Modified fructan accumulation through overexpression of wheat fructan biosynthesis pathway fusion genes Ta1SST:Ta6SFT.

BACKGROUND: Fructans are water-soluble carbohydrates that accumulate in wheat and are thought to contribute to a pool of stored carbon reserves used in grain filling and tolerance to abiotic stress. RESULTS: In this study, transgenic wheat plants were engineered to overexpress a fusion of two fructan biosynthesis pathway genes, wheat sucrose: sucrose 1-fructosyltransferase (Ta1SST) and wheat sucrose: fructan 6-fructosyltransferase (Ta6SFT), regulated by a wheat ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (TaRbcS) gene promoter. We have shown that T4 generation transgene-homozygous single-copy events accumulated more fructan polymers in leaf, stem and grain when compared in the same tissues from transgene null lines. Under water-deficit (WD) conditions, transgenic wheat plants showed an increased accumulation of fructan polymers with a high degree of polymerisation (DP) when compared to non-transgenic plants. In wheat grain of a transgenic event, increased deposition of particular fructan polymers such as, DP4 was observed. CONCLUSIONS: This study demonstrated that the tissue-regulated expression of a gene fusion between Ta1SST and Ta6SFT resulted in modified fructan accumulation in transgenic wheat plants and was influenced by water-deficit stress conditions.

A Chiral-LC-MS Method for the Simultaneous Quantification of Short-Chain Fatty Acids and D/L-Lactate in the Ruminal Fluid of Dairy Cows.

Short-chain fatty acids (SCFA) and lactate in ruminal fluid are products resulting from the microbial fermentation of substrates and can be used to reflect the composition and activity of the ruminal microbiome. Determination of SCFA and D-/L-lactate in ruminal fluid currently requires two separate protocols, which is time-consuming and costly. In this study, we have optimised and validated a simple and unified 3-nitrophenylhydrazine (3-NPH) derivatisation protocol and a 20 min chiral-LC-MS method for the simultaneous quantification of all SCFA and D- and L-lactate in ruminal fluid. This method, which requires no sample pretreatment or purification shows adequate sensitivity (limit of detection (LOD): 0.01 µg/mL), satisfactory accuracy (recovery: 88-103%), and excellent reproducibility (relative standard deviation (RSD) for repeated analyses < 3% for most analytes). The application of this method to a cohort of 24 animals allowed us to reveal a large inter-cow variation in ruminal SCFA and lactate level, the concentration range for each species, the widespread correlation between different SCFA, and the strong correlation between D- and L-lactate.

Gene expression and RNA splicing explain large proportions of the heritability for complex traits in cattle.

Many quantitative trait loci (QTLs) are in non-coding regions. Therefore, QTLs are assumed to affect gene regulation. Gene expression and RNA splicing are primary steps of transcription, so DNA variants changing gene expression (eVariants) or RNA splicing (sVariants) are expected to significantly affect phenotypes. We quantify the contribution of eVariants and sVariants detected from 16 tissues (n = 4,725) to 37 traits of ∼120,000 cattle (average magnitude of genetic correlation between traits = 0.13). Analyzed in Bayesian mixture models, averaged across 37 traits, cis and trans eVariants and sVariants detected from 16 tissues jointly explain 69.2% (SE = 0.5%) of heritability, 44% more than expected from the same number of random variants. This 69.2% includes an average of 24% from trans e-/sVariants (14% more than expected). Averaged across 56 lipidomic traits, multi-tissue cis and trans e-/sVariants also explain 71.5% (SE = 0.3%) of heritability, demonstrating the essential role of proximal and distal regulatory variants in shaping mammalian phenotypes.

Clinical and prognostic significance of CCPG1 in hepatocellular carcinoma.

