Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.

The relationship of exercise and cancer-related fatigue in patients with advanced liver cancer: a cross-sectional study.

There is increasing interest in understanding exercise as a potential treatment for cancer-related fatigue (CRF); however, rarely research has been conducted on more aggressive cancers with short survival, such as liver cancer. The purpose of this study was to provide educational ideas for insufficient exercise and provide clues for the design of effective and safe exercise intervention programs with high compliance in patients of advanced liver cancer in the future. Participants were recruited from a tertiary cancer hospital using convenience sampling. All participants were asked to complete self-report questionnaires that assessed their medical and demographic variables, exercise habits and CRF during their hospitalization in the interventional department. Spearman's correlation analysis and Nonparametric test was used to explore correlations between exercise subgroups and CRF. The Baron and Kenny's Approach was used to investigate the mediating effect of exercise index between P-EX and CRF. 207 out of 255 participants were enrolled in this study, with an average age of 55.4 years. The CRF score was 33 (28, 36), and 93.2% had insufficient exercise. Exercise frequency (≥ 3 Times/week) (Z = 4.34, p = 0.037) and maintaining exercise trend (Z = 15.85, p = 0.001) had a positive effect on CRF. P-EX had a great impact on exercise index and affecting CRF directly. Participants in the study showed serious fatigue and insufficient exercise. Exercise education can be initiated earlier, particularly those without regular exercise experience. Sustained light exercise, compliant with exercise habits and interests, three times a week may be a practical way to reduce the risk of CRF in advanced liver cancer.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum).

BACKGROUND: Base editors (BEs) display diverse applications in a variety of plant species such as Arabidopsis, rice, wheat, maize, soybean, and cotton, where they have been used to mediate precise base pair conversions without the collateral generation of undesirable double-stranded breaks (DSB). Studies of single-nucleotide polymorphisms (SNPs) underpinning plant traits are still challenging, particularly in polyploidy species where such SNPs are present in multiple copies, and simultaneous modification of all alleles would be required for functional analysis. Allotetraploid cotton has a number of homoeologous gene pairs located in the A and D sub-genomes with considerable SNPs, and it is desirable to develop adenine base editors (ABEs) for efficient and precise A-to-G single-base editing without DSB in such complex genome. RESULTS: We established various ABE vectors based on different engineered adenosine deaminase (TadA) proteins fused to Cas9 variants (dCas9, nCas9), enabling efficient A to G editing up to 64% efficiency on-target sites of the allotetraploid cotton genome. Comprehensive analysis showed that GhABE7.10n exhibited the highest editing efficiency, with the main editing sites specifically located at the position A5 (counting the PAM as positions 21-23). Furthermore, DNA and RNA off-target analysis of cotton plants edited with GhABE7.10n and GhABE7.10d by whole genome and whole-transcriptome sequencing revealed no DNA off-target mutations, while very low-level RNA off-target mutations were detected. A new base editor, namely GhABE7.10dCpf1 (7.10TadA + dCpf1), that recognizes a T-rich PAM, was developed for the first time. Targeted A-to-G substitutions generated a single amino acid change in the cotton phosphatidyl ethanolamine-binding protein (GhPEBP), leading to a compact cotton plant architecture, an ideotype for mechanized harvesting of modern cotton production. CONCLUSIONS: Our data illustrate the robustness of adenine base editing in plant species with complex genomes, which provides efficient and precise toolkit for cotton functional genomics and precise molecular breeding.

Effect of the food form and structure on lipid digestion and postprandial lipaemic response.

As the food matrix is a determinant of the rate of fat digestion and absorption, it is important for the modulation of postprandial triglyceridaemia. High postprandial triglyceride levels are associated with an increase in inflammation, oxidative stress, an imbalance in the lipoprotein profile and an increase in the risk of developing chronic diseases. This study was designed to assess the in vitro digestion patterns and the postprandial lipaemic responses to test foods with the same nutrient composition but differing in the form and structure. A liquid, a semi-solid and a solid test food with the same nutrient and energy composition were designed. The digestion profiles of the three foods were assessed using a dynamic in vitro model. The foods were also consumed by healthy young adults who donated blood samples after an overnight fast and again 0.5, 1, 2, 3, 4 and 6 h after consuming each of the test foods and who were also assessed for appetite sensations. The solid food showed phase separation during gastric digestion and a lower release of fatty acids during intestinal digestion than the liquid and semi-solid foods. During the postprandial feeding experiments, the solid food caused a lower increase in serum triglycerides than the liquid food and produced higher fullness and satisfaction. In conclusion, the food form and structure modulated fat release, postprandial triglyceridaemia and appetite sensations independent of the nutrient and energy content. Thus, manipulation of the food structure and form may be used in designing strategies for improving metabolic markers and satiety.

