Facile synthesis of core\shell Fe3O4@mSiO2(Hb) and its application for organic wastewater treatment.

Treatment of organic wastewater is a challenging task. Biological techniques using biocatalysts have shown their benefits in organic wastewater treatment. In this research, a novel biocatalyst was developed by encapsulation of Fe3O4 microspheres and haemoglobin (Hb) with mesoporous silica, named Fe3O4@mSiO2(Hb). Fe3O4@mSiO2(Hb) exhibited typical mesoporous characteristics (mesoporous silica), magnetic feature (Fe3O4) and peroxidase activity (Hb). The results showed that the immobilization of Hb into Fe3O4@mSiO2 did not affect its activity. In addition, Fe3O4@mSiO2(Hb) exhibited a higher efficiency in the peroxidation of aromatic compounds than free Hb. The peroxidase activity of the synthesized biocatalyst was estimated to be 120 Ug-1, which was almost four times greater than that of previously reported immobilized Hb. Also, the Km of Fe3O4@mSiO2(Hb) was similar to that of the free Hb and it was estimated to be 4.3 × 10-4 µM, indicating that the activity of the Hb in the immobilized enzyme was not affected after immobilization. The immobilized enzyme was also found to be stable, recyclable and reusable. Taken together, these results indicate that the Fe3O4@mSiO2(Hb) has good potential to be used for treating organic wastewater containing aromatic compounds. The magnetically separable novel biocatalyst developed in this study provided not only a more suitable microenvironment for retaining the activity of Hb, but also demonstrated enhanced stability and activity under unfavorable conditions.

A Study of the Interaction, Morphology, and Structure in Trypsin-Epigallocatechin-3-Gallate Complexes.

Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.

Molecular Characterization, Gene Evolution and Expression Analysis of the F-Box Gene Family in Tomato (Solanum lycopersicum).

F-box genes play an important role in the growth and development of plants, but there are few studies on its role in a plant's response to abiotic stresses. In order to further study the functions of F-box genes in tomato (Solanum lycopersicum, Sl), a total of 139 F-box genes were identified in the whole genome of tomato using bioinformatics methods, and the basic information, transcript structure, conserved motif, cis-elements, chromosomal location, gene evolution, phylogenetic relationship, expression patterns and the expression under cold stress, drought stress, jasmonic acid (JA) treatment and salicylic acid (SA) treatment were analyzed. The results showed that SlFBX genes were distributed on 12 chromosomes of tomato and were prone to TD (tandem duplication) at the ends of chromosomes. WGD (whole genome duplication), TD, PD (proximal duplication) and TRD (transposed duplication) modes seem play an important role in the expansion and evolution of tomato SlFBX genes. The most recent divergence occurred 1.3042 million years ago, between SlFBX89 and SlFBX103. The cis-elements in SlFBX genes' promoter regions were mainly responded to phytohormone and abiotic stress. Expression analysis based on transcriptome data and qRT-PCR (Real-time quantitative PCR) analysis of SlFBX genes showed that most SlFBX genes were differentially expressed under abiotic stress. SlFBX24 was significantly up-regulated at 12 h under cold stress. This study reported the SlFBX gene family of tomato for the first time, providing a theoretical basis for the detailed study of SlFBX genes in the future, especially the function of SlFBX genes under abiotic stress.

Development and evaluation of a novel loop mediated isothermal amplification coupled with TaqMan probe assay for detection of genetically modified organism with NOS terminator.

In this study, we aim to develop a novel loop mediated isothermal amplification (LAMP) coupled with TaqMan (LAMP-TaqMan) method for quick qualitative detection of genetically modified organism (GMOs). We designed four LAMP primers and one TaqMan probe for the LAMP-TaqMan detection method to detect the nopaline synthase gene (NOS) terminator in GMOs. This assay enabled the amplification of DNA within ~20 min at a constant temperature of 65 °C. This assay detected as few as five copies of target sequences, which had a high specificity similar to the TaqMan qPCR method. Furthermore, the LAMP-TaqMan detection method was successfully used to amplify and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel LAMP-TaqMan assay has been developed, which has the similar sensitivity but takes less time than the TaqMan qPCR method. This method offers a novel approach for rapid detection of GMOs in foods.

