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.

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.

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.

BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice.

Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-D'D3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.

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.

Quantifying the contribution of sequence variants with regulatory and evolutionary significance to 34 bovine complex traits.

Many genome variants shaping mammalian phenotype are hypothesized to regulate gene transcription and/or to be under selection. However, most of the evidence to support this hypothesis comes from human studies. Systematic evidence for regulatory and evolutionary signals contributing to complex traits in a different mammalian model is needed. Sequence variants associated with gene expression (expression quantitative trait loci [eQTLs]) and concentration of metabolites (metabolic quantitative trait loci [mQTLs]) and under histone-modification marks in several tissues were discovered from multiomics data of over 400 cattle. Variants under selection and evolutionary constraint were identified using genome databases of multiple species. These analyses defined 30 sets of variants, and for each set, we estimated the genetic variance the set explained across 34 complex traits in 11,923 bulls and 32,347 cows with 17,669,372 imputed variants. The per-variant trait heritability of these sets across traits was highly consistent (r > 0.94) between bulls and cows. Based on the per-variant heritability, conserved sites across 100 vertebrate species and mQTLs ranked the highest, followed by eQTLs, young variants, those under histone-modification marks, and selection signatures. From these results, we defined a Functional-And-Evolutionary Trait Heritability (FAETH) score indicating the functionality and predicted heritability of each variant. In additional 7,551 cattle, the high FAETH-ranking variants had significantly increased genetic variances and genomic prediction accuracies in 3 production traits compared to the low FAETH-ranking variants. The FAETH framework combines the information of gene regulation, evolution, and trait heritability to rank variants, and the publicly available FAETH data provide a set of biological priors for cattle genomic selection worldwide.

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.

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.

CircBACH1 (hsa_circ_0061395) promotes hepatocellular carcinoma growth by regulating p27 repression via HuR.

In recent years, an increasing number of circular RNAs (circRNAs) have been discovered in hepatocellular carcinoma (HCC). However, the functions of most circRNAs require further investigation. Here, we found that circBACH1 was significantly upregulated in HCC tissues and that high circBACH1 levels were closely associated with poor prognosis. In addition, circBACH1 could promote HCC growth by accelerating cell cycle progression in vitro and in vivo. We next investigated the cellular and molecular mechanisms and discovered that circBACH1 inhibited p27 translation, which influenced cell cycle progression. Moreover, we revealed that circBACH1 could combine directly with HuR using RNA immunoprecipitation assays, pull-down assays, and electrophoretic mobility shift assays. The combination of these molecules facilitated HuR translocation from the nucleus to the cytoplasm according to the fluorescence in situ hybridization and immunofluorescence results. Finally, silencing HuR abrogated circBACH1's inhibition of p27 translation and abolished the circBACH1-induced effect on HCC proliferation. In sum, circBACH1 plays a significant role as an oncogene through the circBACH1/HuR/p27 axis in HCC development.

Oligosaccharides in goats' milk-based infant formula and their prebiotic and anti-infection properties.

Human milk contains an abundant supply and diverse array of oligosaccharides that are known to impart significant health benefits to the nursing infant including establishment and maintenance of a healthy gut microflora, immune development and protection against gastrointestinal infections. When breastfeeding is not possible or insufficient, infant formulas are commonly used as an alternative. However, limited information is available about the presence of naturally occurring oligosaccharides in these infant formulas and their likely health benefits. The present study examined the presence of naturally occurring oligosaccharides in commercial goats' milk-based stage 1 and stage 2 infant formulas and their prebiotic and anti-infection properties. LC/MS was used to detect and quantify oligosaccharides and their prebiotic potential was assessed by their ability, at concentrations present in reconstituted ready-to-use infant formula, to promote the growth of Bifidobacterium animalis subsp. lactis BB12, B. longum BB536, Lactobacillus acidophilus 4461 and L. casei 2607 in vitro. For anti-infection properties, the ability of goat milk oligosaccharides to prevent the adhesion of Escherichia coli NCTC 10418 and a Salmonella typhimurium isolate to Caco-2 cells was investigated. The results showed the presence of fourteen quantifiable oligosaccharides in stage 1 and stage 2 goats' milk-based infant formula. This was similar to the number of oligosaccharides detected in the fresh goats' milk. Of these, five were structurally similar to those found in human milk. These oligosaccharides were shown to significantly enhance the growth of bifidobacteria and lactobacilli and reduce the adhesion of E. coli NCTC 10418 and S. typhimurium to Caco-2 cells. Together, these results suggest that oligosaccharides naturally present in goats' milk-based infant formula exhibit strong prebiotic and anti-pathogen adhesion properties and may confer gut health benefits to infants.

