Search

MiR-202-5p is identified as the maker microRNA (miRNA) of germplasm in fish. However, the role of miR-202-5p in gonadal development is still unclear. Here we investigated the expression patterns of miR-202-5p in Japanese flounder (Paralichthys olivaceus). Real-time PCR displayed that miR-202-5p was specifically expressed in bisexual gonads, and its transcription in the testis was relatively abundant than that in the ovary. Moreover, it was gradually increased in testis from stage II to stage IV. Fluorescent in situ hybridization (FISH) illustrated that miR-202-5p was co-localized with vasa (a marker gene of germ cells) in testis, and it was predominately expressed in spermatogonia and spermatocytes, implying that miR-202-5p might participate in spermatogenesis. Considering the important role of retinoic acid (RA) on regulating meiosis during spermatogenesis, we treated Japanese flounders by injecting all-trans retinoic acid (ATRA). MiR-202-5p in testis was shown to be up-regulated, and ccnd1, a key cell cycle regulator, was found to be down-regulated after ATRA treatment. Furthermore, ccnd1 was demonstrated to be a direct regulatory target of miR-202-5p using Dual-luciferase assay. In the primary testis cell from Japanese flounder, we further confirmed that ccnd1 was down-regulated by miR-202-5p. The results suggest that miR-202-5p exerts a crucial role in gonadal development by inhibiting ccnd1 in Japanese flounder.

As a key member of the miRNA family, the role and target gene of the let-7 family in the gonad of Japanese flounder (Paralichthys olivaceus) is unclear. Chromobox homolog 2 (CBX2) is one of the core components of the polycomb group complex (PcG) and significantly influences gonadal development. The deletion of CBX2 can lead to sex reversal in mammals. Therefore, exploring the relationship between the let-7 family and cbx2 is crucial to clarify the role played by the let-7 family in the gonad of Japanese flounder. We predicted and verified the target interaction between the let-7 family and cbx2. The results showed that cbx2 was a direct target of let-7d, let-7e, let-7g, let-7j, and let-7b. Among them, let-7d, let-7e, let-7g, and let-7j exhibited an extremely significant targeting relationship with cbx2 (p 0.001). Taking let-7g as an example, we further investigated the regulatory role between let-7g and cbx2 in the gonad by miRNA overexpression and inhibition experiments in primary testis cells. The results revealed that let-7g could negatively regulate cbx2 at the level of primary testis cells. And the expression of sf1 (steroidogenic factor 1) was also significantly decreased after the interference of cbx2 siRNA. This suggests that the let-7 family may be involved in the Japanese flounder gonadal development via targeting cbx2.

As a regulatory molecule of post-transcriptional gene expression, microRNA (miRNA) is a class of endogenous, non-coding small molecule RNAs. MiRNA detection is essential for biochemical research and clinical diagnostics but challenging due to its low abundance, small size, and sequence similarities. In this chapter, traditional methods of detecting miRNA like polymerase chain reaction (PCR), DNA microarray, and northern blotting are introduced briefly. These approaches are usually used to detect miRNA in vitro. Some novel strategies for sensing miRNAs in vivo, including hybridization probe assays, strand-displacement reaction (SDR), entropy-driven DNA catalysis (EDC), catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), DNAzyme-mediated assays, and CRISPR-mediated assays are elaborated in detail. This chapter describes the principles and designs of these detection technologies and discusses their advantages as well as their shortcomings, providing guidelines for the further development of more sensitive and selective miRNA sensing strategies in vivo.

Chromobox homolog 2 (CBX2), a key member of the polycomb group (PcG) family, is essential for gonadal development in mammals. A functional deficiency or genetic mutation in cbx2 can lead to sex reversal in mice and humans. However, little is known about the function of cbx2 in gonadal development in fish. In this study, the cbx2 gene was identified in medaka, which is a model species for the study of gonadal development in fish. Transcription of cbx2 was abundant in the gonads, with testicular levels relatively higher than ovarian levels. In situ hybridization (ISH) revealed that cbx2 mRNA was predominately localized in spermatogonia and spermatocytes, and was also observed in oocytes at stages I, II, and III. Furthermore, cbx2 and vasa (a marker gene) were co-localized in germ cells by fluorescent in situ hybridization (FISH). After cbx2 knockdown in the gonads by RNA interference (RNAi), the sex-related genes, including sox9 and foxl2, were influenced. These results suggest that cbx2 not only plays a positive role in spermatogenesis and oogenesis but is also involved in gonadal differentiation through regulating the expression levels of sex-related genes in fish.