This study aimed to examine the clinical and prognostic significance of cell-cycle progression gene 1 (CCPG1) in hepatocellular carcinoma (HCC). We firstly analyzed CCPG1 expression in various cancers using The Cancer Genome Atlas and the Genotype-Tissue Expression project databases. The relative expression levels of CCPG1 were determined in 164 paired HCC and adjacent tissues using immunohistochemistry. The correlation between CCPG1 and clinicopathological characteristics of HCC was analyzed. Cox proportional models were used to identify the prognostic factors for overall survival (OS) and disease-free survival (DFS). The expression of CCPG1 was lower in HCC tissues than in adjacent non-tumor liver tissues. The expression of CCPG1 was significantly correlated with tumor number (p = 0.02) and tumor differentiation (p = 0.04) in HCC. Lower expression of CCPG1 in HCC patients was associated with poor OS and DFS (p < 0.01). Relative low expression of CCPG1 in HCC is significantly correlated with the poor prognosis of HCC patients after surgical resection, suggesting its possible role as a potential prognostic marker for HCC.

circFAM134B is a key factor regulating reticulophagy-mediated ferroptosis in hepatocellular carcinoma.

Ferroptosis is an important mode of regulated cell death (RCD). Its inhibition is closely related to therapeutic resistance and poor prognosis in hepatocellular carcinoma (HCC). Previous reports have demonstrated ferroptosis as a biological process highly dependent on selective autophagy, such as ferritinophagy, lipophagy, and clockophagy. Our study also revealed a role for ER-phagy-mediated ferroptosis in HCC cells treated with multi-targeted tyrosine kinase inhibitors (TKIs). In the current study, we found that the homologous circular RNA (circRNA) of the family with sequence similarity 134, member B (FAM134B), hsa_circ_0128505 (was abbreviated as circFAM134B in the present study), was identified to specifically target ER-phagy to promote lenvatinib (LV)-induced ferroptosis using reactive oxygen species (ROS), Fe2+, malondialdehyde (MDA), and western blot (WB) assays in HCC cells. RNA pull-down and mass spectrometry analyses suggested that circFAM134B and FAM134B mRNA were enriched with several common interacting proteins. Among them, poly (A) binding protein cytoplasmic 4 (PABPC4) was identified as the most enriched binding partner. It was proven to be a novel antagonist against the nonsense-mediated mRNA decay (NMD) mechanism. We then applied RNA immunoprecipitation (RIP), RNA pull-down, luciferase reporter, and NMD reporter gene assays to further explore the exact role and underlying mechanism of circFAM134B-PABPC4-FAM134B axis in HCC cells. circFAM134B was confirmed as a sponge that competitively interacted with PABPC4, thereby influencing FAM134B mRNA nonsense decay. Our results provide novel evidences and strategies for the comprehensive treatment of HCC.

SMRT Sequencing Technology Was Used to Construct the Batocera horsfieldi (Hope) Transcriptome and Reveal Its Features.

Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae) is an important forest pest in China that mainly infests timber and economic forests. This pest primarily causes plant tissue to necrotize, rot, and eventually die by feeding on the woody parts of tree trunks. To gain a deeper understanding of the genetic mechanism of B. horsfieldi, this study employed single-molecule real-time sequencing (SMRT) and Illumina RNA-seq technologies to conduct full-length transcriptome sequencing of the insect. Total RNA extracted from male and female adults was mixed and subjected to SMRT sequencing, generating a complete transcriptome. Transcriptome analysis, prediction of long non-coding RNA (lncRNA), coding sequences (CDs), analysis of simple sequence repeats (SSR), prediction of transcription factors, and functional annotation of transcripts were performed in this study. The collective 20,356,793 subreads (38.26 G, clean reads) were generated, including 432,091 circular consensus sequences and 395,851 full-length non-chimera reads. The full-length non-chimera reads (FLNC) were clustered and redundancies were removed, resulting in 39,912 consensus reads. SSR and ANGEL software v3.0 were used for predicting SSR and CDs. In addition, four tools were used for annotating 6058 lncRNAs, identifying 636 transcription factors. Furthermore, a total of 84,650 transcripts were functionally annotated in seven different databases. This is the first time that the full-length transcriptome of B. horsfieldi has been obtained using SMRT sequencing. This provides an important foundation for investigating the gene regulation underlying the interaction between B. horsfieldi and its host plants through gene editing in the future and provides a scientific basis for the prevention and control of B. horsfieldi.

Different influences of phylogenetically conserved and independent floral traits on plant functional specialization and pollination network structure.

