activities.

We have designed 2-domain anticancer peptides with RGD-based KLAK bi-functional short motifs (linear and cyclic analogues). RGD tripeptide acts as tumor blood vessel 'homing' motif while KLAK tetrapeptide internalized in mitochondria and causes cell apoptosis. All three peptides (RGDKLAK; HM, cyclic-RGDKLAK; HMC-1, and RGD-cyclic-KLAK; HMC-2) were conjugated with fluorescein isothiocyanate isomer-I (5-FITC; F) for in-vivo and in-vitro optical imaging studies. These fluorescent-peptide (FL-peptide) analogues were analyzed to possess αvß3-integrin targeting affinity, high uptake in in-vitro cell binding assays followed by in-vivo tumor xenograft mice studies. Pharmacological profile reveals that F-HMC-1 analogue exhibited selectively and specifically higher affinity for αvß3-integrin than other analogues in U87MG cells in comparison with HeLa cells. The subcutaneous U87MG tumor xenograft mice models clearly visualized the uptake of F-HMC-1 in tumor tissue in contrast with normal tissues with tumor-to-normal tissue ratio (T/NT = 15.9 ±â€¯1.1) at 2 h post-injection. These results suggested that F-HMC-1 peptide has potential diagnostic applications for targeting αvß3-integrin assessed by optical imaging study in U87MG tumor xenograft mice models.

Label-free fluorometric detection of chymotrypsin activity using graphene oxide/nucleic-acid-stabilized silver nanoclusters hybrid materials.

Pancreatic function tests are used to determine the presence of chronic pancreatitis, particularly in the early stage of the disease. Chymotrypsin is an indicator of pancreatic function and is thus related to pancreatic diseases. A new fluorescent biosensing method for assay of chymotrypsin activity was developed using DNA (dC12)-templated silver nanoclusters and graphene oxide (GO). A peptide probe was also designed using chymotrypsin-cleavable amino acid sequence and a cysteine terminus. The peptide probe formed Ag-S bond to dC12-AgNCs to enhance the fluorescence of dC12-AgNCs. After the addition of GO, the peptide was adsorbed to the negative GO surface and the fluorescence of dC12-AgNCs was quenched by FRET. The peptide was then degraded into amino acid fragments upon addition of chymotrypsin; these fragments were released from the GO surface, and the FRET was terminated. The developed label-free method features lower cost and higher sensitivity to chymotrypsin activity assay compared with conventional fluorescence analysis. The method can be used to analyze chymotrypsin (as low as 3ng/mL, signal/noise =3) across a dynamic range of 0.0-50.0ng/mL. The proposed biosensing strategy can also be extended to other proteases by using different peptide substrates.

Thiolated DNA-templated silver nanoclusters with strong fluorescence emission and a long shelf-life.

Thiolated DNA (DNA-SH) was employed as a template in the synthesis and stabilization of AgNCs (DNA-SH-AgNCs). Resulting from the synergistic protective effect of specific Ag+-DNA interactions and Ag-S bonding, DNA-SH-AgNCs exhibited high quantum yields and resistance to oxidation.

Mimicking the Active Sites of Organophosphorus Hydrolase on the Backbone of Graphene Oxide to Destroy Nerve Agent Simulants.

Recent global military events, such as the conflict in Syria, have emphasized the need to find effective strategies to rapidly destroy organophosphorus-based nerve agents. In this work, we designed active site-engineered graphene oxide (GO) via polymerization (polymer bead-GOs) as organophosphorus hydrolase (OPH) mimetic hotspots for the rapid degradation of nerve agents. This hybrid catalyst has a high total turnover frequency value of 0.65 s-1 and good stability (94.8% activity maintained after 5 cycles). Mechanism investigations suggested that the high catalytic performance could be attributed to the synergistic effect among the clusters of imidazole and the presence of - COOH groups on the GO surface and Zn2+.