Fluorescence Biosensing Based on Bifurcated DNA Scaffold-Aggregated Ag Nanocluster via Responsive Conformation Switch of Quasi-Molecular Beacon.

To address the limitations of typical hairpin-structural molecular beacons, exploring the ability of a quasi-molecular beacon (qMB) to create label-free fluorescence biosensors is intriguing and remains a challenge. Herein, we propose the first example of modular qMB with the feature of a stimulation-responsive conformation switch to develop an aggregated Ag nanocluster (aAgNC) in a bifurcated DNA scaffold for fluorescently sensing a specific initiator (I*). This qMB was well designed to program four functional modules: I*-recognizable element adopting metastable stem-loop bihairpin structure and two DNA splits (exposed C3GT4 and locked C4AC4T) of aAgNC template that is separated by a tunable hairpin spacer for the customized combination of selective recognition and signaling readout. When presenting I* in an assay route, the specific hybridization induces the directional disassembly of the bihairpin unit, on which the qMB is configurationally switched to liberate the locked split. Thus, the bifurcated parent template pair of C3GT4/C4AC4T is proximal, affording in situ nucleation and clustering of emissive aAgNC. By collecting the fluorescence signal, the quantitative detection of I* is achieved. Benefiting from the ingenious programming of qMB, the recognizing and signaling integration actuates the construction of a facile and convenient fluorescent biosensor featuring rapid reaction kinetics, a wide linear range, high sensitivity, and specificity. This would provide a new paradigm to exploit versatile qMB-based biosensing platforms via stimulation-responsive conformation switches for developing various DNA-scaffolded Ag clusters.

Stimulus-Responsive Four-Stranded DNA Nanoring Assembly to Host Multiple Nanosilver Clusters for Cooperatively Enhanced Fluorescence Biosensing.

Exploring the ability of four-stranded DNA nanorings (fsDNRs) to host multiple nanosilver clusters (NAgCs) for cooperatively amplifiable fluorescence biosensing to a specific initiator (tI*) is fascinating. By designing three DNA single strands and three analogous stem-loop hairpins, we developed a functional fsDNR through sequential cross-opening and overlapped hybridization. Note that a substrate strand (SS) was programmed with six modules: two severed splits (sT and sT') of NAgCs template, two sequestered segments by a middle unpaired spacer, and a partition for tI*-recognizable displacement, while sT and sT' were also tethered in two ends of three hairpins. At first, a triple dsDNA complex with stimulus-responsiveness was formed to guide the specific binding to tI*, while the exposed toehold of the SS activated the forward cascade hybridization of three hairpins, until the ring closure in the tailored self-assembly pathway for forming the fsDNR. The resulting four duplexes forced each pair of sT/sT' to be merged as the parent template in four nicks, guiding the preferential synthesis of four clusters in the shared fsDNR, thereby cooperatively amplifying the green fluorescence signal for sensitive assay of tI*. Meanwhile, the topological conformation of fsDNR can be stabilized by the as-formed cluster adducts to rivet the pair of two splits in the nicks. Benefitting from the self-enhanced effect of multiple emitters, this label-free fluorescent sensing strategy features simplicity, rapidity, and high on-off contrast, without involving complicated nucleic acid amplifiers.

Dual-specificity phosphatase 1 inhibits Singapore grouper iridovirus replication via regulating apoptosis in Epinephelus coioides.

The dual-specificity phosphatase (DUSP) family plays key roles in the maintenance of cellular homeostasis and apoptosis etc. In this study, the DUSP member DUSP1 of Epinephelus coioides was characterized: the length was 2371 bp including 281 bp 5' UTR, 911 bp 3' UTR, and a 1125 bp open reading frame encoding 374 amino acids. E. coioides DUSP1 has two conserved domains, a ROHD and DSPc along with a p38 MAPK phosphorylation site, localized at Ser308. E. coioides DUSP1 mRNA can be detected in all of the tissues examined, and the subcellular localization showed that DUSP1 was mainly distributed in the nucleus. Singapore grouper iridovirus (SGIV) infection could induce the differential expression of E. coioides DUSP1. Overexpression of DUSP1 could inhibit SGIV-induced cytopathic effect (CPE), the expressions of SGIV key genes, and the viral titers. Overexpression of DUSP1 could also regulate SGIV-induced apoptosis, and the expression of apoptosis-related factor caspase 3. The results would be helpful to further study the role of DUSP1 in viral infection.