The effective identification and quantification of pathogenic bacteria is essential for addressing serious public health issues. Here, we demonstrate a simple and universal impedimetric sensor for highly selective and sensitive detection of pathogenic bacteria based on the recognition by a bacteria-imprinted polypyrrole (BIP) film. The BIP film was facilely prepared via one-step electro-polymerization followed by in situ removal of the bacterial template. The film structure is novel with noncavity-like imprinted sites situated at the surface of the polypyrrole (PPy) matrix, which are more accessible for the target bacteria and should enhance the mass transfer and the binding kinetics. A limit of quantitation low to 103 CFU/mL was achieved within 1 h for the detection of E. coli O157:H7, which is comparable to the antibody-based assays. Moreover, the sensor displayed remarkable selectivity, especially regarding the specific identification of bacterial serotypes. When employed to analyze E. coli O157:H7 in real drinking water, apple juice, and milk samples, the sensor showed recoveries from 96.0% to 107.9% with relative standard derivations (RSDs) less than 4%. The BIP-based sensing strategy provides a universal approach for specific, selective, and rapid detection of pathogenic bacteria. As compared to conventional biosensors based on biomolecular recognition, this sensor shows clear advantages including easy-of-preparation, robustness, and low cost, which may hold great potential in fields of food/public safety monitoring.

The sensitive detection of Pb2+ is of significant importance for food safety, environmental monitoring, and human health care. To this end, a novel fluorescent biosensor, DNAzyme-functionalized R-phycoerythrin (DNAzyme-R-PE), was presented for Pb2+ analysis. The biosensor was prepared via the immobilization of Iowa Black&reg, FQ-modified DNAzyme&ndash, substrate complex onto the surface of SPDP-functionalized R-PE. The biosensor produced a minimal fluorescence signal in the absence of Pb2+. However, Pb2+ recognition can induce the cleavage of substrate, resulting in a fluorescence restoration of R-PE. The fluorescence changes were used to measure sensitively Pb2+ and the limit of detection was 0.16 nM with a linear range from 0.5&ndash, 75 nM. Furthermore, the proposed biosensor showed excellent selectivity towards Pb2+ even in the presence of other metal ions interferences and was demonstrated to successfully determine Pb2+ in spiked lake water samples.

A non-invasive fluorescent probe, morpholino molecular beacon (MO-MB), was designed for RNA visualization in vivo. Featuring negligible toxicity, stability, and high target specificity in living embryos, MO-MB is superior to conventional probes and has the potential for specific RNA visualization in basic biological and clinical research.

Development of simple, rapid and highly sensitive platforms for heavy metal ions on-site visual monitoring is extremely challenging and appealing for addressing environmental pollution and public health issues. Herein, we present an extended GR-5 DNAzyme-based autonomous isothermal cascade machine (EGD-AICM) for visible detection of trace Pb2+ in a single tube system. A home-designed GR-5 complex is assembled from an extended GR-5 DNAzyme (GR-5E, integrating GR-5 DNAzyme with a DNA track) and its corresponding extended substrate (GR-5S). Once Pb2+ triggers GR-5S cleavage, the machine automatically initiates isothermal strand displacement amplification to synthesize numerous EAD2 DNA copies. The released copies further integrate with hemin to form horseradish peroxidase (HRP)-mimicking DNAzymes, which efficiently promotes H2O2-mediated oxidation of ABTS and generates amplified colorimetric signals. EGD-AICM enables label-free and one-tube determination of Pb2+ with a short assay time (∼50 min), excellent selectivity and high sensitivity (LOD = 35.25 pM). In addition, the proposed EGD-AICM has been employed to measure Pb2+ in real water and shrimp samples with satisfactory accuracy and precision. Such a straightforward and economical EGD-AICM platform demonstrates great potential for routinely screening metal ions in environmental monitoring, food safety assessment, and public safety fields.

A simple, fast, fluorescent biosensor for DNA detection was developed based on a poly (diallyldimethylammonium chloride) (PDADMAC) -mediated R-phycoerythrin (R-PE) /DNA hybrid system. In the presence of PDADMAC, R-PE can efficiently absorb BHQ2-labeled single-stranded DNA (BHQ2-ssDNA) form stable R-PE/ssDNA complex and its fluorescence is significantly quenched by the labeled BHQ2 of the ssDNA through fluorescence resonance energy transfer (FRET) between R-PE and BHQ2. Upon the addition of complementary target DNA, BHQ2-ssDNA was released from the R-PE surface due to the formation of rigid double-strand DNA and the fluorescence of R-PE, correspondingly, is recovered. The proposed fluorescent biosensor presents high selectivity and sensitivity with a detection limit of 0.17 nM. This approach provides a promising platform for nucleic acid detection and molecular sensing.

