A seed-mediated and double shell strategy to realize large-size ZnSe/ZnS/ZnS quantum dots for high color purity blue light-emitting diodes.

Environment-friendly high color purity blue zinc selenide (ZnSe) quantum dot-based light-emitting diodes (QLEDs) are promising candidates in next-generation display applications. However, due to the large bandgap of ZnSe (2.7 eV), the reported electroluminescence (EL) wavelengths of ZnSe QLEDs are mainly located within the range from purple to violet blue, and preparing blue emitting (>445 nm) ZnSe QLEDs remains challenging. Herein, we report a seed-mediated and double shell strategy to synthesize large-sized blue ZnSe/ZnS/ZnS core/shell/shell quantum dots (QDs). The as-prepared QDs possess excellent features including narrow full widths at half-maximum (11-19 nm), tunable emission wavelengths (410-451 nm), and high photoluminescence quantum yields (≥50%). Using ZnSe/ZnS/ZnS QDs as emitters in an inverted device, a color saturated blue QLED with an EL wavelength of 446 nm, a maximum luminance of 106 cd m-2, a current efficiency of 0.94 cd A-1, and an EQE of 2.62% is successfully fabricated. These results indicate that blue ZnSe QLEDs have great potential for future display technologies.

Lewis acids promoted organic pollutants degradation in aqueous solution with peroxymonosulfate and MnO2: New insights into the activation mechanism.

Nonredox metal ions have been widely recognized to be important in a wide range of biological and chemical oxidations as Lewis acids (LA). However, the role of LA in peroxymonosulfate (PMS) activation for wastewater treatment has not been considered until now. This study shows that oxidizing power of PMS can be promoted after binding nonredox metal ions such as Ca2+ as LA, leading to the easier reduction of the oxidant to radicals and substantial enhancement of dye degradation by employing manganese oxides OMS-2 as model catalysts. Increased with Lewis acidity of the metal ion, the rate of PMS decomposition enhanced linearly, while the dye degradation rate first increased and then declined due to the formation of a larger amount of dioxygen. The interactions between Ca2+ and PMS were further investigated by Raman, cyclic voltammetry and XPS; and the detailed mechanism of PMS activation was proposed. The performance of Ca2++OMS-2/PMS system under different conditions was also studied. The findings indicate the importance of LA in PMS activation reaction and their role must be considered in other transition metal oxides/PMS systems. It will be also helpful to design new and highly active catalysts for the reactions.

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array.

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.

Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides.

We report a rational strategy to design and construct multiple small perturbation mutagenesis (SPM) libraries using massively parallel synthesis of oligonucleotides on a microchip for affinity maturation of an engineered anti-ErbB2 antibody chA21. On the basis of a comprehensive analysis of the sequence and structural relationships of six complementary determination regions (CDRs) in the Kabatman database, a computational algorithm was developed to introduce single-site and double-site mutations into variable CDR positions using ambiguous nucleotides. The six SPM libraries were composed of 419 degenerate oligonucleotides that can be expanded into 161,832 unique CDR sequences with a high coverage ratio of 95% natural amino acid diversity. We used Illumina next-generation sequencing to demonstrate that the synthetic CDR library sequences, as well as relative quantities per sequence, can be controlled precisely by adjusting reaction chamber assignment and input nucleoside composition. The microchip-synthesized oligonucleotides were used for construction of single-chain antibody fragment (scFv) phage libraries through one-step mutagenic PCR of double-stranded plasmids with >10(6)E. coli transformants. A variant with combinatorial mutations from four individual CDRs achieved more than 19-fold affinity increase. The strategy described herein should be broadly applicable to affinity and selectivity studies of antibodies and other proteins.

A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks.