Age, growth, reproduction and status of resource development of Ptychidio jordani, a critically endangered freshwater fish in the Hongshui River, China.

The age, growth, reproduction and resource development status of Ptychidio jordani, as a critically endangered freshwater fish in the Hongshui River, China, was studied in this work. A total of 525 specimens were collected monthly using the cages and gillnets from October 2021 to September 2022 in the Hongshui River. The scale was used for age determination, and the maximum age for both female and male was estimated to be 5 years and 3 years, respectively. Female and male P. jordani showed different growth patterns, which were expressed as Lt  = 261.3 (1-e-0.4885(t-0.1476) ) and Lt  = 251.2 (1-e-0.4758(t+0.9643) ), respectively. The overall sex ratio was 1:0.47 (female:male). Female attained sex maturity at 2.34 years (192 mm body length). Month variation of the gonad somatic index indicated that the spawning period occurred from April to October. The absolute fecundity was estimated at 9046 ± 3434 eggs per individual, and the relative fecundity was 38.08 ± 15.77 eggs per gram. The exploitation rate of female and male was 0.233 and 0.495, which indicated that P. jordani was not overfishing. This study provided data on the key life-history traits of P. jordani, which has not been known previously and is essential for conservation strategy and policy development.

Ratiometric Fluorescence Biosensing of Tandem Biemissive Ag Clusters Boosted by Confined Catalytic DNA Assembly.

The reaction kinetics and yield of traditional DNA assembly with a low local concentration in homogeneous solution remain challenging. Exploring confined catalytic DNA assembly (CCDA) is intriguing to boost the reaction rate and efficacy for creating rapid and sensitive biosensing platforms. A rolling circle amplification (RCA) product containing multiple tandem repeats is a natural scaffold capable of guiding the periodic assembly of customized functional probes at precise sites. Here, we present a RCA-confined CCDA strategy to speed up amplifiable conversion for ratiometric fluorescent sensing of a sequence-specific inducer (I*) by using string green-/red-Ag clusters (sgAgCs and srAgCs) as two counterbalance emitters. Upon recognition of I*, CCDA events are operated by two toehold-mediated strand displacements and localized in repetitive units, thereby releasing I* for recycled signal amplification in the as-grown RCA concatemer. The local concentration of reactive species is increased to facilitate rapider dsDNA complex assembly and more efficient input-output conversion, on which the clustering template sequences of sgAgCs and srAgCs are blocked and opened, enabling srAgCs synthesis but opposite to sgAgCs. Thus, the fluorescence emission of srAgCs goes up, while sgAgCs go down. With the resultant ratio featuring inherent built-in correction, rapid, sensitive, and accurate quantification of I* at the picomolar level is achieved. Benefiting from efficient RCA confinement to enhance reaction kinetics and conversion yield, this CCDA-based strategy provides a new paradigm for developing simple and diverse biosensing methodologies.

Modular DNA Tetrahedron Nanomachine-Guided Dual-Responsive Hybridization Chain Reactions for Discernible Bivariate Assay and Cell Imaging.

Engineering of multivariate biosensing and imaging platforms involved in disease plays a vital role in effectively discerning cancer cells from normal cells and facilitating reliable targeted therapy. Multiple biomarkers such as mucin 1 (MUC1) and nucleolin are typically overexpressed in breast cancer cells compared to normal human breast epithelium cells. Motivated by this knowledge, a dual-responsive DNA tetrahedron nanomachine (drDT-NM) is constructed through immobilizing two recognition modules, MUC1 aptamer (MA) and a hairpin H1* encoding nucleolin-specific G-rich AS1411 aptamer, in two separate vertexes of a functional DT architecture tethering two localized pendants (PM and PN). When drDT-NM identifiably binds bivariate MUC1 and nucleolin, two independent hybridization chain reactions (HCRM and HCRN) as amplification modules are initiated with two sets of four functional hairpin reactants. Among them, one hairpin for HCRM is dually ended by fluorescein and quencher BHQ1 to sense MUC1. The responsiveness of nucleolin is executed by operating HCRN utilizing another two hairpins programmed with two pairs of AS1411 splits. In the shared HCRN duplex products, the parent AS1411 aptamers are cooperatively merged and folded into G-quadruplex concatemers to embed Zn-protoporphyrin IX (ZnPPIX/G4) for fluorescence signaling readout, thereby achieving a highly sensitive intracellular assay and discernible cell imaging. The tandem ZnPPIX/G4 unities also act as imaging agents and therapeutic cargos for efficient photodynamic therapy of cancer cells. Based on drDT-NM to guide bispecific HCR amplifiers for adaptive bivariate detection, we present a paradigm of exquisitely integrating modular DNA nanostructures with nonenzymatic nucleic acid amplification, thus creating a versatile biosensing platform as a promising candidate for accurate assay, discernible cell imaging, and targeted therapy.

