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...
Minjuan Wang is an associate professor of Educational Technology at San Diego State University. Her research specialties focus on the sociocultural facets of online learning, mobile learning and technological interventions in language education. She has published peer-reviewed articles in Educational Technology Research and Development, Computers and Education, Educational Media International, TechTrends, and the British Journal of Educational Technology. She has also published book chapters on engaged learning in online problem solving, Cybergogy for interactive learning online, informal learning via the Internet, and effective learning in multicultural and multilingual classrooms. AbstractChinese classrooms, whether on school grounds or online, have long suffered from a lack of interactivity. Many online classes simply provide recorded instructor lectures, which only reinforces the negative effects of passive nonparticipatory learning. At Shanghai Jiaotong University, researchers and developers actively seek technologic interventions that can greatly increase interactivity in large blended classes. They developed a cutting-edge mobile learning system that can deliver live broadcasts of real-time classroom teaching to students with mobile devices. Their system allows students to customise means of content-reception based on when and where they tune into the broadcast. The system also supports short text messaging and instant polls. Through these venues, students can ask questions and make suggestions in real time, and the instructor can address them immediately. This article describes this system in detail, and also reports results from a formal implementation of the system in a blended English classroom of 1000 students (with about 800 being online). As the data reveal, m-learning activities can much better engage students in the learning process. Students in this class changed from passive learners to truly engaged learners who are behaviourally, intellectually and emotionally involved in their learning tasks.
A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-␣, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.
Little is known about the regulatory effect of microbiota on the proliferation and regeneration of ISCs. Here, we found that L. reuteri stimulated the proliferation of intestinal epithelia by increasing the expression of R-spondins and thus activating the Wnt/β-catenin pathway. The proliferationstimulating effect of Lactobacillus on repair is further enhanced under TNF -induced intestinal mucosal damage, and the number of Lgr5 + cells is maintained. Moreover, compared to the effects of C. rodentium on the induction of intestinal inflammation and crypt hyperplasia in mice, L. reuteri protected the intestinal mucosal barrier integrity by moderately modulating the Wnt/β-catenin signaling pathway to avoid overactivation. L. reuteri had the ability to maintain the number of Lgr5 + cells and stimulate intestinal epithelial proliferation to repair epithelial damage and reduce proinflammatory cytokine secretion in the intestine and the LPS concentration in serum. Moreover, activation of the Wnt/β-catenin pathway also induced differentiation toward Paneth cells and increased antimicrobial peptide expression to inhibit C. rodentium colonization. The protective effect of Lactobacillus against C. rodentium infection disappeared upon application of the Wnt antagonist Wnt-C59 in both mice and intestinal organoids. This study demonstrates that Lactobacillus is effective at maintaining intestinal epithelial regeneration and homeostasis as well as at repairing intestinal damage after pathological injury and is thus a promising alternative therapeutic method for intestinal inflammation.
We experimentally demonstrate a 16 × 16 non-blocking optical switch fabric with a footprint of 10.7 × 4.4 mm2. The switch fabric is composed of 56 2 × 2 silicon Mach-Zehnder interferometers (MZIs), with each integrated with a pair of TiN resistive micro-heaters and a p-i-n diode. The average on-chip insertion loss at 1560 nm wavelength is ~6.7 dB and ~14 dB for the "all-cross" and "all-bar" states, respectively, with a loss variation of ± 1 dB over all routing paths. The measured rise/fall time of the switch upon electrical tuning is 3.2/2.5 ns. The switching functionality is verified by transmission of 20 Gb/s on-off keying (OOK) and 50 Gb/s quadrature phase-shift keying (QPSK) optical signals.
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