The architecture of cellular proteins connected to form signaling pathways in response to internal and external cues is much more complex than a group of simple protein-protein interactions. Post translational modifications on proteins (e.g., phosphorylation of serine, threonine and tyrosine residues on proteins) initiate many downstream signaling events leading to protein-protein interactions and subsequent activation of signaling cascades leading to cell proliferation, cell differentiation and cell death. As evidenced by a rapidly expanding mass spectrometry database demonstrating protein phosphorylation at specific motifs, there is currently a large gap in understanding the functional significance of phosphoproteins with respect to their specific protein connections in the signaling cascades. A comprehensive map that interconnects phospho-motifs in pathways will enable identification of nodal protein interactions that are sensitive signatures indicating a disease phenotype from the physiological hemostasis and provide clues into control of disease. Using a novel phosphopeptide microarray technology, we have mapped endogenous tyrosine-phosphoproteome interaction networks in breast cancer cells mediated by signaling adaptor protein GRB2, which transduces cellular responses downstream of several RTKs through the Ras-ERK signaling cascade. We have identified several previously reported motif specific interactions and novel interactions. The peptide microarray data indicate that various phospho-motifs on a single protein are differentially regulated in various cell types and shows global downregulation of phosphoprotein interactions specifically in cells with metastatic potential. The study has revealed novel phosphoprotein mediated signaling networks, which warrants further detailed analysis of the nodes of protein-protein interaction to uncover their biomarker or therapeutic potential.

Immune-related microRNAs are abundant in breast milk exosomes.

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P < 10(-16), χ(2) test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.

MicroRNA profiling using µParaflo microfluidic array technology.

The diverse functions of microRNA (miRNA) molecules have drawn broad and intensive interest in various biological fields, biomedical applications, and technology development. Which are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (1). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (2-4). miRNAs are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (Bartel, Cell 116:281-97, 2004). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (He et al., Nature 435:828-833, 2005; Lu et al., Nature 435:834-838, 2005; O'Donnell, et al., Nature 435:839-843, 2005). In this chapter, we describe the methods using µParaflo(®) microfluidic oligonucleotide microarray technology for applications in miRNA profiling. One unique feature of this technology is the flexibility that provides users with the freedom to select sequence content either for focused studies wherein only the most relevant sequences are included or for discovery studies wherein the most updated sequence content such as those newly derived from deep sequencing. This chapter provides detailed information from experimental design to sample preparation, as well as data analysis for a miRNA array experiment.

Repertoire of porcine microRNAs in adult ovary and testis by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs), a large family of short endogenous RNAs known to post-transcriptionally repress gene expression, participate in the regulation of almost every cellular process. Changes in miRNA expression are associated with many pathologies. Ovarian folliculogenesis and testicular spermatogenesis are complex and coordinated biological processes, in which tightly regulated expression and interaction of a multitude of genes could be regulated by these miRNAs. Identification and preliminary characterization of gonad-specific miRNAs would be a prerequisite for a thorough understanding of the role that miRNA-mediated posttranscriptional gene regulation plays in mammalian reproduction. METHOD: Here, we present the identification of a repertoire of porcine miRNAs in adult ovary and testis using deep sequencing technology. A bioinformatics pipeline was developed to distinguish authentic mature miRNA sequences from other classes of small RNAs represented in the sequencing data. RESULTS: Using this approach, we detected 582 precursor hairpins (pre-miRNAs) encoding for 732 mature miRNAs, of which 673 are unique. Statistically, 224 unique miRNAs (out of 673, 33.28%) were identified which had significant differential expression (DE) between ovary and testis libraries (P < 0.001). Most of DE miRNAs located on the X chromosome (X-linked miRNAs) (24 out of 34, 70.59%) significantly up-regulated in ovary versus testis (P < 0.001). Predictably, X-linked miRNAs are expressed in a testis-preferential or testis-specific pattern. To explore the potential for co-expression among genomic location clusters of X-linked miRNAs, we surveyed the relationship between the distance separating miRNA loci and the coordinate expression patterns of 32 high confidence X-linked miRNAs in seven normal pig tissues using the real-time quantitative PCR (q-PCR) approach. Our results show that proximal pairs of miRNAs are generally co-expressed implying that miRNAs within 50 kb of genomic bases are typically derived from a common transcript. CONCLUSIONS: The present study characterizes the miRNA transcriptome of adult porcine gonads, with an emphasis on the co-expression patterns of X-linked miRNAs. Our report should facilitate studies of the organ-specific reproductive roles of miRNAs.

