The N-terminal domain of HMW-GS 1Dx5 (1Dx5-N) contains three cysteine residues (Cys10, Cys25, Cys40), which are the basis of gluten network formation through disulfide bonds. Disulfide linkage in 1Dx5-N was dissected by site-directed mutagenesis and LC-MS/MS, and its contributions to structural and conformational stability of 1Dx5-N and dough functionality were investigated by circular dichroism, intrinsic fluorescence, surface hydrophobicity determination, size exclusion chromatography, nonreducing/reducing SDS-PAGE, atomic force microscopy, and farinographic analysis. Results showed that Cys10 and Cys40 of 1Dx5-N were the active sites for intermolecular linkage. Meanwhile, Cys40 also exhibited the ability to form intrachain disulfide linkage with Cys25. Moreover, Cys10 and Cys40 played a functionally important role in maintaining the structural and conformational stability and high surface hydrophobicity of the N-terminal domain of HMW-GS, which in turn facilitated the formation of HMW polymers and massive disulfide linkage of HMW-GS through hydrophobic interaction. Additionally, the 1Dx5-N mutants in which Cys were replaced by serine (Ser) presented different effects on dough functionality, while only the C25S mutant produced positive effects compared with wild type 1Dx5-N. NaCO-induced β-elimination of cystine might occur in glutenin without heating, which would make it much easier to reduce the nutritional quality of flour products by the cost of lysine. Therefore, these results give a deep understanding of the disulfide linkage of the N-terminal domain of HMW-GS and its functional importance, which will provide a practical guide to effectively generate a superior HMW-GS allele by artificial mutagenesis.
Extracellular P1,P4-diadenosine 5'-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have previously demonstrated specific receptors for Ap4A at the surface of cardiac myocytes (Walker et al., 1993a). In addition, we have isolated a monoclonal antibody (mAb TL4) that recognized the Ap4A receptor and inhibited binding of Ap4A to its receptor (Walker & Hilderman, 1993). As part of our effort to characterize the Ap4A receptor building domain, we screened a random phage peptide library with mAb TL4. After affinity purification of specifically bound phage, we isolated 38 individual phage clones. Twenty-eight of these clones bound mAb TL4 in ELISA and dot blot analyses. Twenty-two of the twenty-eight individual clones contained inserts with an RGS tripeptide sequence. Synthetic RGS peptide specifically inhibits the binding of mAb TL4 to its membrane receptor. Furthermore, the RGS peptide also inhibits [3H]Ap4A binding to its receptor. These data are consistent with the RGS peptide mimicking part of the mAb TL4 recognition site on the Ap4A receptor. The The RGS peptide may be used to help characterize the Ap4A receptor binding domain and to help determine the physiological significance of the interaction between Ap4A and its receptor.
With the continuing development of sequencing technology, genomics has been applied in a variety of biological research areas. In particular, the application of genomics to marine species, which boast a high diversity, promises great scientific and industrial potential. Significant progress has been made in marine genomics especially over the past few years. Consequently, BGI, leveraging its prominent contributions in genomics research, established BGI-Qingdao, an institute specifically aimed at exploring marine genomics. In order to accelerate marine genomics research and related applications, BGI-Qingdao initiated the International Conference on Genomics of the Ocean (ICG-Ocean) to develop international collaborations and establish a focused and coherent global research plan. Last year, the first ICG-Ocean conference was held in Qingdao, China, during which 47 scientists in marine genomics from all over the world reported on their research progress to an audience of about 300 attendees. This year, we would like to build on that success, drafting a report on marine genomics to draw global attention to marine genomics. We summarized the recent progress, proposed future directions, and we would like to enable additional profound insights on marine genomics. Similar to the annual report on plant and fungal research by Kew Gardens, and the White Paper of ethical issues on experimental animals, we hope our first report on marine genomics can provide some useful insights for researchers, funding agencies as well as industry, and that future versions will expand upon the foundation established here in both breadth and depth of knowledge.This report summarizes the recent progress in marine genomics in six parts including: marine microorganisms, marine fungi, marine algae and plants, marine invertebrates, marine vertebrates and genomics-based applications.
The binding properties of the protein-inhibitor complex of human immunodeficiency virus type 1 (HIV-1) protease with the inhibitor TMC-126 are investigated by combining computational alanine scanning (CAS) mutagenesis with binding free-energy decomposition (BFED). The calculated results demonstrate that the flap region (residues 38-58) and the active site region (residues 23-32) in HIV-1 protease contribute 63.72% of the protease to the binding of the inhibitor. In particular, the mechanisms for the interactions of key residues of these species are fully explored and analyzed. Interestingly, the regression analyses show that both CAS and BFED based on the generalized Born model yield similar results, with a correlation coefficient of 0.94. However, compared to CAS, BFED is faster and can decompose the per-residue binding free-energy contributions into backbone and sidechain contributions. The results obtained in this study are useful for studying the binding mechanism between receptor and ligand and for designing potent inhibitors that can combat diseases.
Transposons are mobile genetic elements in the genome. The piggyBac (PB) transposon system is increasingly being used for stem cell research due to its high transposition efficiency and seamless excision capacity. Over the past few decades, forward genetic screens based on PB transposons have been successfully established to identify genes associated with drug resistance and stem cell-related characteristics. Moreover, PB transposon is regarded as a promising gene therapy vector and has been used in some clinically relevant stem cells. Here, we review the recent progress on the basic biology of PB, highlight its applications in current stem cell research, and discuss its advantages and challenges.
A human co-infected with H1N1 and H7N9 subtypes influenza A virus (IAV) causes a complex infectious disease. The identification of molecular-level variations in composition and dynamics of IAV quasispecies will help to understand the pathogenesis and provide guidance for precision medicine treatment. In this study, using single-molecule real-time sequencing (SMRT) technology, we successfully acquired full-length IAV genomic sequences and quantified their genotypes abundance in serial samples from an 81-year-old male co-infected with H1N1 and H7N9 subtypes IAV. A total of 26 high diversity nucleotide loci was detected, in which the A-G base transversion was the most abundant substitution type (67 and 64%, in H1N1 and H7N9, respectively). Seven significant amino acid variations were detected, such as NA:H275Y and HA: R222K in H1N1 as well as PB2:E627K and NA: K432E in H7N9, which are related to viral drug-resistance or mammalian adaptation. Furtherly, we retrieved 25 H1N1 and 22 H7N9 genomic segment haplotypes from the eight samples based on combining high-diversity nucleotide loci, which provided a more concise overview of viral quasispecies composition and dynamics. Our approach promotes the popularization of viral quasispecies analysis in a complex infectious disease, which will boost the understanding of viral infections, pathogenesis, evolution, and precision medicine.
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