Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.Several recent reports have established the feasibility of protein arrays for a variety of applications [1][2][3][4][5][6][7] . To meet the emerging needs of expression proteomics, however, such arrays must yield highly multiplexed, sensitive, quantitative, and reproducible measurements of protein levels. It is also desirable that assays on these arrays utilize small sample volumes and be compatible with hardware and software used by the DNA microarray industry. Microarrays of ordered immobilized capture antibodies and attendant sandwich immunoassays are a straightforward, near-term approach for highly parallel measurement of protein levels. Polyclonal or monoclonal antibodies for several thousand proteins are available, and are being supplemented with affinity probes generated by phage and ribosomal display, affibodies, and aptamers [8][9][10][11] . Indeed, recent studies have described sensitive 12,13 RCA is a useful alternative for on-chip signal amplification [15][16][17] .It permits sensitive and highly multiplexed assays on microarrays because RCA-amplified signals remain localized at the microarray spot ( Fig. 1) 16,17 . When utilized on microarrays of printed proteins, RCA has been shown to allow detection of protein analytes with zeptomole sensitivity and broad dynamic range 16,18,19 . In the present study, we establish the utility of RCA for highthroughput analysis of protein expression on microarrays, providing assays that are highly sensitive, quantitative, and reproducible. We describe highly multiplexed, microarray immunoassays with four steps: sample application and...

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/mL range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.

The limited knowledge of genomic noncoding and regulatory regions has restricted our ability to decipher the genetic mechanisms underlying complex traits in pigs. In this study, we characterized the spatiotemporal landscape of putative enhancers and promoters and their target genes by combining H3K27ac-targeted ChIP-Seq and RNA-Seq in fetal (prenatal days 74-75) and adult (postnatal days 132-150) tissues (brain, liver, heart, muscle and small intestine) sampled from Asian aboriginal Bama Xiang and European highly selected Large White pigs of both sexes. We identified 101,290 H3K27ac peaks, marking 18,521 promoters and 82,769 enhancers, including peaks that were active across all tissues and developmental stages (which could indicate safe harbor locus for exogenous gene insertion) and tissue-and developmental stage-specific peaks (which regulate gene pathways matching tissue-and developmental stage-specific physiological functions). We found that H3K27ac and DNA methylation in the promoter region of the XIST gene may be involved in X chromosome inactivation and demonstrated the utility of the present resource for revealing the regulatory patterns of known causal genes and prioritizing candidate causal variants for complex traits in pigs. In addition, we identified an average of 1,124 super-enhancers per sample and found that they were more likely to show tissue-specific activity than ordinary peaks. We have developed a web browser to improve the accessibility of the results (http://segtp.jxau.edu.cn/pencode/?genome=susScr11).

Camellia oleifera Abel (C. oleifera ) absorb nutrients from surrounding soils and its yield is highly influenced by these nutrients and by fertilizer application. Thus, the soil nutrients play a central role in C. oleifera production. This study investigated the effects of biogas slurry applications on soil nutrients and economic traits of C . oleifera fruits. Five different amounts of biogas slurry (0, 10, 20, 30, or 40 kg/plant/year, three applications per year) were used as fertilizer for C . oleifera plants in 2015 and 2016. The nutrients of rhizosphere soil and the economic traits, including fruit yield, seed rate, and oil yield of C . oleifera fruit, were measured each year. The results showed that fertilization with biogas slurry significantly increased soil organic matter, available nitrogen (N), phosphorus (P), and potassium (K) both in 2015 and 2016. Increases in soil available N, P, and K were maximal in the highest slurry application group followed by the second highest application group. The oil yield correlated with the content of soil available P in both 2015 and 2016, and with soil organic matter in 2015. Fertilization with biogas slurry decreased the saturated fatty acid content in fruit but had no effect on the unsaturated fatty acid content. In conclusion, fertilization with biogas slurry increased rhizosphere soil nutrients and fruit economic traits of C . oleifera and rates of at least30 kg/plant/year had the most positive effects. This study expands the knowledge of fertilization with biogas slurry in C. oleifera production.

Epigenetic regulation of gene expression has been reported in the pathogenesis of metabolic disorders such as diabetes and liver steatosis in humans. However, the molecular mechanisms of fatty liver hemorrhagic syndrome (FLHS) in chickens have been rarely studied. H3K27ac chromatin immunoprecipitation coupled with high-throughput sequencing and high-throughput RNA sequencing was performed to compare genome-wide H3K27ac profiles and transcriptomes of liver tissue between healthy and FLHS chickens. In total, 1,321 differential H3K27ac regions and 443 differentially expressed genes were identified (| log2Fold change| ≥ 1 and P-value ≤ 0.05) between the two groups. Binding motifs for transcription factors involved in immune processes and metabolic homeostasis were enriched among those differential H3K27ac regions. Differential H3K27ac peaks were associated with multiple known FLHS risk genes, involved in lipid and energy metabolism (PCK1, APOA1, ANGPTL4, and FABP1) and the immune system (FGF7, PDGFRA, and KIT). Previous studies and our current results suggested that the high-energy, low-protein (HELP) diet might have an impact on histone modification and chromatin structure, leading to the dysregulation of candidate genes and the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which causes excessive accumulation of fat in the liver tissue and induces the development of FLHS. These findings highlight that epigenetic modifications contribute to the regulation of gene expression and play a central regulatory role in FLHS. The PPAR signaling pathway and other genes implicated in FLHS are of great importance for the development of novel and specific therapies for FLHS-susceptible commercial laying hens.