Microarray-based identification of tomato microRNAs and time course analysis of their response to Cucumber mosaic virus infection.

A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with Cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

MicroRNAome of porcine pre- and postnatal development.

The domestic pig is of enormous agricultural significance and valuable models for many human diseases. Information concerning the pig microRNAome (miRNAome) has been long overdue and elucidation of this information will permit an atlas of microRNA (miRNA) regulation functions and networks to be constructed. Here we performed a comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained during the entire pig lifetime, from the fetal period through adulthood. The sequencing results were analyzed using mammalian miRNAs, the precursor hairpins (pre-miRNAs) and the first release of the high-coverage porcine genome assembly (Sscrofa9, April 2009) and the available expressed sequence tag (EST) sequences. Our results extend the repertoire of pig miRNAome to 867 pre-miRNAs (623 with genomic coordinates) encoding for 1,004 miRNAs, of which 777 are unique. We preformed real-time quantitative PCR (q-PCR) experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing and q-PCR data. This broad survey provides detailed information about multiple variants of mature sequences, precursors, chromosomal organization, development-specific expression, and conservation patterns. Our data mining produced a broad view of the pig miRNAome, consisting of miRNAs and isomiRs and a wealth of information of pig miRNA characteristics. These results are prelude to the advancement in pig biology as well the use of pigs as model organism for human biological and biomedical studies.

Web-based design of peptide microarrays using microPepArray Pro.

Peptide microarrays (peptide arrays) have increasingly become an important research tool for studying protein detection, profiling, and protein-protein interactions, and they have the potential to foster high-throughput protein analysis as DNA arrays did for genomics research a decade ago. Recently, technologies have emerged that allow flexible synthesis of high-density peptide arrays based on specific application needs (e.g., phosphopeptide microarrays). To fully unleash the power of this promising research tool, significant efforts are required to develop computational and informatics resources that facilitate the experimental design and data analysis for a wide range of peptide array-based applications. The design of peptide arrays is inherently more complex than that of DNA arrays. We herein introduce microPepArray Pro, a Web-based general-purpose peptide array design program. microPepArray Pro features strong content design capabilities and maximized user control. The program suits the needs of a diversity of design tasks, works with a variety of peptide array configurations, and is highly expandable: new functionalities can be developed and added to microPepArray Pro with relative ease.

Qualitative and quantitative analysis of peptide microarray binding experiments using SVM-PEPARRAY.

A main objective of analyzing peptide array-based binding experiments is to uncover the relationship between a peptide sequence and the binding outcome. Limited by the peptide array technologies available for applications, few attempts have been made to construct qualitative or quantitative models that depict the peptide sequence:binding strength relationships in peptide microarray-based binding studies. There has been a long history of similar modeling efforts based on low-throughput binding data in the areas of T-cell epitope screening and kinase substrate mapping, however. The keen needs in peptide array applications and the success of the modeling efforts in related fields have prompted us to develop SVM-PEPARRAY, a Web-based program capable of constructing qualitative and quantitative models based on peptide microarray binding datasets using support vector machine (SVM) modeling methods. We expect that such modeling analysis will allow researchers to quickly extract sequence-based biological information from improved peptide array binding results and provide more precise and accurate information about the biological systems investigated.

Microfluidic Reactor Array Device for Massively Parallel In-situ Synthesis of Oligonucleotides.