Plant specialization and pollination network structure play important roles in community assembly. Floral traits can mediate plant-pollinator interactions and thus have important impacts on nestedness and modularity of pollination network. When such traits are phylogenetically conserved, therefore, phylogeny and traits should predict network structure to similar degrees. Moreover, conserved network structures were also found attributed to pollination syndrome or pollination system. However, we still know little about the relation between pollination syndrome and pollination network, especially under a phylogenetic framework. Herein, we established a phylogenetic framework including five floral traits (flower density, floral size, floral shape, floral symmetry, and floral color) and five species-level metrics (species strength, weighted closeness, specialization d', nestedness contribution, and modularity contribution) to test how floral traits could directly or indirectly influence species' specialization and network structure in central China. Phylogenetic signals were found in all floral traits except flower density. Structural equation model and phylogenetic structural equation model results showed that both floral size and floral density affected plant specialization and its contribution to network modularity indirectly. However, compared with phylogenetic independent flower density, phylogenetic conserved floral size had much more complexed influences, having a direct influence both on species' specialization and on modularity contribution. In this nested and modular network, abundant species with larger flowers tend to be more central and had larger values of z. Floral shape, symmetry, and color could act as co-flowering filters in pollination sharing and help to shape network modularity. Our results emphasize that phylogenetically conserved traits partially represent pollination syndrome and are important drivers for modular structure of local pollination network. This study may improve the understanding how the evolutionary history and ecological process drive local network structure and dynamics.

The PTBP1­NCOA4 axis promotes ferroptosis in liver cancer cells.

Polypyrimidine tract­binding protein 1 (PTBP1) plays an important role in tumor immunity, cell proliferation, apoptosis, and autophagy by regulating RNA metabolism. However, the specific function and mechanism of PTBP1 in ferroptosis remain unclear. In the present study, it was investigated whether PTBP1 regulates ferroptosis and the exact mechanism. The iron, malondialdehyde (MDA), and GSH levels were detected in sorafenib (SF)­treated liver cancer cells. si­PTBP1 introduction into SF­treated liver cancer cells resulted in a significant reduction in the levels of MDA and iron. Additionally, a significant recovery of GSH levels was observed after silencing PTBP1. StarBase v2.0 database was used to predict potential transcripts that can physically interact with PTBP1 and nuclear receptor coactivator 4 (NCOA4) mRNA was identified as the most enriched binding partner in the PTBP1­RNA complex. A dual­luciferase assay then demonstrated that PTBP1 directly interacted with NCOA4. PTBP1 silencing did not affect NCOA4 stability following treatment with cycloheximide. A pull­down assay revealed that the PTBP1­binding region was in the 5'­UTR of the NCOA4 mRNA sequence. These results suggest that PTBP1 mediates ferroptosis in liver cancer cells by regulating NCOA4 translation. In vivo experiments reconfirmed the role of the PTBP1­NCOA4 axis in a xenograft transplantation model. It was observed that the mean tumor weight increased after PTBP1 knockout. In conclusion, silencing of PTBP1 decreased the sensitivity of liver cancer cells to ferroptosis after SF treatment and regulated ferritinophagy by mediating NCOA4 translation.

L-Cys-Assisted Conversion of H2/CO2 to Biochemicals Using Clostridium ljungdahlii.

Carbon fixation and conversion based on Clostridium ljungdahlii have great potential for the sustainable production of biochemicals (i.e., 2,3-butanediol, acetic acid, and ethanol). Here, the effects of reducing agents on the production of biochemicals from H2/CO2 using C. ljungdahlii were studied. It was found that the element S and reducing power could significantly affect the production of biochemicals, and cysteine (Cys) was better than sodium sulfide for the production of biochemicals, especially for the production of 2,3-butanediol. Moreover, comparing to the control (i.e., without the addition of Cys), the gene expression profiles indicated that the fdh and adhE1 were significantly upregulated with the addition of Cys, which involved in pathways of the CO2 fixation and ethanol production. Therefore, the irreplaceability of Cys on the production of biochemicals was both caused by its utilization as a reducing agent and its effect on the metabolic pathway. Finally, compared to the control, the production of 2,3-butanediol was increased by 2.17 times under the addition of 1.7 g/L Cys.

Superhydrophobic and highly moisture-resistant PVA@EC composite membrane for air purification.

