Transcriptome-wide study revealed m6A regulation of embryonic muscle development in Dingan goose (Anser cygnoides orientalis).

BACKGROUND: The number of myofiber is determined during the embryonic stage and does not increase during the postnatal period for birds, including goose. Thus, muscle production of adult goose is pre-determined during embryogenesis. Previous studies show N6-methyladenosine (m6A) is an important regulator for skeletal muscle development of birds and miRNAs play as a co-regulator for the skeletal muscle development in birds. Herein, we sequenced m6A and miRNA transcriptomes to investigate the profiles of m6A and their potential mechanism of regulating breast muscle development in Dingan Goose. RESULTS: We selected embryonic 21th day (E21) and embryonic 30th day (E30) to investigate the roles of transcriptome-wide m6A modification combining with mRNAs and miRNAs in goose breast muscle development. In this study, m6A peaks were mainly enriched in coding sequence (CDS) and start codon and397 genes were identified as differentially methylated genes (DMGs). GO and KEGG analysis showed that DMGs were highly related to cellular and metabolic process and that most DMGs were enriched in muscle-related pathways including Wnt signaling pathway, mTOR signaling and FoxO signaling pathway. Interestingly, a negative correlation between m6A methylation level and mRNA abundance was found through the analysis of m6A-RNA and RNA-seq data. Besides, we found 26 muscle-related genes in 397 DMGs. We also detected 228 differentially expressed miRNAs (DEMs), and further found 329 genes shared by the target genes of DEMs and DMGs (m6A-miRNA-genes), suggesting a tightly relationship between DEMs and DMGs. Among the m6A-miRNA-genes, we found 10 genes are related to breast muscle development. We further picked out an m6A-miRNA-gene, PDK3, from the 10 genes to visualize it and the result showed differentially methylated peaks on the mRNA transcript consistent with our m6A-seq results. CONCLUSION: GO and KEGG of DMGs between E21 and E30 showed most DMGs were muscle-related. In total, 228 DEMs were found, and the majority of DMGs were overlapped with the targets of DEGs. The differentially methylated peaks along with an m6A-miRNA-gene, PDK3, showed the similar results with m6A-seq results. Taken together, the results presented here provide a reference for further investigation of embryonic skeletal muscle development mechanism in goose.

Ag@Au Core⁻Shell Porous Nanocages with Outstanding SERS Activity for Highly Sensitive SERS Immunoassay.

A highly sensitive immunoassay of biomarkers has been achieved using 4-mercaptobenzoic acid-labeled Ag@Au core⁻shell porous nanocage tags and α-fetoprotein immuno-sensing chips. The Ag@Au porous nanocages were uniquely synthesized by using an Ag core as a self-sacrificial template and reducing agent, where the slow reaction process led to the formation of a porous Au layer. The size of the remaining Ag core and surface roughness of the Au shell were controlled by adjusting the chloroauric acid concentration. The porous cage exhibited excellent surface-enhanced Raman spectroscopy (SERS) activity, presumably due to a synergetic interaction between newly generated hot spots in the rough Au shell and the retained SERS activity of the Ag core. Using α-fetoprotein as a model analyte for immunoassay, the SERS signal had a wide linear range of 0.20 ng mL-1 to 500.0 ng mL-1 with a detection limit of 0.12 ng mL-1. Without the need of further signal amplification, the as-prepared Ag@Au bimetallic nanocages can be directly used for highly sensitive SERS assays of other biomarkers in biomedical research, diagnostics, etc.

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing.

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.

m6A and miRNA jointly regulate the development of breast muscles in duck embryonic stages.

N6-methyladenosine (m6A) is an abundant internal mRNA modification and plays a crucial regulatory role in animal growth and development. In recent years, m6A modification has been found to play a key role in skeletal muscles. However, whether m6A modification contributes to embryonic breast muscle development of Pekin ducks has not been explored. To explore the role of m6A in embryonic breast muscle development of ducks, we performed m6A sequencing and miRNA sequencing for the breast muscle of duck embryos on the 19th (E19) and 27th (E27) days. A total of 12,717 m6A peaks were identified at E19, representing a total of 7,438 gene transcripts. A total of 14,703 m6A peaks were identified, which overlapped with the transcripts of 7,753 genes at E27. Comparing E19 and E27, we identified 2,347 differential m6A peaks, which overlapped with 1,605 m6A-modified genes (MMGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that MMGs were enriched in multiple muscle- or fat-related pathways, which was also revealed from our analysis of differentially expressed genes (DEGs). Conjoint analysis of m6A-seq and RNA-seq data showed that pathways related to ß-oxidation of fatty acids and skeletal muscle development were significantly enriched, suggesting that m6A modification is involved in the regulation of fat deposition and skeletal muscle development. There were 90 upregulated and 102 downregulated miRNAs identified between the E19 and E27 stages. Through overlapping analysis of genes shared by MMGs and DEGs and the targets of differentially expressed miRNAs (DEMs), we identified six m6A-mRNA-regulated miRNAs. Finally, we found that m6A modification can regulate fat deposition and skeletal muscle development. In conclusion, our results suggest that m6A modification is a key regulator for embryonic breast muscle development and fat deposition of ducks by affecting expressions of mRNAs and miRNAs. This is the first study to comprehensively characterize the m6A patterns in the duck transcriptome. These data provide a solid basis for future work aimed at determining the potential functional roles of m6A modification in adipose deposition and muscle growth.

Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags.

A hemin bio-bar-coded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin bio-bar-code. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an electrochemiluminescent (ECL) immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs), and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon-nanotube-enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in bio-bar-code electrocatalyzed reduction of dissolved oxygen. Using α-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL(-1) to 1 ng mL(-1) and a detection limit of 1.0 fg mL(-1). The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the bio-bar-code. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.

Using Extract From the Stems and Leaves of Yizhi (Alpiniae oxyphyllae) as Feed Additive Increases Meat Quality and Intestinal Health in Ducks.

Yizhi (Alpiniae Oxyphyllae, A. oxyphylla) has been widely used as an important traditional Chinese medicinal herb for centuries. Existing studies have shown that A. oxyphylla has numerous benefits in human and animal health. We hypothesized that extract from the stems and leaves of A. oxyphylla (AOE) as a feed additive may have positive effects on animal health and products. Thus, this study was conducted to evaluate the effects of AOE as a feed additive on growth performance, serum biochemical parameters, intestinal morphology, microbial composition, and meat quality in Jiaji ducks. A total of 240 Jiaji ducks of 42 days old (1675.8 ± 44.2 g, male: female ratio = 1:1) were blocked based on body weight and randomly allocated into four dietary treatments with three replicates that each had 20 duck individuals. The dietary treatments included: basal diet, control group (CK); basal diet supplementation with 30 mg/kg (Y1), 80 mg/kg (Y2), and 130 mg/kg (Y3) AOE, respectively, and lasted for 49 days. The results showed that average daily feed intake from day 42 to day 60 was decreased with the increasing level of AOE (P < 0.05). Compared with the CK group, the groups with AOE supplementation decreased serum LDL-C level (P < 0.05), the addition of 30 mg/kg AOE increased total amino acids, essential amino acids, branched-chain amino acids, nonessential amino acids, and umami taste amino acids (P < 0.05), but decreased selenium and zinc concentrations in breast muscle (P < 0.05). In addition, the supplementation of 30 or 130 mg/kg AOE significantly increased jejunal villus height (P < 0.05) and tended to increase the ratio of villus height to crypt depth in the jejunum (P = 0.092) compared to the CK group. Moreover, the addition of 30 mg/kg AOE showed a higher abundance of genus unclassified Bacteroidales and genus unclassified Ruminococcaceae than the CK group (P < 0.05). Therefore, dietary supplementation with 30 mg/kg AOE increased meat nutrition profile and flavor through promoting amino acid contents in breast muscle, as well as maintained intestine integrity and modulated the microbial composition. In conclusion, AOE as an antibiotic alternative displayed potential in maintaining intestinal health and improving meat quality.

Dietary Supplementation With Didancao (Elephantopus scaber L.) Improves Meat Quality and Intestinal Development in Jiaji Ducks.