We have designed and fabricated a microfluidic reactor array device for massively parallel in-situ synthesis of oligonucleotides (oDNA). The device is made of glass anodically bonded to silicon consisting of three level features: microreactors, microchannels and through inlet/outlet holes. Main challenges in the design of this device include preventing diffusion of photogenerated reagents upon activation and achieving uniform reagent flow through thousands of parallel reactors. The device embodies a simple and effective dynamic isolation mechanism which prevents the intermixing of active reagents between discrete microreactors. Depending on the design parameters, it is possible to achieve uniform flow and synthesis reaction in all of the reactors by proper design of the microreactors and the microchannels. We demonstrated the use of this device on a solution-based, light-directed parallel in-situ oDNA synthesis. We were able to synthesize long oDNA, up to 120 mers at stepwise yield of 98 %. The quality of our microfluidic oDNA microarray including sensitivity, signal noise, specificity, spot variation and accuracy was characterized. Our microfluidic reactor array devices show a great potential for genomics and proteomics researches.

Highly parallel identification of essential genes in cancer cells.

More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process.

Novel microRNA candidates and miRNA-mRNA pairs in embryonic stem (ES) cells.

BACKGROUND: MicroRNAS (miRNAS: a class of short non-coding RNAs) are emerging as important agents of post transcriptional gene regulation and integral components of gene networks. MiRNAs have been strongly linked to stem cells, which have a remarkable dual role in development. They can either continuously replenish themselves (self-renewal), or differentiate into cells that execute a limited number of specific actions (pluripotence). METHODOLOGY/PRINCIPAL FINDINGS: In order to identify novel miRNAs from narrow windows of development we carried out an in silico search for micro-conserved elements (MCE) in adult tissue progenitor transcript sequences. A plethora of previously unknown miRNA candidates were revealed including 545 small RNAs that are enriched in embryonic stem (ES) cells over adult cells. Approximately 20% of these novel candidates are down-regulated in ES (Dicer(-/-)) ES cells that are impaired in miRNA maturation. The ES-enriched miRNA candidates exhibit distinct and opposite expression trends from mmu-mirs (an abundant class in adult tissues) during retinoic acid (RA)-induced ES cell differentiation. Significant perturbation of trends is found in both miRNAs and novel candidates in ES (GCNF(-/-)) cells, which display loss of repression of pluripotence genes upon differentiation. CONCLUSION/SIGNIFICANCE: Combining expression profile information with miRNA target prediction, we identified miRNA-mRNA pairs that correlate with ES cell pluripotence and differentiation. Perturbation of these pairs in the ES (GCNF(-/-)) mutant suggests a role for miRNAs in the core regulatory networks underlying ES cell self-renewal, pluripotence and differentiation.

PepCyber:P~PEP: a database of human protein protein interactions mediated by phosphoprotein-binding domains.

Phosphoprotein-binding domains (PPBDs) mediate many important cellular and molecular processes. Ten PPBDs have been known to exist in the human proteome, namely, 14-3-3, BRCT, C2, FHA, MH2, PBD, PTB, SH2, WD-40 and WW. PepCyber:P approximately PEP is a newly constructed database specialized in documenting human PPBD-containing proteins and PPBD-mediated interactions. Our motivation is to provide the research community with a rich information source emphasizing the reported, experimentally validated data for specific PPBD-PPEP interactions. This information is not only useful for designing, comparing and validating the relevant experiments, but it also serves as a knowledge-base for computationally constructing systems signaling pathways and networks. PepCyber:P approximately PEP is accessible through the URL, http://www.pepcyber.org/PPEP/. The current release of the database contains 7044 PPBD-mediated interactions involving 337 PPBD-containing proteins and 1123 substrate proteins.

A FLIPR-based assay to assess potency and selectivity of inhibitors of the TEC family kinases Btk and Itk.