Electrospun fiber membranes have great potential in the field of air filtration because of their high porosity and small pore size. Conventional air filtration membranes are hydrophilic, leading to weak moisture-barrier properties, which hinders their application in high-humidity environments. In this study, eugenol was added to polyvinyl alcohol and ethyl cellulose (EC) for electrospinning and electrospraying, respectively, of superhydrophobic bilayer composite fiber membranes to efficiently filter particulate matter (PM) in air. Owing to its surface microstructure, electrosprayed EC increased the water contact angle of the PVA membrane from 142.8 to 151.1°. More importantly, the composite air-filter membrane showed a low filtration pressure drop (168.1 Pa) and exhibited high filtration efficiencies of 99.74 and 99.77% for PM1.0 and PM2.5, respectively, and their respective quality factors were 0.0351 and 0.0358 Pa-1. At the same time, the filtration performance of the air filtration membrane remained above 99% at high air humidity. This work reports composite membranes that can effectively capture PM of various sizes and thus may provide a reference for the manufacturing of green air filters for high-humidity environments.

A bioinspired, strong, all-natural, superhydrophobic cellulose-based straw.

The promotion of cellulose-based paper straws is one of the important ways to improve white pollution nowadays. However, developing composite straws that are simultaneously highly biocompatible, safe, and non-toxic and that overcome the low water stability and physical strength problems caused by the inherent hydrophilicity of the raw material cellulose has become an important challenge in the development process. In this study, a new all-natural superhydrophobic straw (CFS) made of a composite of cellulose nanofibers and stearic acid was introduced. Stearic acid is a saturated fatty acid derived from plant and animal oils. Inspired by the specific hydrophobicity of sugarcane cane peel, a green straw with both superhydrophobicity (water contact angle up to 153°) and remarkable mechanical strength (tensile strength up to 67.15 MPa) was developed by controlling the hydrophobic modification conditions of stearic acid through solvent vaporization. Furthermore, the composite straws under wet conditions had lower water absorption and exhibited excellent wet tensile strength compared to commercial paper straws. In addition, the composite straw without the addition of chemical binders avoids the defects of non-renewable products, fits into the global green development concept, and brings new strategies for the development of cellulose-based materials.

Optimised Method for Short-Chain Fatty Acid Profiling of Bovine Milk and Serum.

Short-chain fatty acids (SCFA, C2-C5) in milk and serum are derived from rumen bacterial fermentation and, thus, have the potential to be used as biomarkers for the health status of dairy cows. Currently, there is no comprehensive and validated method that can be used to analyse all SCFAs in both bovine serum and milk. This paper reports an optimised protocol, combining 3-nitrophenylhydrazine (3-NPH) derivatisation and liquid chromatography-mass spectrometry (LC-MS) analysis for quantification of SCFA and ß-hydroxybutyric acid (BHBA) in both bovine milk and bovine serum. This method is sensitive (limit of detection (LOD) ≤ 0.1 µmol/L of bovine milk and serum), accurate (recovery 84-115% for most analytes) and reproducible (relative standard deviation (RSD) for repeated analyses below 7% for most measurements) with a short sample preparation step. The application of this method to samples collected from a small cohort of animals allowed us to reveal a large variation in SCFA concentration between serum and milk and across different animals as well as the strong correlation of some SCFAs between milk and serum samples.

Comparison of Workflows for Milk Lipid Analysis: Phospholipids.

Milk is a rich source of lipids, with the major components being triglycerides (TAG) and phospholipids (mainly phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)). Liquid chromatography-mass spectrometry (LC-MS) is the predominant technique for lipid identification and quantification across all biological samples. While fatty acid (FA) composition of the major lipid classes of milk can be readily determined using tandem MS, elucidating the regio-distribution and double bond position of the FA remains difficult. Various workflows have been reported on the quantification of lipid species in biological samples in the past 20 years, but no standard or consensus methods are currently available for the quantification of milk phospholipids. This study will examine the influence of several common factors in lipid analysis workflow (including lipid extraction protocols, LC stationary phases, mobile phase buffers, gradient elution programmes, mass analyser resolution and isotope correction) on the quantification outcome of bovine milk phospholipids. The pros and cons of the current LC-MS methods as well as the critical problems to be solved will also be discussed.

