Search Results (20)

Elephant grass (Cenchrus purpureus) is an important forage crop hindered by high lignin content. Although MYB transcription factors (TFs) regulate lignin biosynthesis, their roles in elephant grass remain unclear. In this study, we identified 247 CpMYB TFs through whole-genome bioinformatic analysis of elephant grass and classified them into 23 phylogenetic subgroups. Among them, 233 were mapped to 14 chromosomes, and 14 to unanchored contigs. Gene structure, conserved motifs, and domain analyses revealed subgroup-specific conservation and CpMYB proteins dominated by random coils and α-helices. Gene duplication and selection pressure analyses indicated that segmental duplication predominantly contributed to family expansion. Transcriptome analysis identified 48 CpMYB genes differentially expressed in internodes at least one of three developmental stages, with promoters containing various growth-, phytohormone-, and stress-related cis-elements. Additionally, nine CpMYB genes were consistently differentially expressed across all three stages, and predicted protein–DNA interaction suggested that four of them (CpMYB094, CpMYB131, CpMYB145, and CpMYB148) potentially regulate key lignin biosynthetic genes, including 4-coumarate:CoA ligase 1 (4CL1), hydroxycinnamoyl transferase (HCT), caffeoyl-CoA O-methyltransferase 1/7 (CCoAOMT1/7), and reduced epidermal fluorescence 3 (REF3). However, their regulatory functions require further experimental validation. Overall, this study characterizes the CpMYB family in elephant grass and highlights their potential roles in lignin biosynthesis. Full article

Metabolic dysfunction-associated fatty liver disease (MAFLD) affects approximately one-quarter of the world’s adult population, and no effective therapeutic drugs are available. Poria cocos is a fungus used as a herb and food nutrient for centuries as well as for MAFLD treatment. Exosome-like nanovesicles have many pharmacological activities; however, studies on the effects of Poria cocos-derived exosome-like nanovesicles (PCELNs) on MAFLD are lacking. Therefore, our study aimed at identifying the effects and mechanism of action of PCELNs on MAFLD. PCELNs were isolated by ultracentrifugation and their morphology was characterized, such as particle size, zeta potential, protein distributions, as well as lipid and miRNA compositions. Then, the absorption and distribution of PCELNs were observed in vivo and in vitro. Finally, L02 cell steatosis model induced by fat emulsion and MAFLD mouse model induced by high-fat diet (HFD) were used to evaluate the effect and mechanism of PCELNs on MAFLD. PCELNs were membrane structured vesicles, with a particle size of 161.4 ± 1.7 nm, a zeta potential of −3.20 ± 0.37 mV, and contained a range of proteins, lipids, and miRNAs. PCELNs were absorbed by L02 cells and targeted the liver and spleen after intraperitoneal injection. PCELNs inhibited body weight gain and improved the index of heart, liver, spleen, and various fats, as well as decreased lipid accumulation and lipid level. They also protected mitochondrial ultrastructure and regulated oxidative stress and energy metabolism disorder. Furthermore, PCELNs increased PTEN induced kinase 1 (PINK1), E3 ubiquitin ligase (Parkin) and microtubule associated protein light chain-3 (LC3) protein expression in the liver, reduced oxidized mitochondrial DNA (Ox-mtDNA) content in mitochondria and cytoplasm of the liver, reduced nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3), pro-cysteinyl aspartate specific proteinase-1 (caspase-1), cleared-caspase-1, and mature-interleukin-1β (IL-1β) protein expression in the liver, and reduced the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and interleukin-18 (IL-18) in serum and liver. In conclusion, we demonstrated that PCELNs may alleviate HFD-induced MAFLD by promoting mitochondrial autophagy and inhibiting NLRP3 inflammasome activation. Full article

Concerns have been raised regarding the potential adverse health effects of the ubiquitous herbicide glyphosate. Here, we investigated long-term effects of developmental exposure to a glyphosate-based herbicide (GBH) by analyzing serum melatonin levels and cellular changes in the striatum of adult male rats (90 days old). Pregnant and lactating rats were exposed to 3% GBH (0.36% glyphosate) through drinking water from gestational day 5 to postnatal day 15. The offspring showed reduced serum melatonin levels (43%) at the adult age compared with the control group. The perinatal exposure to GBH also induced long-term oxidative stress-related changes in the striatum demonstrated by increased lipid peroxidation (45%) and DNA/RNA oxidation (39%) together with increased protein levels of the antioxidant enzymes, superoxide dismutase (SOD1, 24%), glutamate–cysteine ligase (GCLC, 58%), and glutathione peroxidase 1 (GPx1, 31%). Moreover, perinatal GBH exposure significantly increased the total number of neurons (20%) and tyrosine hydroxylase (TH)-positive neurons (38%) in the adult striatum. Mechanistic in vitro studies with primary rat pinealocytes exposed to 50 µM glyphosate demonstrated a decreased melatonin secretion partially through activation of metabotropic glutamate receptor 3 (mGluR3), while higher glyphosate levels (100 or 500 µM) also reduced the pinealocyte viability. Since decreased levels of the important antioxidant and neuroprotector melatonin have been associated with an increased risk of developing neurodegenerative disorders, this demonstrates the need to consider the melatonin hormone system as a central endocrine-related target of glyphosate and other environmental contaminants. Full article

DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5′ phosphate and 3′ hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases. Full article

Prevention of muscle atrophy contributes to improved quality of life and life expectancy. In this study, we investigated the effects of laurel, selected from 34 spices and herbs, on dexamethasone (DEX)-induced skeletal muscle atrophy and deciphered the underlying mechanisms. Co-treatment of C2C12 myotubes with laurel for 12 h inhibited the DEX-induced expression of intracellular ubiquitin ligases—muscle atrophy F-box (atrogin-1/MAFbx) and muscle RING finger 1 (MuRF1)—and reduction in myotube diameter. Male Wistar rats were supplemented with 2% laurel for 17 days, with DEX-induced skeletal muscle atrophy occurring in the last 3 days. Laurel supplementation inhibited the mRNA expression of MuRF1, regulated DNA damage and development 1 (Redd1), and forkhead box class O 1 (Foxo1) in the muscles of rats. Mechanistically, we evaluated the effects of laurel on the cellular proteolysis machinery—namely, the ubiquitin/proteasome system and autophagy—and the mTOR signaling pathway, which regulates protein synthesis. These data indicated that the amelioration of DEX-induced skeletal muscle atrophy induced by laurel, is mainly mediated by the transcriptional inhibition of downstream factors of the ubiquitin-proteasome system. Thus, laurel may be a potential food ingredient that prevents muscle atrophy. Full article

MicroRNA166 (miR166) is a highly conserved plant miRNA that plays a crucial role in plant growth and the resistance to various abiotic stresses. However, the miR166s in tea (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. This study identified 30 mature miR166s and twelve pre-miR166s in tea plants. An evolutionary analysis revealed that csn-miR166s originating from the 3′ arm of their precursors were more conserved than the csn-miR166s derived from the 5′ arm of their precursors. The twelve pre-miR166s in tea were divided into two groups, with csn-MIR166 Scaffold364-2 separated from the other precursors. The Mfold-based predictions indicated that the twelve csn-MIR166s formed typical and stable structures comprising a stem-loop hairpin, with minimum free energy ranging from −110.90 to −71.80 kcal/mol. An analysis of the CsMIR166 promoters detected diverse cis-acting elements, including those related to light responses, biosynthesis and metabolism, abiotic stress defenses, and hormone responses. There was no one-to-one relationship between the csn-miR166s and their targets, but most csn-miR166s targeted HD-Zip III genes. Physiological characterization of tea plants under drought stress showed that leaf water content proportionally decreased with the aggravation of drought stress. In contrast, tea leaves’ malondialdehyde (MDA) content proportionally increased. Moreover, the cleavage site of the ATHB-15-like transcript was identified according to a modified 5′ RNA ligase-mediated rapid amplification of cDNA ends. The RT-qPCR data indicated that the transcription of nine csn-miR166s was negatively correlated with their target gene. Full article

The physiologic function of tripartite motif protein 56 (TRIM56), a ubiquitously expressed E3 ligase classified within the large TRIM protein family, remains elusive. Gene knockdown studies have suggested TRIM56 as a positive regulator of the type I interferon (IFN-I) antiviral response elicited via the Toll-like receptor 3 (TLR3) and cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathways, which detect and respond to danger signals—extracellular double-stranded (ds) RNA and cytosolic dsDNA, respectively. However, to what extent these pathways depend on TRIM56 in human cells is unclear. In addition, it is debatable whether TRIM56 plays a part in controlling the expression of IFN-stimulated genes (ISGs) resulting from IFN-I based antiviral treatment. In this study, we created HeLa-derived TRIM56 null cell lines by gene editing and used these cell models to comprehensively examine the impact of endogenous TRIM56 on innate antiviral responses. Our results showed that TRIM56 knockout severely undermined the upregulation of ISGs by extracellular dsRNA and that loss of TRIM56 weakened the response to cytosolic dsDNA. ISG induction and ISGylation following IFN-α stimulation, however, were not compromised by TRIM56 deletion. Using a vesicular stomatitis virus-based antiviral bioactivity assay, we demonstrated that IFN-α could efficiently establish an antiviral state in TRIM56 null cells, providing direct evidence that TRIM56 is not required for the general antiviral action of IFN-I. Altogether, these data ascertain the contributions of TRIM56 to TLR3- and cGAS–STING-dependent antiviral pathways in HeLa cells and add to our understanding of the roles this protein plays in innate immunity. Full article