Fluorescent Features and Applicable Biosensing of a Core-Shell Ag Nanocluster Shielded by a DNA Tetrahedral Nanocage.

The DNA frame structure as a natural shell to stably shield the sequence-templated Ag nanocluster core (csAgNC) is intriguing yet challenging for applicable fluorescence biosensing, for which the elaborate programming of a cluster scaffold inside a DNA-based cage to guide csAgNC nucleation might be crucial. Herein, we report the first design of a symmetric tetrahedral DNA nanocage (TDC) that was self-assembled in a one-pot process using a C-rich csAgNC template strand and four single strands. Inside the as-constructed soft TDC architecture, the template sequence was logically bridged from one side to another, not in the same face, thereby guiding the in situ synthesis of emissive csAgNC. Because of the strong electron-repulsive capability of the negatively charged TDC, the as-formed csAgNC displayed significantly improved fluorescence stability and superb spectral behavior. By incorporating the recognizable modules of targeted microRNAs (miRNAs) in one vertex of the TDC, an updated TDC (uTDC) biosensing platform was established via the photoinduced electron transfer effect between the emissive csAgNC reporter and hemin/G-quadruplex (hG4) conjugate. Because of the target-interrupted csAgNC switching in three states with the spatial proximity and separation to hG4, an "on-off-on" fluorescing signal response was executed, thus achieving a wide linear range to miRNAs and a limit of detection down to picomoles. Without complicated chemical modifications, this simpler and more cost-effective strategy offered accurate cell imaging of miRNAs, further suggesting possible therapeutic applications.

Koumine induces apoptosis in Cyprinus carpio liver cells by regulating JAK-STAT and p53 signaling pathways.

Koumine is an alkaloid with significant anti-anxiety, anticancer cell proliferation, and analgesic activities, and our previous studies have shown that koumine can be used as an immunostimulant in aquaculture, but the molecular mechanism of its effect remains unclear. We fed a basal diet with 0, 0.2, 2, and 20 mg/kg koumine to C. carpio for 10 weeks, and comprehensive studies of the histological and biochemical parameters and transcriptomes of the four groups were performed. Histological results indicated that the number of apoptotic cells in the liver increased with increasing koumine concentration. Compared with those of the control group, the malondialdehyde, superoxide dismutase, catalase, acid phosphatase, alkaline phosphatase, and lactate dehydrogenase levels of the treatment group increased to varying degrees. In total, 100.11 GB of clean data, 4774 DEGs, and 138 differentially expressed genes were obtained from the transcriptome data. Differentially expressed genes were classified into 187 signalling pathways, and the circadian rhythm signalling pathway, the JAK-STAT signalling pathway, the p53 signalling pathway and the PPAR signalling pathway were the top enriched pathways. The qRT-PCR results confirmed that the key genes ifnar1, socs3l, epoa, ghra, cMyc, mcl-1, shisa4, and gtse1 involved in balancing cell proliferation and apoptosis were enriched in these pathways. We discovered that the JAK-STAT and p53 pathways are important targets of koumine. Such information contributes to a better understanding of the potential mechanism by which koumine regulates hepatic immunity as well as to lays the theoretical foundation for its application.

Microbial and metabolic features in renal transplant recipients with post-transplantation diabetes mellitus.

OBJECTIVE: Post-transplantation diabetes mellitus (PTDM) is a common complication in renal transplant recipients (RTRs). Gut microbiome plays important roles in a variety of chronic metabolic diseases, but its association with the occurrence and development of PTDM is still unknown. The present study integrates the analysis of gut microbiome and metabolites to further identify the characteristics of PTDM. METHODS: A total of 100 RTRs fecal samples were collected in our study. Among them, 55 samples were submitted to Hiseq sequencing, and 100 samples were used for non-targeted metabolomics analysis. The gut microbiome and metabolomics of RTRs were comprehensively characterized. RESULTS: The species Dialister invisus was significantly associated with fasting plasma glucose (FPG). The functions of tryptophan and phenylalanine biosynthesis were enhanced in RTRs with PTDM, while the functions of fructose and butyric acid metabolism were reduced. Fecal metabolome analysis indicated that RTRs with PTDM had unique metabolite distribution characteristics, and two differentially expressed specific metabolites were significantly correlated with FPG. The correlation analysis of gut microbiome and metabolites showed that gut microbiome had an obvious effect on the metabolic characteristics of RTRs with PTDM. Moreover, the relative abundance of microbial function is associated with the expression of several specific gut microbiome and metabolites. CONCLUSIONS: Our study identified the characteristics of gut microbiome and fecal metabolites in RTRs with PTDM, and we also found two important metabolites and a bacterium were significantly associated with PTDM, which might be used as novel targets in the research field of PTDM.