A colorimetric nanoprobe-mercury-specific DNA-functionalized gold nanoparticles (Au-MSD) was developed for sensing Hg(2+). The new mercury-sensing concept relies on measuring changes in the inhibition of "non-crosslinking" aggregation of Au-MSD-induced by the folding of mercury-specific DNA strand through the thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination. In the absence of Hg(2+), a high concentration of MgCl(2) (50 mM) results in a rapid aggregation of Au-MSD because of the removal of charge repulsion. When Hg(2+) is present, the particles remain stable due to the folding of MSD functionalized on the particle surface. The assay enables the colorimetric detection of Hg(2+) in the concentration range of 0.1-10 μM Hg(2+) ions with a detection limit of 60 nM, and allows for the selective discrimination of Hg(2+) ions from the other competitive metal ions. Toward the goal for practical applications, the sensor was further evaluated by monitoring Hg(2+) in fish tissue samples.

A new electrochemical aptamer molecular beacon (MB) was designed by the carminic acid (CA) covalently linking at the each end of a special single-stranded stem-loop shaped oligonucleotide and named as CAs-MB. CA is an electrochemically active molecule and two CA molecules at the ends of molecular beacon stem were closed enough to associate each other to be as CA dimer. The dimer was electrochemically inactive. It separated into two CA monomers and produced the electrochemical signal while CAs-MB combined with target. In this protocol, the detection strategy of CAs-MB for thrombin is based on electrochemical active–inactive switching between monomer and dimer forms of CA. In order to enhance the electrochemical signal, magnetic nanobeads (MNB) was applied by connecting CAs-MB with MNB through a duplex of DNA. With the magnetic enrichment, the detection limit for thrombin reached to 42.4 pM. The experiment results showed that this type of electrochemical active–inactive switching aptamer molecular beacon allowed the direct detection of target proteins in the solution with no requirement of removing uncombined CAs-MB. Besides, CAs-MB/MNB can be easily regenerated by using 2 M NaCl solution to cleave the thrombin from the aptasensor.

Polythymine oligonucleotide (PTO)-modified gold electrode (PTO/Au) was developed for selective and sensitive Hg2+ detection in aqueous solutions. This modified electrode was prepared by self-assembly of thiolated polythymine oligonucleotide (5′-SH-T15-3′) on the gold electrode via AuS bonds, and then the surface was passivated with 1-mercaptohexanol solution. The proposed electrode utilizes the specific binding interactions between Hg2+ and thymine to selectively capture Hg2+, thereby reducing the interference from coexistent ions. After exchanging the medium, electrochemical reduction at −0.2 V for 60 s, voltammetric determination was performed by differential pulse voltammetry using 10 mM HEPES; pH 7.2, 1 M NaClO4 as supporting electrolyte. This electrode showed increasing voltammetric response in the range of 0.21 nM Hg2+, with a relative standard deviation of 5.32% and a practical detection limit of 60 pM. Compared with the conventional stripping approach, the modified electrode exhibits good sensitivity and selectivity, and is expected to be a new type of green electrode.

We report a new kind of electrochemical molecular beacon, termed “electrochemically active–inactive switching molecular beacon”, for direct detection of DNA in homogenous solution. The electrochemical molecular beacon consists of a stable stem-loop oligonucleotide carrying two carminic acid moieties (acting as electrochemical reporter) attached at its termini. In a close form, the electrochemical signal is quenched because two carminic acid moieties are close enough to associate into dimer. In the presence of the complementary DNA target, the electrochemical molecular beacon undergoes a conformational transformation from closed (hairpin) to open (linear) structure, which is associated with an increase in electrochemical signal. We found that the electrochemical molecular beacon is as effective as conventional molecular beacon in signaling the presence of complementary target and discriminating targets that differ by a single nucleotide. The proposed electrochemical molecular beacon has a great potential for investigating the interactions of DNA-protein and developing electrochemical real-time polymerase chain reaction. Keywords: DNA, Electrochemical molecular beacon, Molecular recognition, Nucleotides, Probe