Ubiquitin ligase (E3) plays a versatile role in gonadal development and spermatogenesis in mammals, while its function in fish is little reported. In this study, a Z-chromosome linked ubiquitin ligase rchy1 in C. semilaevis (Cs-rchy1) was cloned and characterized. The full-length cDNA was composed of 1962 bp, including 551 bp 5′UTR, 736 bp 3′UTR, and 675 bp ORF encoding a 224-amino-acid (aa) protein. Cs-rchy1 was examined among seven different tissues and found to be predominantly expressed in gonads. In testis, Cs-rchy1 could be detected from 40 days post hatching (dph) until 3 years post hatching (yph), but there was a significant increase at 6 months post hatching (mph). In comparison, the expression levels in ovary were rather stable among different developmental stages. In situ hybridization showed that Cs-rchy1 was mainly localized in germ cells, that is, spermatid and spermatozoa in testis and stage I, II and III oocytes in ovary. In vitro RNA interference found that Cs-rchy1 knockdown resulted in the decline of sox9 and igf1 in ovarian cell line and down-regulation of cyp19a in the testicular cell line. These data suggested that Cs-rchy1 might participate in gonadal differentiation and gametogenesis, via regulating steroid hormone synthesis. Full article

Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3′ hydroxyl and a 5′ phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD⁺ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC₅₀) in the micromolar to millimolar range (11–2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases. Full article

Wheat pre-harvest sprouting (PHS) causes serious losses in wheat yield. In this study, precise mapping was carried out in the chromosome segment substitution lines (CSSL) F₂ population generated by a direct cross of Zhoumai 18 (PHS-sensitive) and Aegilops tauschii accession T093 (highly PHS-resistant). Three Ae. tauschii-derived quantitative trait loci (QTLs), QDor.3D.1, QDor.3D.2, and QDor.3D.3, were detected on chromosome 3DL using four simple sequence repeats (SSR) markers and 10 developed Kompetitive allele-specific PCR (KASP) markers. Alongside these QTL results, the RNA-Seq and qRT-PCR analysis revealed expression levels of TraesCS3D01G466100 in the QDor.3D.2 region that were significantly higher in CSSLs 495 than in Zhoumai 18 during the seed imbibition treatment. The cDNA sequencing results of TraesCS3D01G466100 showed two single nucleotide polymorphisms (SNPs), resulting in two changed amino acid substitutions between Zhoumai 18 and line 495, and the 148 nt amino acid substitution of TraesCS3D01G466100, derived from Ae. tauschii T093, which may play an important role in the functioning of ubiquitin ligase enzymes 3 (E3) according to the homology protein analysis, which could lead to differential PHS-resistance phenotypes. Taken together, our results may foster a better understanding of the mechanism of PHS resistance and are potentially valuable for marker-assisted selection in practical wheat breeding efforts. Full article

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice. Full article

KLHL and the related KBTBD genes encode components of the Cullin-E3 ubiquitin ligase complex and typically target tissue-specific proteins for degradation, thereby affecting differentiation, homeostasis, metabolism, cell signaling, and the oxidative stress response. Despite their importance in cell function and disease (especially, KLHL40, KLHL41, KBTBD13, KEAP1, and ENC1), previous studies of epigenetic factors that affect transcription were predominantly limited to promoter DNA methylation. Using diverse tissue and cell culture whole-genome profiles, we examined 17 KLHL or KBTBD genes preferentially expressed in skeletal muscle or brain to identify tissue-specific enhancer and promoter chromatin, open chromatin (DNaseI hypersensitivity), and DNA hypomethylation. Sixteen of the 17 genes displayed muscle- or brain-specific enhancer chromatin in their gene bodies, and most exhibited specific intergenic enhancer chromatin as well. Seven genes were embedded in super-enhancers (particularly strong, tissue-specific clusters of enhancers). The enhancer chromatin regions typically displayed foci of DNA hypomethylation at peaks of open chromatin. In addition, we found evidence for an intragenic enhancer in one gene upregulating expression of its neighboring gene, specifically for KLHL40/HHATL and KLHL38/FBXO32 gene pairs. Many KLHL/KBTBD genes had tissue-specific promoter chromatin at their 5′ ends, but surprisingly, two (KBTBD11 and KLHL31) had constitutively unmethylated promoter chromatin in their 3′ exons that overlaps a retrotransposed KLHL gene. Our findings demonstrate the importance of expanding epigenetic analyses beyond the 5′ ends of genes in studies of normal and abnormal gene regulation. Full article