Morphological and anatomical characteristics of exserted stigma sterility and the location and function of SlLst (Solanum lycopersicum Long styles) gene in tomato.

KEY MESSAGE: Anatomical changes in and hormone roles of the exserted stigma were investigated, and localization and functional analysis of SlLst for the exserted stigma were performed using SLAF-BSA-seq, parental resequencing and overexpression of SlLst in tomato. Tomato accession T431 produces stigmas under relatively high temperatures (> 27 °C, the average temperature in Harbin, China, in June-August), so pollen can rarely reach the stigma properly. This allows the percentage of male sterility exceed 95%, making the use of this accession practical for hybrid seed production. To investigate the mechanism underlying the exserted stigma male sterility, the morphological changes of, anatomical changes of, and comparative endogenous hormone (IAA, ABA, GA3, ZT, SA) changes in flowers during flower development of tomato accessions DL5 and T431 were measured. The location and function of genes controlling exserted stigma sterility were analyzed using super SLAF-BSA-seq, parental resequencing, comparative genomics and the overexpression of SlLst in tomato. The results showed that an increase in cell number mainly caused stigma exsertion. IAA played a major role, while ABA had an opposite effect on stigma exertion. Moreover, 26 candidate genes related to the exserted stigma were found, located on chromosome 12. The Solyc12g027610.1 (SlLst) gene was identified as the key candidate gene by functional analysis. A subcellular localization assay revealed that SlLst is targeted to the nucleus and cell membrane. Phenotypic analysis of SlLst-overexpressing tomato showed that SlLst plays a crucial role during stigma exsertion.

Proteomic Analysis Reveals Proteins and Pathways Associated with Lactation in Bovine Mammary Epithelial Cell-Derived Exosomes.

Milk-derived exosomes have been reported, which are involved in many biological processes. The exosomes derived from mammary glands are not known yet, and their relationship with mammary gland lactation and the origin of milk-derived exosomes are largely unclear. The present study aimed to investigate the proteome of exosomes derived from bovine mammary epithelial cells (BMECs) and compare them with milk-derived exosomes in the database. BMEC-derived exosomes were successfully separated from the culture supernatant of BMECs by a combined ultracentrifugation approach, and the purity of exosomes was identified by western blot analysis. Liquid chromatography with tandem mass spectrometry identified 638 proteins in BMEC-derived exosomes. The MS data were deposited into the PUBLIC repository ProteomeXchange, dataset identifier(s): https://www.iprox.org/page/PSV023.html;?url=1590961453176tKpa. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these proteins were associated with specific biological processes and molecular functions of metabolism. Cross comparison of these proteins with the protein database of milk exosomes showed that 77 common expressed proteins (CEPs) were in both BMEC- and milk-derived exosomes. The KEGG pathway analysis for these CEPs showed that they were mainly involved in signaling pathways associated with milk biosynthesis in BMECs. Among these CEPs, six proteins have been previously reported to be associated with the lactation function. The western blot analysis detected that expression of these six proteins in BMEC-derived exosomes was increased after the stimulation of methionine and ß-estradiol on BMECs. In summary, the proteome of BMEC-derived exosomes reveals that they are associated with milk biosynthesis in BMECs and might be a source of milk-derived exosomes.

A Biocontrol Strain of Pseudomonas aeruginosa CQ-40 Promote Growth and Control Botrytis cinerea in Tomato.