Sodium chloride decreases cadmium accumulation and changes the response of metabolites to cadmium stress in the halophyte Carpobrotus rossii.

Background and Aims: Salinity affects the bioavailability of cadmium (Cd) in soils and Cd accumulation in plants, but the associated mechanisms remain unclear. This study aimed to assess the metabolic response to NaCl and Cd and the relationship between metabolites and Cd accumulation in the halophyte Carpobrotus rossii, which has potential for Cd phytoextraction. Methods: Plants were grown in nutrient solution with 0-400 mm NaCl in the presence of 5 or 15 µm Cd, with varied or constant solution Cd2+ activity. Plant growth and Cd uptake were measured, and the accumulation of peptides, and organic and amino acids in plant tissues were assessed. Key Results: The addition of NaCl to Cd-containing solutions improved plant growth along with 70-87 % less shoot Cd accumulation, resulting from decreases in Cd root uptake and root-to-shoot translocation irrespective of Cd2+ activity in solutions. Moreover, Cd exposure increased the concentration of phytochelatins, which correlated positively with Cd concentrations in plants regardless of NaCl addition. In comparison, Cd inhibited the synthesis of organic acids in shoots and roots in the absence of NaCl, but increased it in shoots in the presence of NaCl. While Cd increased the concentrations of amino acids in plant shoots, the effect of NaCl on the synthesis of amino acids was inconsistent. Conclusions: Our data provide the first evidence that NaCl decreased Cd shoot accumulation in C. rossii by decreasing Cd root uptake and root-to-shoot translocation even under constant Cd2+ activity. The present study also supports the important role of peptides and organic acids, particular of phytochelatins, in Cd tolerance and accumulation although the changes of those metabolites was not the main reason for the decreased Cd accumulation.

Characterization of glutathione S-transferases in the detoxification of metolachlor in two maize cultivars of differing herbicide tolerance.

Glutathione S-transferases (GSTs) have been widely studied in relation to their role in herbicide tolerance and detoxification. However, a detailed characterization of GSTs from herbicide tolerant and sensitive maize cultivars is still lacking. In this study, we determined the mechanism of differential tolerance between two maize cultivars which had 4-fold difference tolerance to metolachlor. The metabolism rate of metolachlor was more rapid in the tolerant cultivar (Zea mays L. cv Nongda86) than the susceptible one (Zea mays L. cv Zhengda958). Addition of the GST inhibitor ethacrynic acid reduced the metabolism of metolachlor indicating the involvement of GSTs in the differential detoxification of metolachlor. The expression profiles of 32 GST isozymes were measured using quantitative RT-PCR. The results showed the expression of GST genes were slightly up-regulated in Nongda86, but severely inhibited in Zhengdan958 24h after metolachlor treatment. The genes GSTI, GSTIII, GSTIV, GST5, GST6 and GST7, which can detoxify chloroacetanilide herbicides, were all expressed higher in Nongda86 compared to Zhendgan958. The result of GST activity was consistent with the gene expression profiles. Collectively, higher-level expression of GST genes, leading to higher GST activity and faster herbicide detoxification, appears to be responsible for the difference in tolerance to metolachlor in two maize cultivars.

Long non-coding RNA HNF1A-AS1 functioned as an oncogene and autophagy promoter in hepatocellular carcinoma through sponging hsa-miR-30b-5p.

Long non-coding RNAs (lncRNAs) had been proved to be pivotal regulators in carcinogenesis. On the basis of competitive endogenous RNAs (ceRNAs) system, lncRNAs significantly expanded their regulating networks. In our research, we aimed to figure out the exact role of lncRNA HNF1A-AS1 in the pathogenesis of hepatocellular carcinoma (HCC), in a ceRNA-dependent way. First, we revealed: HNF1A-AS1 was frequently overexpressed in HCC tissues and cell lines and its relative high expression was closely related to larger tumor size, multiple tumor lesions, poor differentiation and advanced TNM stage. Then we found: HNF1A-AS1 functioned as an oncogene in tumor growth and apoptosis through sponging tumor-suppressive hsa-miR-30b-5p (miR-30b) and de-repressing Bcl-2. Further experiments identified: HNF1A-AS1-miR-30b axis significantly promoted autophagy under starvation and ATG5 was first proved to be a target of miR-30b. In summary, we identified HNF1A-AS1-miR-30b axis as a key regulator in hepatocarcinogenesis, which may be promising biomarkers and therapeutic targets in the future.