Bruton's tyrosine kinase (Btk) and interleukin-2-inducible T cell kinase (Itk) are members of the TEC family of nonreceptor tyrosine kinases and are expressed primarily in B and T cells, respectively. Both kinases are critically involved in lymphocyte development and signal transduction. In particular, Btk and Itk regulate calcium mobilization subsequent to antigen receptor stimulation. Small molecule antagonists that specifically inhibit either Btk or Itk may allow for selective modulation of B cell or T cell activity and may be useful in treating inflammatory and autoimmune conditions. We have developed a medium-throughput fluorescent imaging plate reader (FLIPR)- based calcium flux assay that can be used to assay potential Btk and Itk inhibitors. This assay takes advantage of Btk-deficient DT40 (DT40-Btk-/-) chicken B cells, which are unable to mobilize calcium in response to cross-linking of their B cell receptor (BCR). Ectopic expression of TEC family kinases can restore antigen receptor signaling in these cells. We have generated stable DT40-Btk-/- lines expressing either wild-type human Btk (huBtk) or a chimeric Btk-Itk kinase (huBtk-Itk) molecule-a Btk protein whose kinase domain has been replaced by the kinase domain of Itk. Expression of either huBtk or huBtk-Itk in DT40-Btk-/- cells restores calcium flux in response to BCR engagement. Using Btk- and Itk-selective inhibitors, we show that inhibition of calcium responses in huBtk-Itk-DT40-Btk-/- cells and huBtk-DT40-Btk-/- cells is dependent on the Itk or Btk kinase domain, respectively. Thus, the FLIPR assay described here can be used to assess, compare, and rank the potency and selectivity of inhibitors of Itk and Btk kinases.

microParaflo biochip for nucleic acid and protein analysis.

We describe in this chapter the use of oligonucleotide or peptide microarrays (arrays) based on microfluidic chips. Specifically, three major applications are presented: (1) microRNA/small RNA detection using a microRNA detection chip, (2) protein binding and function analysis using epitope, kinase substrate, or phosphopeptide chips, and (3) protein-binding analysis using oligonucleotide chips. These diverse categories of customizable arrays are based on the same biochip platform featuring a significant amount of flexibility in the sequence design to suit a wide range of research needs. The protocols of the array applications play a critical role in obtaining high quality and reliable results. Given the comprehensive and complex nature of the array experiments, the details presented in this chapter is intended merely as a useful information source of reference or a starting point for many researchers who are interested in genome- or proteome-scale studies of proteins and nucleic acids and their interactions.

Predicting DNA duplex stability on oligonucleotide arrays.

DNA duplex stability on oligonucleotide microarray was calculated using recently developed electrostatic theory of on-array hybridization thermodynamics. In this method, the first step is to finding the enthalpy and entropy of duplex formation in solution. This standard calculation was done with nearest-neighbor scheme and on-line software. Next the defined parameters and the array's single characteristic, the surface density of probes, are used to predict on-array duplex melting behavior. Reasonable accords of calculated and experimental melting curves for in situ synthesized microfluidic array were observed. The proposed method could be useful in microarray design and hybridization optimization. However, lack of melting curve measurements for different microarray platforms makes more experiments desirable to determine the method's accuracy.

B-cell depletion inhibits arthritis in a collagen-induced arthritis (CIA) model, but does not adversely affect humoral responses in a respiratory syncytial virus (RSV) vaccination model.

We report the development of a mouse B cell-depleting immunoconjugate (anti-CD22 monoclonal antibody [mAb] conjugated to calicheamicin) and its in vivo use to characterize the kinetics of CD22+ B-cell depletion and reconstitution in murine primary and secondary lymphoid tissues. The effect of B-cell depletion was further studied in a murine collagen-induced arthritis (CIA) model and a respiratory syncytial virus (RSV) vaccination model. Our results show that (1) the immunoconjugate has B-cell-specific in vitro and in vivo cytotoxicity; (2) B-cell reconstitution starts in the bone marrow and spleen around day 30 after depletion and is completed in all tissues tested by day 50; (3) B-cell depletion inhibits the development of clinical and histologic arthritis in the CIA model; (4) depletion of type II collagen antibody levels is not necessary for clinical and histologic prevention of CIA; and (5) B-cell depletion does not adversely affect memory antibody responses after challenge nor clearance of infectious virus from lungs in the RSV vaccination model. These results demonstrate for the first time that only B-cell reduction but not type II collagen antibody levels correlate with the prevention of arthritis and represent key insights into the role of CD22-targeted B-cell depletion in mouse autoimmunity and vaccination models.