Botrytis cinerea infection can be very devastating for tomato production, as it can result in a large-scale reduction in tomato fruit production and fruit quality after harvest. Thus, it negatively affects tomato yield and quality. In this study, a biocontrol bacteria CQ-4 was isolated and screened from the rhizosphere soil of tomato plants. Morphological, physiological, and biochemical characteristics and 16S rDNA sequence analysis revealed that it belongs to the species Pseudomonas aeruginosa, which has a strong antagonistic effect against Botrytis cinerea. In addition, the bacterium's antibacterial spectrum is relatively extensive, and antagonistic tests have shown that it also has varying degrees of inhibition on other 12 plant diseases. The growth promotion test showed that the strain has a clear promotion effect on tomato seed germination and seedling growth. The growth-promoting effect on plant height, stem thickness, dry and fresh weight and main root length of tomato seedlings was significantly improved after the seeds were soaked in a bacterial solution of 2.5 × 108 cfu mL-1 concentration. This did not only maintain the nutritional quality of tomato fruits, but also prevents them from rotting. In vitro and pot experiments showed that the strain CQ-4 can effectively control tomato gray mold, and the control effects on tomato leaves and fruits reached 74.4% and 66.0%, respectively. Strain CQ-4 induce plants to up-regulate the activities of four disease-resistant defense enzymes. The peak enzymatic activities of Phenylalanine Ammonia Lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and Superoxide Dismutase (SOD) were increased by 35.6%, 37.6%, 46.1%, and 38.4%, respectively, as compared with the control group. This study found that the strain can solubilize phosphorus, fix nitrogen, and produce cellulase, protease, ferrophilin, and other antibacterial metabolites, but it does not produce chitinase, glucanase, and HCN (hydrocyanic acid). This research screened out an excellent Pseudomonas aeruginosa strain that can stably and effectively control tomato gray mold, and it provided theoretical basis for further development and the application of biological agents.

GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway.

BACKGROUND: Glucose-regulated protein 78 (GRP78) is a member of the HSP70 protein family and a key endoplasmic reticulum chaperone. It has been revealed to play important roles both in the maturation, folding and transport of proteins and in cellproliferation. However, its involvement in milk biosynthesis or the proliferation of bovine primary mammary epithelial cells (BMECs) has yet to be established. METHODS: The expressions of GRP78 in BMECs stimulated with methionine, leucine, estrogen and prolactin were determined using western blotting and immunofluorescence assays. To explore the function of GRP78 in BMECs, the protein was overexpressed or knocked down, respectively using an overexpression vector or an siRNA mixture transfected into cells cultured in vitro. Flow cytometry was used to analyze cell proliferation and cell activity. The contents of lactose and triglyceride (TG) secreted from the treated BMECs were measured using lactose and TG assay kits, respectively. Western blotting analysis was used to measure the ß-casein content and the protein levels of the signaling molecules known to be involved in milk biosynthesis and cell proliferation. RESULTS: GRP78overexpression significantly stimulated milk protein and milk fat synthesis, enhanced cell proliferation, positively regulated the phosphorylation of mammalian target of rapamycin (mTOR), and increased the amount of protein of cyclinD1andsterol regulatory element-binding protein 1c (SREBP-1c). GRP78 knockdown after siRNA transfection had the opposite effects. We further found that GRP78 was located in the cytoplasm of BMECs, and that stimulating methionine, leucine, estrogen and prolactin expression led to a significant increase in the protein expression of GRP78 in BMECs. CONCLUSIONS: These data reveal that GRP78 is an important regulator of milk biosynthesis and the proliferation of BMECs through the mTOR signaling pathway.

Leucine Promotes Milk Synthesis in Bovine Mammary Epithelial Cells via the PI3K-DDX59 Signaling.

Leucine is an essential amino acid in the milk production of bovine mammary glands, but the regulatory roles and molecular mechanisms of leucine are still not known well. This study investigated the roles of leucine on milk synthesis and explored the corresponding mechanism in bovine mammary epithelial cells (BMECs). Leucine (0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM) was added to BMECs that were cultured in FBS-free OPTI-MEM medium. Leucine significantly promoted milk protein and milk fat synthesis and also increased phosphorylation of mTOR signaling protein and the protein expression levels of SREBP-1c, with the most significant effects at 0.75 mM concentration. Leucine increased the expression and nuclear localization of DDX59, and loss and gain of gene function experiments further reveal that DDX59 mediates the stimulation of leucine on the mRNA expression variation of mTOR and SREBP-1c genes. PI3K inhibition experiment further detected that leucine upregulated expression of DDX59 and its downstream signaling via PI3K activation. ChIP-qPCR analysis further proved the binding of DDX59 to the promoter regions of mTOR and SREBP-1c. In summary, these data prove that DDX59 positively regulates the mTOR and SREBP-1c signaling pathways leading to synthesis of milk, and leucine regulates these two signaling pathways through the PI3K-DDX59 signaling.