Didancao (Elephantopus scaber L.) has been used as a traditional herbal medicine and has exhibited a beneficial role in animal health. This study aimed to investigate the effects of dietary supplementation with E. scaber on growth performance, meat quality, intestinal morphology, and microbiota composition in ducks. A total of 480 Jiaji ducks (42 days old, male:female ratio = 1:1) were randomly assigned to one of four treatments. There were six replicates per treatment, with 20 ducks per replicate. The ducks in the control group (Con) were fed a basal diet; the three experimental groups were fed a basal diet supplementation with 30 (T1), 80 (T2), and 130 mg/kg (T3) of E. scaber. After a 48-day period of supplementation, growth performance, meat quality, intestinal morphology, and microbiota composition were evaluated. The results showed that no differences were observed in the final body weight, average daily feed intake, and average daily gain among the four groups. Compared with that in the Con group, the feed conversion in the T1 and T2 groups was increased significantly; the T2 group was shown to decrease the concentration of alanine aminotransferase in serum; the T3 group was lower than the Con group in the concentration of aspartate aminotransferase and was higher than the Con group in the concentration of high-density lipoprotein-cholesterol. The highest concentration of creatinine was observed in the T1 group. The T2 group was higher than the Con group in the contents of Phe, Ala, Gly, Glu, Arg, Lys, Tyr, Leu, Ser, Thr, Asp, and total amino acids in the breast muscle. Moreover, the T2 group was higher than the Con group in the contents of meat C18:2n-6 and polyunsaturated fatty acid. The concentration of inosinic acid in the T1, T2, and T3 groups was significantly higher than that in the Con group. However, the Con group was higher than the T2 or T3 group in the Zn content. The T2 group was lower than the Con group in the jejunal crypt depth. The T3 group was higher than the Con group in the ileal villus height and the ratio of villus height to crypt depth. In addition, the T3 group had a trend to significantly increase the abundance of Fusobacteria. Compared with the Con group, the T1 and T2 groups displayed a higher abundance of Subdoligranulum. Collectively, dietary supplementation with 80 mg/kg of E. scaber improves meat quality and intestinal development in ducks.

Genetic characteristics of Jiaji Duck by whole genome re-sequencing.

Jiaji Duck (JJ) is a Muscovy duck species that possesses many superior characteristics, and it has become an important genetic resource in China. However, to date, its genetic characteristics and genetic relationship with other duck breeds have not been explored yet, which greatly limits the utilization of JJ. In the present study, we investigated the genome sequences of 15 individual ducks representing five different duck populations, including JJ, French Muscovy duck (FF), mallard (YD), hong duck (HD) and Beijing duck (BD). Moreover, we investigated the characteristics of JJ-specific single nucleotide polymorphisms (SNPs) and compared the genome sequences of JJ vs. YD and JJ vs. BD using integrated strategies, including mutation detection, selective screening, and Gene Ontology (GO) analysis. More than 40 Gb of clean data were obtained for each population (mean coverage of 13.46 Gb per individual). A total number of 22,481,367 SNPs and 4,156,829 small insertion-deletions (Indels) were identified for the five duck populations, which could be used as molecular markers in breeding and utilization of JJ. Moreover, we identified 1,447,932 JJ-specific SNPs, and found that genes covering at least one JJ-specific SNP mainly involved in protein phosphorylation and dephosphorylation, as well as DNA modification. Phylogenetic tree and principal components analysis (PCA) revealed that the genetic relationship of JJ was closest to FF, while it was farthest to BD. A total of 120 and 111 genes were identified as positive selection genes for JJ vs. BD and JJ vs. YD, respectively. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the positive selection genes for JJ vs. BD ducks mainly involved in pigmentation, muscle contraction and stretch, gland secretion, and immunology, while the positive selection genes obtained from JJ vs. YD ducks mainly involved in embryo development, muscle contraction and stretch, and gland secretion. Taken together, our findings enabled us to better understand the characteristics of JJ and provided a molecular basis for the breeding and hybrid utilization of JJ in the future.

Highly sensitive and selective electrochemical detection of Hg(2+) through surface-initiated enzymatic polymerization.

A Hg(2+) electrochemical biosensor is developed by integrating thymine-Hg(2+)-thymine (T-Hg(2+)-T) base pairs for the high selectivity with surface-initiated enzymatic polymerization (SIEP) for signal amplification. The fabrication begins with the covalent conjugation of capture DNA probe labeled with thiol at its 3'terminal onto the gold electrode. The presence of Hg(2+) leads to DNA hybridization, in which complementary DNA was captured onto the biosensor surface, which subsequently catalyzed the addition of deoxynucleotides (dNTP) containing biotinlated 2'-deoxyadenosine 5'-triphosphate (biotin-dATP) by terminal deoxynucleotidyl transferase (TdT). The binding between biotin and strepavidin leads to the attachment of a large number of strepavidin functionalized silver nanoparticles (strepavidin-AgNPs), which could generate electrochemical stripping signal of silver to monitor the concentration of Hg(2+) in KCl solution. Through utilizing the T-Hg(2+)-T selectivity and SIEP amplification, this assay method can detect aqueous Hg(2+) with a wide linear range from 0.05 nM to 100 nM and a detection limit of 0.024 nM. The application of this sensor in the analysis of drinking water demonstrates that the proposed method works well for real samples.

Cascade signal amplification for electrochemical immunosensing by integrating biobarcode probes, surface-initiated enzymatic polymerization and silver nanoparticle deposition.