Microbial volatile organic compounds (VOCs) are cell metabolites that affect many physiological functions of prokaryotic and eukaryotic organisms. Earlier we have demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. Cyanobacteria are very sensitive to ketone action. To investigate the possible molecular mechanisms of the ketone 2-nonanone influence on cyanobacterium Synechococcus elongatus PCC 7942, we applied a genetic approach. After Tn5-692 transposon mutagenesis, several 2-nonanone resistant mutants have been selected. Four different mutant strains were used for identification of the impaired genes (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732, Synpcc7942_0726) that encode correspondingly: 1) a murein-peptide ligase Mpl that is involved in the biogenesis of cyanobacteria cell wall; 2) a putative ABC transport system substrate-binding proteins MlaD, which participates in ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane by aberrantly localized phospholipids transport from outer to inner membranes of bacterial cells; 3) a conserved hypothetical protein that is encoding by gene belonging to phage gene cluster in Synechococcus elongatus PCC 7942 genome; 4) a protein containing the VRR-NUC (virus-type replication-repair nuclease) domain present in restriction-modification enzymes involved in replication and DNA repair. The obtained results demonstrated that 2-nonanone may have different targets in Synechococcus elongatus PCC 7942 cells. Among them are proteins involved in the biogenesis and functioning of the cyanobacteria cell wall (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732) and protein participating in stress response at DNA restriction-modification level (Synpcc7942_0726). This paper is the first report about the genes that encode protein products, which can be affected by 2-nonanone. Full article

Carthamus tinctorius L. (safflower), an economic crop and herb, has been extensively studied for its diverse chemical constituents and pharmacological effects, but the mechanism of safflower pigments (SP) leading to different colors of florets has not been clarified. In the present study, we compared the contents of SP in two varieties of safflower with white and red florets, named Xinhonghua No. 7 (WXHH) and Yunhong No. 2 (RYH). The results showed the contents of SP in RYH were higher than WXHH. To investigate genes related to SP, we obtained six cDNA libraries of florets from the two varieties by transcriptome sequencing. A total of 225,008 unigenes were assembled and 40 unigenes related to safflower pigment biosynthesis were annotated, including 7 unigenes of phenylalanine ammonia-lyase (PAL), 20 unigenes of 4-coumarate-CoA ligase (4CL), 1 unigene of trans-cinnamate 4-monooxygenase (C4H), 7 unigenes of chalcone synthase (CHS), 4 unigenes of chalcone isomerase (CHI), and 1 unigene of flavanone 3-hydroxylase (F3H). Based on expression levels we selected 16 differentially expressed unigenes (DEGs) and tested them using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), which was consistent with the sequencing results. Consequently, we speculated that in WXHH, 3 PALs, 3 4CLs, 1 C4H, 1 CHS, and 1 CHI, which were down-regulated, and 1 F3H, which was up-regulated, may play a key role in the formation of white florets. Full article

Mitochondria plays privotal role in diverse pathways that regulate cellular function and survival, and have emerged as a prime focus in aging and age-associated motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Accumulating evidence suggests that many amyloidogenic proteins, including MND-associated RNA/DNA-binding proteins fused in sarcoma (FUS) and TAR DNA binding protein (TDP)-43, are strongly linked to mitochondrial dysfunction. Animal model and patient studies have highlighted changes in mitochondrial structure, plasticity, replication/copy number, mitochondrial DNA instability, and altered membrane potential in several subsets of MNDs, and these observations are consistent with the evidence of increased excitotoxicity, induction of reactive oxygen species, and activation of intrinsic apoptotic pathways. Studies in MND rodent models also indicate that mitochondrial abnormalities begin prior to the clinical and pathological onset of the disease, suggesting a causal role of mitochondrial dysfunction. Our recent studies, which demonstrated the involvement of specific defects in DNA break-ligation mediated by DNA ligase 3 (LIG3) in FUS-associated ALS, raised a key question of its potential implication in mitochondrial DNA transactions because LIG3 is essential for both mitochondrial DNA replication and repair. This question, as well as how wild-type and mutant MND-associated factors affect mitochondria, remain to be elucidated. These new investigation avenues into the mechanistic role of mitochondrial dysfunction in MNDs are critical to identify therapeutic targets to alleviate mitochondrial toxicity and its consequences. In this article, we critically review recent advances in our understanding of mitochondrial dysfunction in diverse subgroups of MNDs and discuss challenges and future directions. Full article