Association between a p73 gene polymorphism and genetic susceptibility to non-small cell lung cancer in the South of China.

BACKGROUND: This study aimed to identify any association between the p73 gene G4C14-to-A4T14 polymorphism and risk of non-small cell lung cancer (NSCLC) in the south of China. MATERIALS AND METHODS: We genotyped the p73 gene polymorphism of peripheral blood DNA from 168 patients with NSCLC and 195 normal controls using HRM (high resolution melting) and PCR-CTPP (polymerase chain reaction with confronting two-pair primers). RESULTS: The results of genotyping by HRM and PCR-CTPP were consistent with direct sequencing, the p73 genotype distribution in 168 lung cancer patients being as follows: GC/GC 101 cases (60.1%), GC/ AT 59 cases (35.1%), AT/AT 8 cases (4.8%). The carriers of AT/AT genotype had a significantly reduced risk of NSCLC (OR=0.370; 95%CI: 0.170-0.806; p=0.010) as compared with non-carriers. However, we found no relations between p73 genotypes and histological type (p=0.798, x2=0.452), tumor stage (p=0.806, x2=0.806), or lymph node metastasis (p=0.578, x2=1.098). CONCLUSIONS: Our findings suggest that the p73 G4C14-to-A4T14 polymorphism may be a modifier of NSCLC susceptibility in the Chinese population.

Effect of calcium on the kinetics of free fatty acid release during in vitro lipid digestion in model emulsions.

The effects of different calcium salts on in vitro lipid digestion were examined by determining the free fatty acids released from various oil-in-water emulsions. The kinetics of the total and individual free fatty acids released by lipolysis were monitored by the pH-stat method and gas chromatography, respectively. The rate and the extent of free fatty acid release increased with an increase in the added calcium concentration, but the increase was dependent on the emulsifying agent. The effect of calcium was diminished when the emulsion contained phosphate. Soluble calcium salts, such as calcium gluconate, calcium acetate and CaCl2, had greater effects on the rate and extent of free fatty acid release than did insoluble salts, such as CaO and CaSO4, suggesting that the ionic state of calcium plays a critical role in lipid digestion in emulsions. The addition of calcium did not alter the profiles of the individual free fatty acids released. This study provides useful information for food formulation with respect to lipid digestion.

Inhibition of Hepatitis B virus replication by phospholipid scramblase 1 in vitro and in vivo.

Human Phospholipid scramblase 1 (PLSCR1) is an α/ß interferon-inducible protein that mediates antiviral activity against RNA viruses including vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). In the present study, we investigated the antiviral activity of PLSCR1 protein against HBV (Hepatitis B virus). Firstly, PLSCR1 mRNA and protein expression was found to be downregulated in HepG2 cells after HBV infection. Then by performing co-transient-transfection experiments in cells and hydrodynamics-based transfection experiments in mice using a HBV expression plasmid and a PLSCR1 expression plasmid, we found that PLSCR1 inhibited HBV replication in vitro and in vivo through a significant reduction in the synthesis of viral proteins, DNA replicative intermediates and HBV RNAs. We also demonstrated that the antiviral action of PLSCR1 against HBV occurs, partly at least, by activating the Jak/Stat pathway. In conclusion, our results suggest that the expression of PLSCR1 is involved in HBV replication and that PLSCR1 has antiviral activity against HBV.

Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface.

Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells.

Binding of 2-nonanone and milk proteins in aqueous model systems.

Interactions of the model flavor compound 2-nonanone with individual milk proteins, whey protein isolate (WPI), and sodium caseinate in aqueous solutions were investigated. A method to quantify the free 2-nonanone was developed using headspace solid-phase microextraction followed by gas chromatography with flame ionization detection. Binding constants (K) and numbers of binding sites (n) for 2-nonanone on the individual proteins were calculated. The 2-nonanone binding capacities decreased in the order bovine serum albumin > beta-lactoglobulin > alpha-lactalbumin > alpha s1-casein > beta-casein, and the binding to WPI was stronger than the binding to sodium caseinate. All proteins appeared to have one binding site for 2-nonanone per molecule of protein at the flavor concentrations investigated, except for bovine serum albumin, which possessed two classes of binding sites. The binding mechanism is believed to involve predominantly hydrophobic interactions.