A cascade signal amplification strategy through combining surface-initiated enzymatic polymerization (SIEP) and the subsequent deposition of strepavidin functionalized silver nanoparticles (AgNPs) was proposed. The first step of constructing the electrochemical immunosensor involves covalently immobilizing capture antibody on a chitosan modified glass carbon electrode, which then catalyzes DNA addition of deoxynucleotides (dNTP) at the 3'-OH group by terminal deoxynucleotidyl transferase (TdT), leading to the formation of long single-stranded DNAs labeled with numerous biotins. Following the deposition of numerous strepavidin functionalized AgNPs on those long DNA chains, electrochemical stripping signal of silver was used to monitor the immunoreaction in KCl solution. Using α-fetoprotein as a model analyte, this amplification strategy could detect fetoprotein down to 0.046pg/mL with a wide linear range from 0.1pg/mL to 1.0ng/mL. The achieved high sensitivity and good reproducibility suggest that this cascade signal amplification strategy has great potential for detecting biological samples and possibly clinical application.

Nanogold/mesoporous carbon foam-mediated silver enhancement for graphene-enhanced electrochemical immunosensing of carcinoembryonic antigen.

Nanogold functionalized mesoporous carbon foam (Au/MCF) coupling with a signal amplification by C-Au synergistic silver enhancement was designed for sensitive electrochemical immunosensing of biomarker. The Au/MCF was prepared by in situ growth of nanogold on carboxylated MCF and used as a tracing tag to label signal antibody via the inherent interaction between protein and nanogold. The immunosensor was prepared by covalently immobilizing capture antibody on an electrochemically reduced graphene oxide/chitosan film modified glassy carbon electrode. Through a sandwich-type immunoreaction, Au/MCF tags were captured on the immunoconjugates to induce a silver deposition process. The electrochemical stripping signal of the deposited silver was used to monitor the immunoreaction. The Au/MCF-mediated silver enhancement along with the graphene-promoted electron transfer led to high detection sensitivity of carcinoembryonic antigen. Under optimal conditions, the proposed immunoassay method showed wide linear range from 0.05 pg mL(-1) to 1 ng mL(-1) and a detection limit down to 0.024 pg mL(-1). The newly designed amplification strategy holds great potential for ultrasensitive electrochemical biosensing of other analytes.

The selective formation of graphene ranging from two-dimensional sheets to three-dimensional mesoporous nanospheres.

This research presents a template-free solvothermal method which offers selective preparation of graphene ranging from two-dimensional sheets to 3-dimensional nanospheres. The thus prepared nanospheres have size-defined mesopores with a huge surface area and, after doping with nitrogen, exhibited stronger electrocatalytic activity toward oxygen reduction than commercial Pt/C catalysts.

Signal amplification for electrochemical immunosensing by in situ assembly of host-guest linked gold nanorod superstructure on immunocomplex.

An amplification strategy for signal tracing was developed by introducing a host-guest binding reaction into the assembly process of gold nanorods (AuNRs) superstructure. The amplification pathway firstly used a thio-ß-cyclodextrin (SH-ß-CD) functionalized gold nanoparticles to label signal antibody, and then in situ assembled multi-layer SH-ß-CD end-functionalized AuNRs to sandwich immunocomplex on immunosensor surface by using 4,4,4,4-(21H, 23H-porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) as a bridge to achieve simple and convenient host-guest reaction. The built end-to-end AuNRs superstructure showed excellent performance for the signal amplification in connection with the electrochemical biosensing by preoxidation and then voltammetric analysis of gold element. Using α-fetoprotein as an analyte, the immunosensor was constructed by covalently binding capture antibody to chitosan-carbon nanotubes-poly(diallyldimethylammonium chloride) modified electrode. The superstructure rich in AuNRs brought an enhanced detection sensitivity of protein, which could detect α-fetoprotein in a linear range from 0.5 pg mL(-1) to 0.5 ng mL(-1) with a detection limit down to 0.032 pg mL(-1). The immunoassay exhibited good stability and acceptable reproducibility and accuracy. The in situ superstructure assembly could be extended to other labeled recognition systems, providing a promising novel avenue for signal amplification and potential applications in bioanalysis and clinical diagnostics.

Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid) complex as a carrier.

A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.

Signal amplification by adsorption-induced catalytic reduction of dissolved oxygen on nitrogen-doped carbon nanotubes for electrochemiluminescent immunoassay.

A signal amplification strategy was proposed for quantum dot-based electrochemiluminescence by an adsorption-induced catalytic reduction of dissolved oxygen at the sidewall of nitrogen-doped carbon nanotubes, which led to a 'signal-on' sandwich immunoassay with a linear range of 6 orders of magnitude.