Cloning and Functional Characterization of 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase (LiMCT) Gene in Oriental Lily (Lilium 'Sorbonne').

2-C-methyl-D-erythritol-phosphate cytidylyltransferase (MCT) is a key enzyme in the MEP pathway of monoterpene synthesis, catalyzing the generation of 4- (5'-pyrophosphate cytidine)-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol-4-phosphate. We used homologous cloning strategy to clone gene, LiMCT, in the MEP pathway that may be involved in the regulation of floral fragrance synthesis in the Lilium oriental hybrid 'Sorbonne.' The full-length ORF sequence was 837 bp, encoding 278 amino acids. Bioinformatics analysis showed that the relative molecular weight of LiMCT protein is 68.56 kD and the isoelectric point (pI) is 5.12. The expression pattern of LiMCT gene was found to be consistent with the accumulation sites and emission patterns of floral fragrance monoterpenes in transcriptome data (unpublished). Subcellular localization indicated that the LiMCT protein is located in chloroplasts, which is consistent with the location of MEP pathway genes functioning in plastids to produce isoprene precursors. Overexpression of LiMCT in Arabidopsis thaliana affected the expression levels of MEP and MVA pathway genes, suggesting that overexpression of the LiMCT in A. thaliana affected the metabolic flow of C5 precursors of two different terpene synthesis pathways. The expression of the monoterpene synthase AtTPS14 was elevated nearly fourfold in transgenic A. thaliana compared with the control, and the levels of carotenoids and chlorophylls, the end products of the MEP pathway, were significantly increased in the leaves at full bloom, indicating that LiMCT plays an important role in regulating monoterpene synthesis and in the synthesis of other isoprene-like precursors in transgenic A. thaliana flowers. However, the specific mechanism of LiMCT in promoting the accumulation of isoprene products of the MEP pathway and the biosynthesis of floral monoterpene volatile components needs further investigation.

Influence of enzymatic modification on the basis of improved extrusion cooking technology (IECT) on the structure and properties of corn starch.

Enzymatic modification can directly affect the structure and properties of starch, but generally causes high energy consumption in drying process. Improved extrusion cooking technology (IECT) itself is a starch modification technology. In this work, a co-extrusion method of starch with 42 % moisture and enzyme was adopted to reveal the effects of different enzyme dosages on the structure and properties of corn starch. After enzyme treatment on the basis of IECT, starch granules were broken into fragments without the occurrence of clear Maltese cross. R1047/1022 and R995/1022 values, peak intensity of Raman spectra and gelatinization temperature decreased, and the full width at half maximum at 480 cm-1 of Raman spectra raised. Moreover, the bound water proportion decreased from 87.44 % to 85.84 % âˆ¼ 78.67 %, and the maximum light transmittance and dextrose equivalent values increased to 34.13 % and 26.14, respectively. The solubility of starch granules was all above 60 %. Findings supported that the mechanochemical effect of IECT on starch was conducive to the enzymatic modification.

Configurational patterns for COVID-19 related social media rumor refutation effectiveness enhancement based on machine learning and fsQCA.

Infodemics are intertwined with the COVID-19 pandemic, affecting people's perception and social order. To curb the spread of COVID-19 related false rumors, fuzzy-set qualitative comparative analysis (fsQCA) is used to find configurational pathways to enhance rumor refutation effectiveness. In this paper, a total of 1,903 COVID-19 related false rumor refutation microblogs on Sina Weibo are collected by a web crawler from January 1, 2022 to April 20, 2022, and 10 main conditions affecting rumor refutation effectiveness index (REI) are identified based on "three rules of epidemics". To reduce data redundancy, five ensemble machine learning models are established and tuned, among which Light Gradient Boosting Machine (LGBM) regression model has the best performance. Then five core conditions are extracted by feature importance ranking of LGBM. Based on fsQCA with the five core conditions, REI enhancement can be achieved through three different pathway elements configurations solutions: "Highly influential microblogger * high followers' stickiness microblogger", "high followers' stickiness microblogger * highly active microblogger * concise information description" and "high followers' stickiness microblogger * the sentiment tendency of the topic * concise information description". Finally, decision-making suggestions for false rumor refutation platforms and new ideas for improving false rumor refutation effectiveness are proposed. The innovation of this paper reflects in exploring the REI enhancement strategy from the perspective of configuration for the first time.

Identification of a novel peptide ligand for the cancer-specific receptor mutation EGFRvIII using high-throughput sequencing of phage-selected peptides.

We report here the selection and characterization of a novel peptide ligand using phage display targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer: ovarian cancer, breast cancer and glioblastoma, but not in normal tissues. A 12-mer random peptide library was screened against EGFRvIII. Phage-selected peptides were sequenced in high-throughput by next generation sequencing (NGS), and their diversity was studied to identify highly abundant clones expected to bind with the highest affinities to EGFRvIII. The enriched peptides were characterized and their binding capacity towards stable cell lines expressing EGFRvIII, EGFR wild type (EGFR WT), or a low endogenous level of EGFR WT was confirmed by flow cytometry analysis. The best peptide candidate, VLGREEWSTSYW, was synthesized, and its binding specificity towards EGFRvIII was validated in vitro. Additionally, computational docking analysis suggested that the identified peptide binds selectively to EGFRvIII. The novel VLGREEWSTSYW peptide is thus a promising EGFRvIII-targeting agent for future applications in cancer diagnosis and therapy.

FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats.

FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.

Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation.

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.