Identification and quantification of triacylglycerols containing n-3 long-chain polyunsaturated fatty acids in bovine milk.

The n-3 long-chain polyunsaturated fatty acids (LC-PUFA) are low-abundance components in milk fat, but have great potential in promoting human health. A comprehensive survey on triacylglycerol (TAG) molecular species in milk that contain at least one type of n-3 LC-PUFA, namely eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid, was conducted in this work using HPLC-linear trap quadrupole-Orbitrap and HPLC-triple quadrupole mass spectrometry techniques. A total of 51 TAG species that contain n-3 LC-PUFA have been identified in bovine milk and their structures assigned. The TAG species containing docosahexaenoic acid were found in much smaller number and at much lower abundance compared with the other 2 types of TAG. An HPLC-triple quadrupole mass spectrometry-based method was developed, which provides relative quantification of all these TAG species in a run of 36 min. Application of this method to the quantification of n-3 LC-PUFA-incorporated TAG in 32 individual animal milk samples allowed us to determine variation between animals, identify strong metabolic relationships between TAG species, and reveal negative effect of a grape marc supplement on the accumulation of eicosapentaenoic acid in milk.

Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum by down-regulation of a Cinnamoyl CoA reductase gene.

C4 grasses are favoured as forage crops in warm, humid climates. The use of C4 grasses in pastures is expected to increase because the tropical belt is widening due to global climate change. While the forage quality of Paspalum dilatatum (dallisgrass) is higher than that of other C4 forage grass species, digestibility of warm-season grasses is, in general, poor compared with most temperate grasses. The presence of thick-walled parenchyma bundle-sheath cells around the vascular bundles found in the C4 forage grasses are associated with the deposition of lignin polymers in cell walls. High lignin content correlates negatively with digestibility, which is further reduced by a high ratio of syringyl (S) to guaiacyl (G) lignin subunits. Cinnamoyl-CoA reductase (CCR) catalyses the conversion of cinnamoyl CoA to cinnemaldehyde in the monolignol biosynthetic pathway and is considered to be the first step in the lignin-specific branch of the phenylpropanoid pathway. We have isolated three putative CCR1 cDNAs from P. dilatatum and demonstrated that their spatio-temporal expression pattern correlates with the developmental profile of lignin deposition. Further, transgenic P. dilatatum plants were produced in which a sense-suppression gene cassette, delivered free of vector backbone and integrated separately to the selectable marker, reduced CCR1 transcript levels. This resulted in the reduction of lignin, largely attributable to a decrease in G lignin.

Synthesis, preliminary bioevaluation and computational analysis of caffeic acid analogues.

A series of caffeic acid amides were designed, synthesized and evaluated for anti-inflammatory activity. Most of them exhibited promising anti-inflammatory activity against nitric oxide (NO) generation in murine macrophage RAW264.7 cells. A 3D pharmacophore model was created based on the biological results for further structural optimization. Moreover, predication of the potential targets was also carried out by the PharmMapper server. These amide analogues represent a promising class of anti-inflammatory scaffold for further exploration and target identification.

Recent progress in polar metabolite quantification in plants using liquid chromatography­mass spectrometry.

Metabolite analysis or metabolomics is an important component of systems biology in the post-genomic era. Although separate liquid chromatography (LC) methods for quantification of the major classes of polar metabolites of plants have been available for decades, a single method that enables simultaneous determination of hundreds of polar metabolites is possible only with gas chromatography­mass spectrometry (GC­MS) techniques. The rapid expansion of new LC stationary phases in the market and the ready access of mass spectrometry in many laboratories provides an excellent opportunity for developing LC­MS based methods for multi-target quantification of polar metabolites. Although various LC­MS methods have been developed over the last 10 years with the aim to quantify one or more classes of polar compounds in different matrices, currently there is no consensus LC­MS method that is widely used in plant metabolomics studies. The most promising methods applicable to plant metabolite analysis will be reviewed in this paper and the major problems encountered highlighted. The aim of this review is to provide plant scientists, with limited to moderate experience in analytical chemistry, with up-to-date and simplified information regarding the current status of polar metabolite analysis using LC­MS techniques.

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne).

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.