Regio-distribution and double bond locations of unsaturated fatty acids in phospholipids of bovine milk.

Hundreds of phospholipid (PL) species with defined fatty acid (FA) composition have been identified previously in bovine milk using liquid chromatography tandem mass spectrometry (LC-MS/MS). Paterno-Buchi photochemical reaction coupled with LC-MS/MS was applied in this study to further unravel the regio-distribution and double bond (DB) locations of FAs. Using SPE-purified PLs and 2-acetylpyridine as the photochemical derivatization reagent, we were able to reveal the non-specific regio-distribution of unsaturated FAs and the widespread occurrence of regioisomers in milk PLs. Although Δ9 and Δ9,12 were found to be the predominant DB location(s) for C18:1 and C18:2 respectively, other DB positional isomers such as C18:1Δ11, C18:1Δ12 and C18:1Δ13 and C18:2Δ9,11 were widely detected in PL structures, implying that the minor isomers of C18:1 and C18:2 equally participate in the synthesis of PLs. Our study provides novel information on the fine structure of milk PLs and further underlines the complexity of milk lipid composition.

Targeting FAM134B-mediated reticulophagy activates sorafenib-induced ferroptosis in hepatocellular carcinoma.

Ferroptosis is a kind of cell death closely related to selective autophagy, such as ferritinophagy, lipophagy, clockophagy and chaperone-mediated autophagy. However, the role of reticulophagy, which specifically degrades endoplasmic reticulum (ER) fragments (also known as ER-phagy), in ferroptosis regulation is still unclear. In this study, we found that sorafenib (ferroptosis inducer) can effectively activate the receptor protein FAM134B-mediated ER-phagy, and FAM134B knockdown not only blocked ER-phagy but also significantly strengthened cellular sensitivity to ferroptosis without affecting macroautophagy. In vivo experiments also yielded similar results. These evidences provided new clues for ferroptosis regulation. Subsequently, bioinformatic analysis combined with RNA binding protein immunoprecipitation and polyribosome fractionation preliminarily indicated that PABPC1 can interact with FAM134B mRNA and promote its translation. Taken together, this study revealed the role of the PABPC1-FAM134B-ER-phagy pathway on ferroptosis, providing important evidence for novel anti-cancer strategies.

Bovine Milk Triacylglycerol Regioisomer Ratio Shows Remarkable Inter-Breed and Inter-Cow Variation.

Regioisomers (or positional isomers) of triacylglycerols (TAGs) of milk are known to show differential outcome in relation to human absorption. Quantitation of TAG regioisomers remains a big challenge due to the lack of facile chromatographic separation technique. The feasibility of using fragment ion intensity ratio to determine the ratio of co-eluting AAB/ABA-type regioisomer pairs was confirmed in this study. The ability of C30 stationary phase in resolving interfering TAG isomers was demonstrated for the first time. This allowed us to reveal the complexity of using fragment ion intensity to quantify 1,2-olein-3-palmitin (OOP), 1,3-olein-2-palmitin (OPO), 1,2-olein-3-stearin (OOS), and 1,3-olein-2-stearin (OSO) regioisomers in milk samples. A novel algorithm was proposed to consider the contribution of OPO/OOP and OSO/OOS double bond (DB)-isomers and to eliminate the interference of isobaric ions from other isomers, an aspect overlooked in previous studies. This liquid chromatography-mass spectrometry method that requires no pre-fractioning and a moderate chromatographic separation time of 36 min is simple and, thus, suitable for screening a large number of samples for genetic analysis of this trait. Preliminary results using a small cohort of animals showed that OPO/OOP ratio differs significantly between Jersey and Holstein cows, and a large variation was also observed across individual Holstein cows.

Comparative transcriptomic and metabolomic analyses reveal the protective effects of silicon against low phosphorus stress in tomato plants.