The gut microbiome and metabolome in kidney transplant recipients with normal and moderately decreased kidney function.

BACKGROUND: The kidney transplant recipients (KTRs) were diagnosed with Chronic Kidney Disease after transplantation (CKD-T). CKD-T can be affected by the microbial composition and metabolites. The present study integrates the analysis of gut microbiome and metabolites to further identify the characteristics of CKD-T. METHODS: We collected 100 fecal samples of KTRs and divided them into two groups according to the stage progression of CKD-T. Among them, 55 samples were analyzed by Hiseq sequencing, and 100 samples were used for non-targeted metabolomics analysis. The gut microbiome and metabolomics of KTRs were comprehensively characterized. RESULTS: As well as significant differences in gut microbiome diversity between the CKD G1-2T group and CKD G3T group. Eight flora including Akkermansia were found to be enriched in CKD G3T group. As compared with CKD G1-2T group, the relative abundance of some amino acid metabolism, glycerophospholipid metabolism, amino acid biosynthesis, carbohydrate metabolism and purine metabolism in CKD G3T group were differential expressed significantly. In addition, fecal metabolome analysis indicated that CKD G3T group had a unique metabolite distribution characteristic. Two differentially expressed metabolites, N-acetylornithine and 5-deoxy-5'-(Methylthio) Adenosine, were highly correlated with serum creatinine, eGFR and cystatin C. The enrichment of gut microbial function in CKD-T is correlated with the expression of gut metabolites. CONCLUSION: Gut microbiome and metabolites in the progression of CKD-T display some unique distribution and expression characteristics. The composition of the gut microbiome and their metabolites appears to be different between patients with CKD G3T and those with CKD G1-2T.

Circular RNA expression profiles and CircSnd1-miR-135b/c-foxl2 axis analysis in gonadal differentiation of protogynous hermaphroditic ricefield eel Monopterus albus.

BACKGROUND: The expression and biological functions of circular RNAs (circRNAs) in reproductive organs have been extensively reported. However, it is still unclear whether circRNAs are involved in sex change. To this end, RNA sequencing (RNA-seq) was performed in gonads at 5 sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) of ricefield eel, and the expression profiles and potential functions of circRNAs were studied. RESULTS: Seven hundred twenty-one circRNAs were identified, and the expression levels of 10 circRNAs were verified by quantitative real-time PCR (qRT-PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Then, the sequence length, category, sequence composition and the relationship between the parent genes of the circRNAs were explored. A total of 147 circRNAs were differentially expressed in the sex change process, and GO and KEGG analyses revealed that some differentially expressed (such as novel_circ_0000659, novel_circ_0004005 and novel_circ_0005865) circRNAs were closely involved in sex change. Furthermore, expression pattern analysis demonstrated that both circSnd1 and foxl2 were downregulated in the process of sex change, which was contrary to mal-miR-135b. Finally, dual-luciferase reporter assay and RNA immunoprecipitation showed that circSnd1 and foxl2 can combine with mal-miR-135b and mal-miR-135c. These data revealed that circSnd1 regulates foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. CONCLUSION: Our results are the first to demonstrate that circRNAs have potential effects on sex change in ricefield eel; and circSnd1 could regulate foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. These data will be useful for enhancing our understanding of sequential hermaphroditism and sex change in ricefield eel or other teleosts.

Target Deoxyribonucleic Acid-Recycled Lighting-Up Amplifiable Ratiometric Fluorescence Biosensing of Bicolor Silver Nanoclusters Hosted in a Switchable Deoxyribonucleic Acid Construct.

Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellow- and red-silver nanocluster (yH-AgNC and rH-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins (yH and rH), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in yH is recognizable to a DNA trigger (TOC) related to SARS-CoV-2. With the help of an enhancer strand (G15E) tethering G-rich bases (G15) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with yH and rH, in which the harbored yH-AgNC close to G15 is lighted-up. Upon introducing TOC, its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing TOC-repeatable recycling amplification through competitive strand displacement. Consequently, the harbored rH-AgNC is also placed adjacent to G15 for turning on its red fluorescence, while the yH-AgNC is retainable. As demonstrated, the intensity ratio dependent on varying TOC is reliable with high sensitivity down to 0.27 pM. By lighting-up dual-cluster emitters using one G15 enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.

Target DNA-Activating Proximity-Localized Catalytic Hairpin Assembly Enables Forming Split-DNA Ag Nanoclusters for Robust and Sensitive Fluorescence Biosensing.

Proximity-localized catalytic hairpin assembly (plCHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment (T*). Using template-integrated green Ag nanoclusters (igAgNCs) as emitters, herein, we report the first design of a T*-activated plCHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of T*. To this end, the T*-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of igAgNCs are separately overhung contiguous to the paired stems of H1 and another hairpin (H2). The hybridization among H1, H2, and two single-stranded linkers (L1 and L2) allows the stable construction of 3WJA. Upon presenting the input T*, the 3WJA-localized plCHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and T* is rationally displaced and repeatably recycled, analogous to a specific catalyst, inducing more hairpin assembly events. Resultantly, the hybridized products enable the collective combination of two splits in the parent scaffold for hosting igAgNCs, outputting T*-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the plCHA operation, achieving rapider reaction kinetics via T*-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various plCHA-based circuits capable of modulating the fluorescence emission of igAgNCs for applicable biosensing and bioanalysis.

A Reciprocal-Amplifiable Fluorescence Sensing Platform via Replicated Hybridization Chain Reaction for Hosting Concatenated Multi-Ag Nanoclusters as Signal Reporter.

Exploring the replication of hybridization chain reaction HCR (rHCR) for reciprocal amplification is intriguing in biosensing and bioanalysis. Herein, we develop a rHCR-based fluorescence platform that is manipulated by the combination of a specific DNA trigger (T) and a T-analogous amplicon (T*), thereby concatenating multi green-emissive Ag nanoclusters (mgAgNCs) for amplifiable signal readout. Four well-designed hairpins (H1 recognizing T, H2, H3, and H4) with sequential complements are executed to operate rHCR. The termini of H1/H3 are merged to hybridize an inhibiting strand (I). The parent scaffold for mgAgNCs is separated into two splits (C4AC4T and C3GT4) that are individually overhung in H2/H4. The presence of T activates the first HCR amplifier through cross-hybridization of four reactive hairpins for forming HCR duplexes. The next invasion of a complex (T*·I) drives I to hybridize the tandem repeats in H1/H3, so that the displaced T* functions as T to catalyze the second amplifier rHCR for feeding back more hairpin assemblies with rapid reaction kinetics. In the shared rHCR polymers, the parent scaffolds (C4AC4TC3GT4) in H2/H4 are collectively concatenated for the preferential clustering of mgAgNCs adducts, which cooperatively emit enormous T-responsive fluorescence signal. Because of the localization of T in HCR products, an alternative amplicon T* is introduced to drive rHCR progress via DNA strand displacement, generating more nucleating sites of emitters. Thus, the rational combination of nonenzymatic rHCR and label-free fluorescent concatemers would create a reciprocal signal amplification, achieving a simplified, rapid, and highly sensitive assay down to femtomolar concentrations.

Modulating the Fluorescence of Silver Nanoclusters Wrapped in DNA Hairpin Loops via Confined Strand Displacement and Transient Concatenate Ligation for Amplifiable Biosensing.

It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.

Molecular cloning, inducible expression and function analysis of Epinephelus coioides Sec6 response to SGIV infection.

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

Identification and characterization of lncRNAs and the interaction of lncRNA-mRNA in Epinephelus coioides induced with Singapore grouper iridovirus infection.

