Ultrasensitive fluorescence microRNA biosensor by coupling hybridization-initiated exonuclease I protection and tyramine signal amplification.

Tyramine signal amplification (TSA) has made its mark in immunoassay due to its excellent signal amplification ability and short reaction time, but its application in nucleic acid detection is still very limited. Herein, an ultrasensitive microRNA (miRNA) biosensor by coupling hybridization-initiated exonuclease I (Exo I) protection and TSA strategy was established. Target miRNA is complementarily hybridized to the biotin-modified DNA probe to form a double strand, which protects the DNA probe from Exo I hydrolysis. Subsequently, horseradish peroxidase (HRP) is attached to the duplex via the biotin-streptavidin reaction and catalyzes the deposition of large amounts of biotin-tyramine in the presence of hydrogen peroxide (H2O2), followed by the conjugation of signal molecule streptavidin-phycoerythrin (SA-PE), which generates an intense fluorescence signal upon laser excitation. This method gave broad linearity in the range of 0.1 fM - 10 pM, yielding a detection limit as low as 74 aM. An increase in sensitivity of 4 orders of magnitude was observed compared to the miRNA detection without TSA amplification. This biosensor was successfully applied to the determination of miR-21 in breast cancer cells and human serum. By further design of specific DNA probes and coupling with the Luminex xMAP technology, it could be easily extended to multiplex miRNA assay, which possesses great application potential in clinical diagnosis.

A chemiluminescent sensor for imaging endogenous hydrogen polysulfides in a living system.

By constructing 2-(benzoylthio)benzoate and a 2-fluoro-4-nitrobenzoate structure in an adamantylidene-dioxetane system, we designed and synthesized two novel chemiluminescent probes for the detection of H2Sn from other RSS. Under the same conditions, the maximum luminescence emission intensity of the probe CL-HP2 could reach 150 times that of the probe CL-HP1, and the chemiluminescence signal still existed at low concentrations. Therefore, CL-HP2 was more suitable for H2Sn detection as a chemiluminescent probe. The probe CL-HP2 exhibited a good linear relationship with Na2S4 in a wide range (0.025-10 mM). Interestingly, a good linear relationship (R2 = 0.997) was also observed at low concentrations (0-100 µM) with a LOD as low as 0.23 µM. CL-HP2 has been effectively employed to visualize endogenous H2Sn within living cells. Moreover, it has been applied for live imaging of bacterially infected murine models and the ferroptosis process in tumor-bearing mouse models.

Radical nucleophilic substitution/cyclization: A novel strategy for selective and ultrafast fluorescence imaging of cysteine levels in ferroptosis process.

Diphenylisoindolo[2,1-a]quinoline can be used to detect cysteine among homocysteine, glutathione, and other 19 natural amino acids. Unlike other reported probes, the response mechanism involves sulfhydryl radical nucleophilic substitution and cyclization, and thus the differences in ring-formation kinetics enable high selectivity. After treated with Cys, the response process was completed rapidly and the maximum fluorescence intensity (at 496 nm) was reached extremely fast (<1 s) when excited at 380 nm in MeCN-PBS buffer (10.0 mM, pH = 7.4, 3:7 (v/v)). The quantum yield after the reaction was increased almost 7 times to be 0.02 from 0.003. Fluorescence intensity displayed a good quantitative linear relationship in the range 1-10 µM Cys with a detection limit of 270 nM. Furthermore, the probe was demonstrated for real-time monitoring of intracellular cysteine levels within HepG2 cells in ferroptosis process.

[Characterization and immunoprotective effect of SjIrV1, a 66 kDa calcium-binding protein from Schistosoma japonicum].

Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.

Cloning, expression, and preliminary characterization of the dysferlin tegument protein in Schistosoma japonicum.

The schistosomal tegument is a dynamic host-interactive layer. Proteins exposed to the host on the tegumental surface are important for completion of the parasitic lifecycle. Dysferlin is a member of the ferlin family and is involved in plasma membrane repair. Based on the results of a proteomics study of tegument surface proteins of Schistosoma japonicum in our laboratory, dysferlin was identified as a tegumental protein of S. japonicum. The gene encoding S. japonicum dysferlin (SjDF), which codes for several Ca(2+) binding sites, was cloned, expressed in Escherichia coli, and characterized. Western blot analysis revealed that recombinant SjDF had good immunogenicity. Real-time RT-PCR analysis showed that SjDF was upregulated mainly in adult worms and the transcription level in 42-day-old female worms was significantly higher than that in males. Immunofluorescence analysis revealed that SjDF was mainly distributed in the tegument at various developmental stages. Experimental mice were treated with praziquantel and at 35days post-infection, we noted that damage to the tegument and subtegument worsened and did not recover at 36h post-treatment in the high-dose group and was accompanied by downregulation of SjDF mRNA, while the damage was less severe and recovered by this time in the low-dose group, and accompanied by upregulation of SjDF. Our results suggested that SjDF is a tegumental protein that may be important in schistosomal development and may participate in the repair process in muscle and tegument, and could present a viable vaccine candidate for schistosomiasis.

Characterization Analysis of Schistosoma japonicum Plasma Membrane Repair Relative Gene Myoferlin.

Myoferlin is a member of the ferlin family of proteins, which are involved in plasma membrane repair, and has been identified as one of the tegument proteins of Schistosoma japonicum. The tegument proteins are potential candidates for vaccines and new drug targets. In this study, myoferlin of S. japonicum (SjMF) was cloned, expressed and characterized, the potential of SjMF recombinant protein (rSjMF) as a vaccine candidate was evaluated, and the effect of praziquantel on SjMF was detected by Real-time PCR. Immunofluorescence showed that this protein was mainly distributed on the surface of worms at different stages. Sequence analysis revealed that the SjMF open reading frame was conserved at all stages of the S. japonicum life cycle. And SjMF transcription was upregulated in 42-day-old worms, and was significantly higher in female worms. Western blotting revealed that rSjMF showed strong immunogenicity. The cytokine profile and IgG isotype analysis demonstrated that rSjMF plus ISA206 immunization induced a mixed T helper (Th)1/Th2 response. Purified rSjMF emulsified with ISA206 adjuvant significantly reduced worm burden from 21.8% to 23.21% and liver egg number from42.58% to 28.35%. Besides, SjMF transcription was downregulated when worms were exposed to low-dose praziquantel (PZQ) and upregulated when PZQ was degraded, accompanied by recovery of damaged tegument. When worms were exposed to high-dose PZQ, SjMF transcription was downregulated all the time and the damaged tegument did not recover. These findings indicated that SjMF is a potential vaccine against S. japonicum and provides the basis for further investigations into the biological function of SjMF.