Phosphorus (P) is an essential nutrient controlling plant growth and development through the regulation of basic metabolic processes. Soil P deficiency is one of the major limiting factors for sustainable crop production worldwide. Previous studies have demonstrated that silicon (Si), as a beneficial element, promotes plant nutrition, growth, development, and responses to low P (LP) stress; however, the molecular mechanisms underlying Si-mediated LP tolerance remain largely unclear. Here, we found that LP + Si treatment increased the net photosynthetic rate and shoot fresh weight by 34.3%, and 121.3%, respectively compared with LP alone. RNA-sequencing and metabolomic analyses were subsequently performed with tomato plants grown under control and P depleted conditions with or without Si amendment. RNA-sequencing showed that Si supply alters not only the expression of genes involved in the metabolism of carbon (C), nitrogen (N), and P but also phosphorylation processes and metabolism of glutathione and reactive active oxygen in tomato roots. Si also affected the expression of genes encoding major transcription factors such as WRKY and MYB under LP stress. Moreover, a set of genes encoding the enzymes or regulators of organic acid (OA) metabolism or secretion were differentially expressed in Si-treated P deficient roots compared with those in LP stress alone. Furthermore, the metabolomic analysis showed that the levels of several OAs were significantly elevated in Si-treated P deficient roots. Taken together, these results indicate that exogenous Si increases the secretion of OAs by modulating C/N metabolism in LP-treated tomato roots and thereby improving plant growth under LP stress.

Analysis of Glucosinolate Content and Metabolism Related Genes in Different Parts of Chinese Flowering Cabbage.

Glucosinolates (GSLs) are important secondary metabolites that play important defensive roles in cruciferous plants. Chinese flowering cabbage, one of the most common vegetable crops, is rich in GSLs and thus has the potential to reduce the risk of cancer in humans. Many genes that are involved in GSL biosynthesis and metabolism have been identified in the model plant Arabidopsis thaliana; however, few studies investigated the genes related to GSL biosynthesis and metabolism in Chinese flowering cabbage. In the present study, the GSL composition and content in three different organs of Chinese flowering cabbage (leaf, stalk, and flower bud) were determined. Our results showed that the total GSL content in flower buds was significantly higher than in stalks and leaves, and aliphatic GSLs were the most abundant GSL type. To understand the molecular mechanisms underlying the variations of GSL content, we analyzed the expression of genes encoding enzymes involved in GSL biosynthesis and transport in different tissues of Chinese flowering cabbage using RNA sequencing; the expression levels of most genes were found to be consistent with the pattern of total GSL content. Correlation and consistency analysis of differentially expressed genes from different organs with the GSL content revealed that seven genes (Bra029966, Bra012640, Bra016787, Bra011761, Bra006830, Bra011759, and Bra029248) were positively correlated with GSL content. These findings provide a molecular basis for further elucidating GSL biosynthesis and transport in Chinese flowering cabbage.

RNA binding protein DAZAP1 promotes HCC progression and regulates ferroptosis by interacting with SLC7A11 mRNA.

RNA-binding proteins (RBPs) closely regulate the whole lifecycle of most RNA molecules, from the very early stage of transcription to RNA decay. Dysregulation of RBPs significantly affects the fate of cancer-related transcripts. Therefore, it is imperative to fully understand the complicated RBP-RNA regulatory networks in malignant diseases and to explore novel therapeutic targets. The RBP DAZAP1 (deleted in azoospermia-associated protein 1), originally identified as an important protein in spermatogenesis, had rarely been studied in the context of carcinogenesis. The role of DAZAP1 in hepatocellular carcinoma (HCC) was unveiled in this study. The relative expression of DAZAP1 was significantly upregulated in HCC and was positively associated with several key malignant characteristics and poor postoperative survival in patients. DAZAP1 knockdown by small interfering RNA markedly inhibited HCC cell proliferation, migration and invasion. Furthermore, DAZAP1 significantly reduced cellular sensitivity to sorafenib (SF), which had been proven to be an inducer of ferroptosis by targeting the system Xc- (composed of a light chain, xCT/SLC7A11, and a heavy chain, 4F2 heavy chain). At the mechanistic level, DAZAP1 was identified as a potent inhibitor of ferroptosis and an efficient binding partner of SLC7A11 mRNA. Further study revealed that DAZAP1 interacted with the 3'UTR (untranslated region) of SLC7A11 mRNA and positively regulated its stability. In our work, we clarified novel functions of DAZAP1 and preliminarily revealed its underlying mechanism in ferroptosis, which may be conducive to the exploration of biomarkers and therapeutic targets in HCC patients.