DEAD-box helicase 6 (DDX6) is a new negative regulator for milk synthesis and proliferation of bovine mammary epithelial cells.

Milk synthesis of bovine mammary gland is a complex biological process that is regulated by hormones and nutrients, but the mechanism of these regulations still needs further research. DEAD-box helicase 6 (DDX6) is an important member of the RNA helicase family, involved in the regulation of mRNA storage and translation in different systems, but its physiological role and mechanism are largely unclear. In this study, we describe DDX6 as a potentially novel negative regulator for milk synthesis and proliferation of bovine mammary epithelial cells (BMECs). Treatment of BMECs with amino acids (methionine or leucine) or hormones (estrogen or prolactin) decreased the expression of DDX6. DDX6 expression was lower in mammary tissues of lactation period than in mammary tissues of puberty and dry period. Notably, overexpressing DDX6 in BMECs significantly decreased milk synthesis, cell proliferation, and protein levels of p-mTOR, SREBP-1c, and cyclin D1, while inhibiting DDX6 had the opposite effect. Taken together, these results reveal that DDX6 is a new negative regulator to control milk synthesis and proliferation of BMECs.

Computational identification and characterization of novel microRNA in the mammary gland of dairy goat (Capra hircus).

Many studies have indicated that microRNAs (miRNAs) influence the development of the mammary gland by posttranscriptionally affecting their target genes. The objective of this research was to identify novel miRNAs in the mammary gland of dairy goats with a bioinformatics approach that was based on expressed sequence tag (EST) and genome survey sequence (GSS) analyses. We applied all known major mammals, miRNAs to search against the goat EST and GSS databases for the first time to identify new miRNAs. We, then, validated these newly predicted miRNAs with stem-loop reverse transcription followed by a SYBR Green polymerase chain reaction assay. Finally, 29 mature miRNAs were identified and verified, and of these, 14 were grouped into 13 families based on seed sequence identity and 85 potential target genes of newly verified miRNAs were subsequently predicted, most of which seemed to encode the proteins participating in regulation of metabolism, signal transduction, growth and development. The predicting accuracy of the new miRNAs was 70.37%, which confirmed that the methods used in this study were efficient and reliable. Detailed analyses of the sequence characteristics of the novel miRNAs of the goat mammary gland were performed. In conclusion, these results provide a reference for further identification of miRNAs in animals without a complete genome and thus improve the understanding of miRNAs in the caprine mammary gland.

Effects of low temperature on mRNA and small RNA transcriptomes in Solanum lycopersicoides leaf revealed by RNA-Seq.

The plant low temperature tolerance mechanisms have been studied in the Arabidopsis, tomato, Solanum commersonii, Solanum tuberosum, Chorispora bungeana, and Chinese cabbage at the transcriptional level. Some genome-wide works to identify cold-regulated genes, but no comprehensive research of the Solanum lycopersicoides transcriptome under low temperature stress have been performed. S. lycopersicoides is more freeze-tolerant than the cultivated tomato. We analyzed the low temperature transcriptomes and small RNA fractions of S. lycopersicoides leaf tissue using an Illumina platform for high-throughput RNA sequencing (RNA-seq). There were 59,286 unigenes obtained using de novo assembly, and 2052 down-regulated and 2409 up-regulated unigenes were identified in response to chilling. The expression of six cold-regulated genes was confirmed by qPCR. Some biological processes were showed, by gene ontology term enrichment analysis of the cold-regulated genes, including 'response to stimulus', 'signaling', and 'cell killing' in the response of S. lycopersicoides to chilling. In addition, we identified a total of 952 novel miRNA candidates that may regulate relevant target genes. Our data indicated that certain miRNAs (e.g., sly-miR156a, sly-miR397, and unconservative_SL2.50ch00_21686) play roles in response to low temperature stress. Sequencing of mRNAs and miRNAs revealed new genes and allowed us to have new assumptions for a low temperature tolerance mechanism.