Singapore grouper iridovirus (SGIV) is a highly pathogenic double-stranded DNA virus, and the fatality rate of SGIV-infected grouper is more than 90%. Up to now, there is no effective methods to control the disease. Long non-coding RNAs (lncRNAs) might play an important role in individual growth and development, immune regulation and other life processes. In this study, lncRNAs were identified in Epinephelus coioides, an important economic aquaculture marine fish in China and Southeast Asia, and the regulatory relationships of lncRNAs and mRNA response to SGIV infection were analyzed. A total of 11,678 lncRNAs were identified and classified from the spleen and GS (grouper spleen) cells. 105 differentially expressed lncRNAs (DElncRNAs) were detected during SGIV infection. The lncRNAs and the regulated mRNAs were analyzed using co-expression network, lncRNA target gene annotation and GO enrichment. At 24 and 48 h after SGIV infection, 118 and 339 lncRNA-mRNA pairs in GS cells were detected, and 728 and 688 differentially expressed lncRNA-mRNA pairs in spleen were obtained, respectively. GO and KEGG were used to predict the DE lncRNAs' target genes, and deduce the DE lncRNAs-affected signaling pathways. In GS cells, lncRNAs might participate in cell part, binding and catalytic activity; and lncRNAs might be involved in immune system process and transcription factor activity in spleen. These data demonstrated that lncRNAs could regulate the expression of immune-related genes response to viral infection, and providing a new insight into understanding the complexity of immune regulatory networks mediated by lncRNAs during viral infection in teleost fish.

Epinephelus coioides PCSK9 affect the infection of SGIV by regulating the innate immune response.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1ß, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.

Expression Patterns and Gonadotropin Regulation of the TGF-ß II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus.

Bmpr2 plays a central role in the regulation of reproductive development in mammals, but its role during ovarian development in fish is still unclear. To ascertain the function of bmpr2 in ovarian development in the ricefield eel, we isolated and characterized the bmpr2 cDNA sequence; the localization of Bmpr2 protein was determined by immunohistochemical staining; and the expression patterns of bmpr2 in ovarian tissue incubated with FSH and hCG in vitro were analyzed. The full-length bmpr2 cDNA was 3311 bp, with 1061 amino acids encoded. Compared to other tissues, bmpr2 was abundantly expressed in the ovary and highly expressed in the early yolk accumulation (EV) stages of the ovary. In addition, a positive signal for Bmpr2 was detected in the cytoplasm of oocytes in primary growth (PG) and EV stages. In vitro, the expression level of gdf9, the ligand of bmpr2, in the 10 ng/mL FSH treatment group was significantly higher after incubation for 4 h than after incubation for different durations. However, bmpr2 expression in the 10 ng/mL FSH treatment group at 2 h, 4 h and 10 h was significantly lower. Importantly, the expression level of bmpr2 and gdf9 in the 100 IU/mL hCG group had similar changes that were significantly decreased at 4 h and 10 h. In summary, Bmpr2 might play a pivotal role in ovarian growth in the ricefield eel, and these results provide a better understanding of the function of bmpr2 in ovarian development and the basic data for further exploration of the regulatory mechanism of gdf9 in oocyte development.

Antibody-Responsive Ratiometric Fluorescence Biosensing of Biemissive Silver Nanoclusters Wrapped in Switchable DNA Tweezers.

Exploring the ratiometric fluorescence biosensing of DNA-templated biemissive silver nanoclusters (AgNCs) is significant in bioanalysis, yet the design of a stimuli-responsive DNA device is a challenge. Herein, using the anti-digoxin antibody (anti-Dig) with two identical binding sites as a model, a tweezer-like DNA architecture is assembled to populate fluorescent green- and red-AgNCs (g-AgNCs and r-AgNCs), aiming to produce a ratio signal via specific recognition of anti-Dig with two haptens (DigH). To this end, four DNA probes are programmed, including a reporter strand (RS) dually ended with a g-/r-AgNC template sequence, an enhancer strand (ES) tethering two same G-rich tails (G18), a capture strand (CS) labeled with DigH at two ends, and a help strand (HS). Initially, both g-AgNCs and r-AgNCs wrapped in the intact RS are nonfluorescent, whereas the base pairing between RS, ES, CS, and HS resulted in the construction of DNA mechanical tweezers with two symmetric arms hinged by a rigid "fulcrum", in which g-AgNCs are lighted up due to G18 proximity ("green-on"), and r-AgNCs away from G18 are still dark ("red-off"). When two DigHs in proximity recognize and bind anti-Dig, the conformation switch of these tweezers resultantly occurs, taking g-AgNCs away from G18 for "green-off" and bringing r-AgNCs close to G18 for "red-on". As such, the ratiometric fluorescence of r-AgNCs versus g-AgNCs is generated in response to anti-Dig, achieving reliable quantization with a limit of detection at the picomolar level. Based on the fast stimulated switch of unique DNA tweezers, our ratiometric strategy of dual-emitting AgNCs would provide a new avenue for a variety of bioassays.