Combined Use of a Solid-Phase Hexapeptide Ligand Library with Liquid Chromatography and Two-Dimensional Difference Gel Electrophoresis for Intact Plasma Proteomics

The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies

Enhanced luminescence efficiency in Eu-doped GaN superlattice structures revealed by terahertz emission spectroscopy

Eu-doped Gallium nitride (GaN) is a promising candidate for GaN-based red light-emitting diodes, which are needed for future micro-display technologies. Introducing a superlattice structure comprised of alternating undoped and Eu-doped GaN layers has been observed to lead to an order-of-magnitude increase in output power; however, the underlying mechanism remains unknown. Here, we explore the optical and electrical properties of these superlattice structures utilizing terahertz emission spectroscopy. We find that ~0.1% Eu doping reduces the bandgap of GaN by ~40 meV and increases the index of refraction by ~20%, which would result in potential barriers and carrier confinement within a superlattice structure. To confirm the presence of these potential barriers, we explored the temperature dependence of the terahertz emission, which was used to estimate the barrier potentials. The result revealed that even a dilutely doped superlattice structure induces significant confinement for carriers, enhancing carrier recombination within the Eu-doped regions. Such an enhancement would improve the external quantum efficiency in the Eu-doped devices. We argue that the benefits of the superlattice structure are not limited to Eu-doped GaN, which provides a roadmap for enhanced optoelectronic functionalities in all rare-earth-doped semiconductor systems.Murakami F., Takeo A., Mitchell B., et al. Enhanced luminescence efficiency in Eu-doped GaN superlattice structures revealed by terahertz emission spectroscopy. Communications Materials 4, 100 (2023); https://doi.org/10.1038/s43246-023-00428-6

The Application of Restriction Landmark Genome Scanning Method for Surveillance of Non-Mendelian Inheritance in F1 Hybrids

We analyzed inheritance of DNA methylation in reciprocal F1 hybrids (subsp. japonica cv. Nipponbare × subsp. indica cv. Kasalath) of rice (Oryza sativa L.) using restriction landmark genome scanning (RLGS), and detected differing RLGS spots between the parents and reciprocal F1 hybrids. MspI/HpaII restriction sites in the DNA from these different spots were suspected to be heterozygously methylated in the Nipponbare parent. These spots segregated in F1 plants, but did not segregate in selfed progeny of Nipponbare, showing non-Mendelian inheritance of the methylation status. As a result of RT-PCR and sequencing, a specific allele of the gene nearest to the methylated sites was expressed in reciprocal F1 plants, showing evidence of biased allelic expression. These results show the applicability of RLGS for scanning of non-Mendelian inheritance of DNA methylation and biased allelic expression

Trapping DNA Replication Origins from the Human Genome

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks

Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells

BACKGROUND: Many lines of evidence suggest that poly(ADP-ribose) polymerase-1 (Parp-1) is involved in transcriptional regulation of various genes as a coactivator or a corepressor by modulating chromatin structure. However, the impact of Parp-1-deficiency on the regulation of genome-wide gene expression has not been fully studied yet. RESULTS: We employed a microarray analysis covering 12,488 genes and ESTs using mouse Parp-1-deficient (Parp-1(-/-)) embryonic stem (ES) cell lines and the livers of Parp-1(-/- )mice and their wild-type (Parp-1(+/+)) counterparts. Here, we demonstrate that of the 9,907 genes analyzed, in Parp-1(-/- )ES cells, 9.6% showed altered gene expression. Of these, 6.3% and 3.3% of the genes were down- or up-regulated by 2-fold or greater, respectively, compared with Parp-1(+/+ )ES cells (p < 0.05). In the livers of Parp-1(-/- )mice, of the 12,353 genes that were analyzed, 2.0% or 1.3% were down- and up-regulated, respectively (p < 0.05). Notably, the number of down-regulated genes was higher in both ES cells and livers, than that of the up-regulated genes. The genes that showed altered expression in ES cells or in the livers are ascribed to various cellular processes, including metabolism, signal transduction, cell cycle control and transcription. We also observed expression of the genes involved in the pathway of extraembryonic tissue development is augmented in Parp-1(-/- )ES cells, including H19. After withdrawal of leukemia inhibitory factor, expression of H19 as well as other trophoblast marker genes were further up-regulated in Parp-1(-/- )ES cells compared to Parp-1(+/+ )ES cells. CONCLUSION: These results suggest that Parp-1 is required to maintain transcriptional regulation of a wide variety of genes on a genome-wide scale. The gene expression profiles in Parp-1-deficient cells may be useful to delineate the functional role of Parp-1 in epigenetic regulation of the genomes involved in various biological phenomena

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h

An oxyl/oxo mechanism for dioxygen bond formation in PSII revealed by X-ray free electron lasers

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S0 to S4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S1, S2, and S3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S2, but flipping of D1 Glu189 upon transition to S3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation