Microcin C7 as a Potential Antibacterial-Immunomodulatory Agent in the Postantibiotic Era: Overview of Its Bioactivity Aspects and Applications.

In the postantibiotic era, the pathogenicity and resistance of pathogens have increased, leading to an increase in intestinal inflammatory disease. Bacterial infections remain the leading cause of animal mortality. With increasing resistance to antibiotics, there has been a significant decrease in resistance to both inflammation and disease in animals, thus decreasing production efficiency and increasing production costs. These side effects have serious consequences and have detracted from the development of China's pig industry. Microcin C7 (McC7) demonstrates potent antibacterial activity against a broad spectrum of pathogens, stable physicochemical properties, and low toxicity, reducing the likelihood of resistance development. Thus, McC7 has received increasing attention as a potential clinical antibacterial and immunomodulatory agent. McC7 has the potential to serve as a new generation of antibiotic substitutes; however, its commercial applications in the livestock and poultry industry have been limited. In this review, we summarize and discuss the biosynthesis, biochemical properties, structural characteristics, mechanism of action, and immune strategies of McC7. We also describe the ability of McC7 to improve intestinal health. Our aim in this study was to provide a theoretical basis for the application of McC7 as a new feed additive or new veterinary drug in the livestock and poultry breeding industry, thus providing a new strategy for alleviating resistance through feed and mitigating drug resistance. Furthermore, this review provides insight into the new functions and anti-infection mechanisms of bacteriocin peptides and proposes crucial ideas for the research, product development, and application of bacteriocin peptides in different fields, such as the food and medical industries.

New N-Terminal Fatty-Acid-Modified Melittin Analogs with Potent Biological Activity.

Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation.

Comprehensive Proteome and Acetylome Analysis of Needle Senescence in Larix gmelinii.

Leaf senescence is essential for the growth and development of deciduous trees in the next season. Larix gmelinii, a deciduous coniferous tree, exhibits its most distinctive feature by turning yellow in the autumn and eventually shedding its leaves, resulting in significant changes in its appearance during the fall. Lysine acetylation plays an important role in diverse cellular processes; however, limited knowledge is available regarding acetylations in the needle senescence of L. gmelinii. In this study, the proteomics and acetylated modification omics of two phenotypic leaves, yellow and green (senescent and non-senescent) needles, were analyzed before autumn defoliation. In total, 5022 proteins and 4469 unique acetylation sites in 2414 lysine acylated proteins were identified, and this resulted in the discovery of 1335 differentially expressed proteins (DEPs) and 605 differentially expressed acetylated proteins (DAPs) in yellow versus green needles. There are significant differences between the proteome and acetylome; only 269 proteins were found to be DEP and DAP, of which 136 proteins were consistently expressed in both the DEP and DAP, 91 proteins were upregulated, and 45 proteins were down-regulated. The DEPs participate in the metabolism of starch and sucrose, while the DAPs are involved in glycolysis and the tricarboxylic acid cycle. Among them, DEPs underwent significant changes in glycolysis and citric acid cycling. Most of the enzymes involved in glycolysis and the citrate cycle were acetylated. DAPs were down-regulated in glycolysis and up-regulated in the citrate cycle. In all, the results of this study reveal the important role of lysine acetylation in the senescence of L. gmelinii needles and provide a new perspective for understanding the molecular mechanism of leaf senescence and tree seasonal growth.

Porcine ß-Defensin 114: Creating a Dichotomous Response to Inflammation.

The immunity-related functions of defensins seem to be dependent on environmental stimuli, the cell type, and the concentration of peptides. However, the function and mechanism of porcine ß-defensin 114 (pBD114) in regulating the inflammatory response to macrophages are unclear. Therefore, the modulatory effects of porcine pBD114 on the inflammatory response were investigated by treating the mouse monocyte macrophage cell line RAW264.7 with different concentrations of pBD114 with or without lipopolysaccharide (LPS). RNA-seq analysis was performed to investigate the mechanisms underlying pBD114's regulation of inflammatory responses in macrophages. In addition, the inflammatory response-modulating effects of pBD114 were also further verified with a mouse assay. The results showed that 100 µg/mL of pBD114 significantly promoted the secretion of TNF-α and IL-10 in RAW264.7. However, the LPS-induced increase in TNFα in the RAW264.7 cell cultures was significantly decreased with 10 µg/mL of pBD114. These results suggest that pBD114 can exhibit pro-inflammatory activities under normal physiological conditions with 100 µg/mL of pBD114, and anti-inflammatory activities during an excessive inflammatory response with 10 µg/mL of pBD114. RNA-seq analysis was performed to gain further insights into the effects of pBD114 on the inflammatory response. Among the pBD114-promoting RAW264.7 pro-inflammatory responses, pBD114 significantly up-regulated 1170 genes and down-regulated 724 genes. KEGG enrichment showed that the differentially expressed genes (DEGs) were significantly enriched in the immune- and signal-transduction-related signaling pathways. Protein-Protein Interaction (PPI) and key driver analysis (KDA) analyses revealed that Bcl10 and Bcl3 were the key genes. In addition, pBD114 significantly up-regulated 12 genes and down-regulated 38 genes in the anti-inflammatory response. KEGG enrichment analysis revealed that the DEGs were mainly enriched in the "Cytokine-cytokine receptor interaction" signaling pathway, and PPI and KDA analyses showed that Stat1 and Csf2 were the key genes. The results of qRT-PCR verified those of RNA-seq. In vivo mouse tests also confirmed the pro- or anti-inflammatory activities of pBD114. Although the inflammatory response is a rapid and complex physiological reaction to noxious stimuli, this study found that pBD114 plays an essential role mainly by acting on the genes related to immunity, signal transduction, signaling molecules, and interactions. In conclusion, this study provides a certain theoretical basis for the research and application of defensins.

Soybean oligosaccharides combined with probiotics reduce faecal odour compound content by improving intestinal microbiota in pigs.

As a potential prebiotic, soybean oligosaccharides (SBOS) can improve animal health by modulating gut microbiota. The aim of this study was to investigate the different effects of supplementing SBOS and supplementing SBOS plus probiotic on the growth and health of pigs. Three groups of growing pigs (n = 12) were fed with basal diet (Control), basal diet + 0.5% SBOS (SBOS), or basal diet +0.5% SBOS + 0.1% compound probiotics (SOP) for 42 days. Results showed that SBOS and SOP treatments had positive effects on the pigs in the experiment, and the latter was more effective. Compared with the control pigs, the average daily gain of SBOS group and SOP group slightly increased, SOP significantly increased the serum levels of growth hormone and thyroid hormone T3. Importantly, serum concentrations of immunoglobulin (IgA, IgG and IgM), total antioxidant capacity and superoxide dismutase in both treatments were increased significantly, SOP group most. Moreover, the faecal odour compounds of pigs, especially skatole, were significantly reduced by the treatments. Additionally, SOP significantly increased the diversity and richness of the faecal microbiota, both the treatments increased genera of norank_f_Muribaculaceae and Ruminococcaceae but reduced Lactobacillus. Correlation analysis indicated that Lactobacillus was significantly positively correlated with odour compounds, while Ruminococcaceae was the opposite. Conclusively, synbiotics combined with SBOS and probiotics had stronger promotion effects on the growth and health of pigs.

Time-Series Clustering of lncRNA-mRNA Expression during the Adipogenic Transdifferentiation of Porcine Skeletal Muscle Satellite Cells.

Skeletal muscle satellite cells (SMSCs), which are multifunctional muscle-derived stem cells, can differentiate into adipocytes. Long-chain non-coding RNA (lncRNA) has diverse biological functions, including the regulation of gene expression, chromosome silencing, and nuclear transport. However, the regulatory roles and mechanism of lncRNA during adipogenic transdifferentiation in muscle cells have not been thoroughly investigated. Here, porcine SMSCs were isolated, cultured, and induced for adipogenic differentiation. The expressions of lncRNA and mRNA at different time points during transdifferentiation were analysed using RNA-seq analysis. In total, 1005 lncRNAs and 7671 mRNAs showed significant changes in expression at differential differentiation stages. Time-series expression analysis showed that the differentially expressed (DE) lncRNAs and mRNAs were clustered into 5 and 11 different profiles with different changes, respectively. GO, KEGG, and REACTOME enrichment analyses revealed that DE mRNAs with increased expressions during the trans-differentiation were mainly enriched in the pathways for lipid metabolism and fat cell differentiation. The genes with decreased expressions were mainly enriched in the regulation of cell cycle and genetic information processing. In addition, 1883 DE mRNAs were regulated by 193 DE lncRNAs, and these genes were related to the controlling in cell cycle mainly. Notably, three genes in the fatty acid binding protein (FABP) family significantly and continuously increased during trans-differentiation, and 15, 13, and 11 lncRNAs may target FABP3, FABP4, and FABP5 genes by cis- or trans-regulation, respectively. In conclusion, these studies identify a set of new potential regulator for adipogenesis and cell fate and help us in better understanding the molecular mechanisms of trans-differentiation.

Extracellular vesicles derived from miR-199a-5p-modified adipose-derived mesenchymal stem cells alleviate immune thrombocytopenia by inhibiting T helper 17 differentiation.

The abnormal differentiation of T helper 17 (Th17) cells is considered a vital promoter of immune thrombocytopenia (ITP) progression. Therefore, this study investigated the role of miR-199a-5p in Th17 differentiation and determined whether extracellular vesicles (EVs) derived from miR-199a-5p-modified adipose-derived mesenchymal stem cells (ADSCs) could relieve ITP by inhibiting Th17 differentiation. The miR-199a-5p level was lessened in the spleen tissues of mice with ITP, while the signal transducer and activator of transcription 3 (STAT3) expression and the population of Th17 in CD4+T cells were boosted. Functionally, miR-199a-5p overexpression lowered IL-17 secretion and the proportion of Th17/CD4+T cells. Further investigation showed that miR-199a-5p directly targeted STAT3 mRNA, and negatively modulated its expression. STAT3 overexpression was found to facilitate Th17 differentiation, which was subsequently abolished by miR-199a-5p overexpression. EVs isolated from miR-199a-5p-modified ADSCs (miR-199a-5p-EVs) highly expressed miR-199a-5p and could restrain CD4+T cells polarized toward a Th17 phenotype in vitro. Administering of miR-199a-5p-EVs elevated platelet counts and decreased the proportion of Th17/CD4+T cells in mice with ITP. Taken together, EVs derived from miR-199a-5p-modified ADSCs vividly repressed Th17 differentiation by transferring miR-199a-5p to CD4+T cells, thus ameliorating experimental ITP.

Transcriptome-based identification and expression profiling of AP2/ERF members in Caragana intermedia and functional analysis of CiDREB3.

BACKGROUND: The AP2/ERF transcription factor family plays important roles in regulation of plant growth and development as well as the response of plants to stress. However, there are currently few studies focusing on the function of the AP2/ERF-type transcription factors in Caragana intermedia Kuang et H. C. Fu. Here, the expression pattern of AP2/ERF transcription factors family in different tissues and under four stress treatments were evaluated, and the function of CiDREB3 was examined. METHODS AND RESULTS: In this study, the genes encoding the AP2/ERF family of transcription factors were screened from the C. intermedia drought transcriptome database and subjected to bioinformatic analysis using the online tool and software. The expression pattern of the members of AP2/ERF transcription factors in C. intermedia were detected via quantitative real-time PCR (qRT-PCR). The function of CiDREB3 on growth, development and drought tolerance was evaluated by transgenic Arabidopsis. As a result, 22 sequences with complete ORFs were obtained and all sequences were divided into 13 sub-groups. Most of the AP2/ERF transcription factors exhibited tissue-specific expression and were induced by cold, heat, NaCl and mannitol treatments. Furthermore, heterologous expression of CiDREB3 altered the morphology of the transgenic Arabidopsis thaliana L. Heynh and improved its drought tolerance during seedlings development. CONCLUSIONS: Taken together, the results of the present study helped to better understand the function of the AP2/ERF family transcription factors in response to multiple abiotic stresses and uncovered the role of CiDREB3 in affecting the morphology and abiotic stress tolerance of Arabidopsis.

Bacillus velezensis DP-2 isolated from Douchi and its application in soybean meal fermentation.

BACKGROUND: Soybean meal (SBM) is the most common protein source used in the poultry and livestock industries. It has high-quality protein, an excellent amino acid (AA) profile, and positive isoflavone properties. However, the antigen proteins in SBM are unsuitable for young animals. The objective of this study was to identify a Bacillus strain that can degrade soybean antigen proteins, and to evaluate the feasibility of its application in SBM fermentation. RESULTS: Bacillus velezensis DP-2 was isolated from Douchi, a fermented Chinese food. It degraded 96.14% and 66.51% of glycinin and ß-conglycinin, and increased the trichloroacetic acid-soluble protein (TCAN) content by 5.46 times in the SBM medium. DP-2 could secrete alkaline protease and neutral protease, with productivities of 5.85 and 5.99 U mL-1 . It had broad-spectrum, antibacterial activities against Rhizopus nigricans HR, Fusarium oxysporum ACCC37404, Penicillium digitatum SQ2, Aspergillus flavus C1, Aspergillus niger ACCC30005, Trichoderma viride YZ1, Candida tropicalis CICC1630, and Salmonella sp. ZY. For SBM fermentation, the optimal inoculum rate, temperature, and fermentation time of DP-2 were 2.21 × 107 CFU g-1 , 37 °C, and 48 h, respectively. The fermented soybean meal (FSBM) was cream-colored and glutinous. Its crude protein (CP), soluble protein, and TCA-N content were improved by 13.45%, 12.53%, and 6.37 times, respectively. The glycinin and ß-conglycinin content were reduced by 78.00% and 43.07%, respectively, compared with raw SBM. CONCLUSIONS: Bacillus velezensis DP-2 has potential as a starter culture for SBM fermentation. © 2020 Society of Chemical Industry.

Microbial and metabolic alterations in gut microbiota of sows during pregnancy and lactation.

The gut microbiota plays a critical role in various physiologic processes; however, maternal microbial and metabolic changes during pregnancy and lactation remain elusive. Using pigs as an animal model, we conducted comparative analyses of gut microbiota and short-chain fatty acid (SCFA) profiles across different stages of gestation, lactation, and the empty (nonpregnancy) phase in 2 distinct breeds of sow, Rongchang (RS) and Landrace (LS). Coriobacteriaceae were found to gradually increase over gestational time irrespective of breed, which was further validated in an independent cohort of sows, indicating that Coriobacteriaceae are likely associated with the progression of pregnancy. Escherichia increased as well. Relative to empty and gestation, lactation was associated with an increase in SCFA producers and a concomitant augmentation in SCFA production in both breeds. A comparison between the 2 breeds revealed that Ruminococcaceae were more abundant in RSs than in LSs, consistent with the strong ability of Rongchang pigs to digest highly fibrous feedstuffs. Taken together, we revealed characteristic structural and metabolic changes in maternal gut microbiota throughout pregnancy, lactation, and the empty phase, which could potentially help improve the pregnancy and lactation outcomes for both animals and humans.-Liu, H., Hou, C., Li, N., Zhang, X., Zhang, G., Yang, F., Zeng, X., Liu, Z., Qiao, S. Microbial and metabolic alterations in gut microbiota of sows during pregnancy and lactation.

Maternal milk and fecal microbes guide the spatiotemporal development of mucosa-associated microbiota and barrier function in the porcine neonatal gut.

BACKGROUND: The early-life microbiota exerts a profound and lifelong impact on host health. Longitudinal studies in humans have been informative but are mostly based on the analysis of fecal samples and cannot shed direct light on the early development of mucosa-associated intestinal microbiota and its impact on GI function. Using piglets as a model for human infants, we assess here the succession of mucosa-associated microbiota across the intestinal tract in the first 35 days after birth. RESULTS: Although sharing a similar composition and predicted functional profile at birth, the mucosa-associated microbiome in the small intestine (jejunum and ileum) remained relatively stable, while that of the large intestine (cecum and colon) quickly expanded and diversified by day 35. Among detected microbial sources (milk, vagina, areolar skin, and feces of sows, farrowing crate, and incubator), maternal milk microbes were primarily responsible for the colonization of the small intestine, contributing approximately 90% bacteria throughout the first 35 days of the neonatal life. Although maternal milk microbes contributed greater than 90% bacteria to the large intestinal microbiota of neonates upon birth, their presence gradually diminished, and they were replaced by maternal fecal microbes by day 35. We found strong correlations between the relative abundance of specific mucosa-associated microbes, particularly those vertically transmitted from the mother, and the expression levels of multiple intestinal immune and barrier function genes in different segments of the intestinal tract. CONCLUSION: We revealed spatially specific trajectories of microbial colonization of the intestinal mucosa in the small and large intestines, which can be primarily attributed to the colonization by vertically transmitted maternal milk and intestinal microbes. Additionally, these maternal microbes may be involved in the establishment of intestinal immune and barrier functions in neonates. Our findings strengthen the notion that studying fecal samples alone is insufficient to fully understand the co-development of the intestinal microbiota and immune system and suggest the possibility of improving neonatal health through the manipulation of maternal microbiota.

Development of Agrobacterium-mediated transient expression system in Caragana intermedia and characterization of CiDREB1C in stress response.

BACKGROUND: The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia. RESULTS: In this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment. CONCLUSIONS: The optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.

Comparison of LncRNA Expression Profiles during Myogenic Differentiation and Adipogenic Transdifferentiation of Myoblasts.

Myoblasts could transdifferentiate into adipocytes or adipocyte-like cells, which have the capability of producing and storing intracellular lipids. Long-chain non-coding RNAs (lncRNAs) have many important physiological functions in eukaryotes, which include regulating gene expression, chromosome silencing, and nuclear transport. However, changes in the expression of lncRNAs in muscle cells during adipogenic transdifferentiation have not been investigated to date. Here, C2C12 myoblasts were seeded and then induced to undergo myogenic and adipogenic transdifferentiation. The expression profiles of lncRNAs in various differentiated cells were analyzed and then compared by digital gene expression (DGE) RNA sequencing. A total of 114 core lncRNAs from 836 differentially expressed lncRNAs in adipogenic cells were identified. Further investigation by in silico analysis revealed that the target genes of core lncRNAs significantly enriched various signaling pathways that were related to glucose and lipid metabolism and muscle growth. The lncRNA-GM43652 gene was a potential regulator of adipogenesis in muscle cells. It showed the highest levels of expression in adipogenic cells, and the knocking down lncRNA-GM43652 negatively influenced lipid deposition in transdifferentiated myoblasts. This study has identified the novel candidate regulators that may be assessed in future molecular studies on adipogenic conversion of muscle cells.

Analysis of Metabolite Accumulation Related to Pod Color Variation of Caragana intermedia.

Caragana intermedia, a leguminous shrub widely distributed in cold and arid regions, is rich in secondary metabolites and natural active substances, with high nutritional and medical values. It is interesting that the pods of C. intermedia often show different colors among individual plants. In this study, 10-, 20- and 30-day-old red and green pods of C. intermedia were used to identify and characterize important metabolites associated with pod color. A total 557 metabolites, which could be classified into 21 groups, were detected in the pod extracts using liquid chromatography coupled with ESI-triple quadrupole-linear ion trap mass spectrometer (LC-ESI-MS/MS). Metabolomics analysis revealed significant differences in 15 groups of metabolites between red and green pods, including amino acids, nucleotide derivatives, flavonoids, and phytohormones. Metabolic pathway analysis showed that the shikimic acid and the phytohormone metabolic pathways were extraordinarily active in red pods, and the difference between red and green pods was obvious. Moreover, red pods showed remarkable flavonoids, cytokinins, and auxin accumulation, and the content of total flavonoids and proanthocyanidins in 30-day-old red pods was significantly higher than that in green pods. This metabolic profile contributes to valuable insights into the metabolic regulation mechanism in different color pods.

MicroRNA-224-5p regulates adipocyte apoptosis induced by TNFα via controlling NF-κB activation.

Tumor necrosis factor (TNF) α can induce cell apoptosis and activate nuclear transcription (NF)-κB in different cell types. Activated NF-κB further promotes or suppresses cellular apoptosis in different cases. The present study explored the effect of activated NF-κB on adipocyte apoptosis induced by TNFα and which microRNAs (miRNAs) were involved in the process. Our findings demonstrated that treatment of differentiated 3T3-L1 adipocytes with TNFα (20 ng/mL) rapidly activated NF-κB and induced moderate apoptosis. Pyrrolidinedithiocarbamic acid (PDTC, 60 µM), a specific NF-κB inhibitor, abated NF-κB activation that rendered the adipocytes vulnerable to TNFα-induced apoptosis. Dozens of miRNAs exhibited significant expression changes following TNFα treatment and the addition of PDTC. In which, miRNA-224-5p (miR-224) was up-regulated by TNFα exposure but down-regulated by PDTC addition. Furthermore, over-expression of miR-224 promoted NF-κB activation and prevented the adipocyte apoptosis induced by TNFα, while miR-224 deficiency showed the opposite effects. The TRAF-associated NF-κB activator (TANK) gene was identified as a direct target of miR-224 by computational and luciferase reporter assays. Additionally, silencing the TANK gene by the small interfering RNA similarly promoted NF-κB activation and attenuated the cellular apoptosis. In conclusion, these findings demonstrate that miR-224 plays an essential role in adipocyte apoptosis caused by TNFα through control of NF-κB activation via targeting the TANK gene.

Identification of the WRKY gene family and functional analysis of two genes in Caragana intermedia.

BACKGROUND: WRKY transcription factors, one of the largest families of transcriptional regulators in plants, play important roles in plant development and various stress responses. The WRKYs of Caragana intermedia are still not well characterized, although many WRKYs have been identified in various plant species. RESULTS: We identified 53 CiWRKY genes from C. intermedia transcriptome data, 28 of which exhibited complete open reading frames (ORFs). These CiWRKYs were divided into three groups via phylogenetic analysis according to their WRKY domains and zinc finger motifs. Conserved domain analysis showed that the CiWRKY proteins contain a highly conserved WRKYGQK motif and two variant motifs (WRKYGKK and WKKYEEK). The subcellular localization of CiWRKY26 and CiWRKY28-1 indicated that these two proteins localized exclusively to nuclei, supporting their role as transcription factors. The expression patterns of the 28 CiWRKYs with complete ORFs were examined through quantitative real-time PCR (qRT-PCR) in various tissues and under different abiotic stresses (drought, cold, salt, high-pH and abscisic acid (ABA)). The results showed that each CiWRKY responded to at least one stress treatment. Furthermore, overexpression of CiWRKY75-1 and CiWRKY40-4 in Arabidopsis thaliana suppressed the drought stress tolerance of the plants and delayed leaf senescence, respectively. CONCLUSIONS: Fifty-three CiWRKY genes from the C. intermedia transcriptome were identified and divided into three groups via phylogenetic analysis. The expression patterns of the 28 CiWRKYs under different abiotic stresses suggested that each CiWRKY responded to at least one stress treatment. Overexpression of CiWRKY75-1 and CiWRKY40-4 suppressed the drought stress tolerance of Arabidopsis and delayed leaf senescence, respectively. These results provide a basis for the molecular mechanism through which CiWRKYs mediate stress tolerance.

Expressions and Regulatory Effects of P38/ERK/JNK Mapks in the Adipogenic Trans-Differentiation of C2C12 Myoblasts.

BACKGROUND/AIMS: Myoblasts and muscle satellite cells have the potential to transdifferentiate into adipocytes or adipocyte-like cells. Previous studies suggest that mitogen-activated protein kinase (MAPK) is critical to adipogenic trans-differentiation of muscle cells. ERK1/2, P38 and JNK are three major MAPK family members; their activation and regulatory functions during adipogenic trans-differentiation of myoblasts are investigated. METHODS: C2C12 myoblasts were cultured and induced for adipogenic trans-differentiation. Activation patterns of MAPKs were assayed using protein microarray and Western blot. Three specific MAPK blockers, U0126, SB20358 and SP600125, were used to block ERK1/2, P38 and JNK during trans-differentiation. Cellular adipogenesis was measured using staining and morphological observations of cells and expression changes in adipogenic genes. RESULTS: Inhibitors reduced phosphorylation of corresponding MAPK and produced unique cellular effects. Suppressing P38 promoted adipogenic trans-differentiation and intensified adipolytic metabolism in differentiated cells. However, inhibition of ERK1/2 had the opposite effects on adipogenesis and no effect on adipolysis. Blocking JNK weakly blocked trans-differentiation but stimulated adipolysis and induced apoptosis. CONCLUSION: Three MAPKs participate in the regulation of myoblast adipogenic trans-differentiation by controlling adipogenic and adipolysis metabolism.

Expression Pattern and Regulatory Role of microRNA-23a in Conjugated Linoleic Acids-Induced Apoptosis of Adipocytes.

BACKGROUND/AIMS: Conjugated linoleic acids (CLAs) are known to induce apoptosis in adipocytes; however, the cellular mechanisms involved remained illdefined. We explored the different apoptotic induction effects of two CLA isomers on adipocytes and then investigated the expression and function of microRNAs (miRNAs) related to the apoptosis. METHODS: TUNEL and FCM assays were used to detect CLAs-induced adipocyte apoptosis. Microarrays were used to compare the differential expression of miRNAs. MiR-23a, a miRNA that showed significant changes in expression in the CLA-treated cells, was selected for the subsequent functional studies via over-expression and knock down in in vivo and in vitro experiments. RESULTS: C9, t11-CLA exhibited a stronger induction of apoptosis in the differentiated 3T3-L1 adipocytes than t10, c12-CLA. However, t10, c12-CLA could rapidly activate NF-κB, which may have caused their different apoptotic effects. MiR-23a was markedly down-regulated by the CLAs treatment and miR-23a over-expression attenuated CLA-induced apoptosis. Apoptosis protease-activating factor 1 (APAF1) was identified as a target gene of miR-23a. In an in vivo experiment endogenous miR-23a was down-regulated in mice fed with a mixture of both CLAs. The mice also exhibited less fat deposition and more apoptotic fat cells in adipose tissue. Moreover, endogenous miR-23a was suppressed in mice via intravenous injection with an antagomir which resulted in decreased body weight, increased number of apoptotic fat cells and increased APAF1 expression in adipose tissue. CONCLUSION: Taken together, our results suggest that miR-23a plays a critical role in CLA-induced apoptosis in adipocytes via controlling APAF1 expression.

Reference gene selection for qRT-PCR in Caragana korshinskii Kom. under different stress conditions.

Caragana korshinskii Kom., which is widely distributed in the northwest China and Mongolia, is an important forage bush belonging to the legume family with high economic and ecological value. Strong tolerance ability to various stresses makes C. korshinskii Kom. a valuable species for plant stress research. In this study, suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) were screened from 11 candidate reference genes, including ACT, GAPDH, EF1α, UBQ, TUA, CAP, TUB, TUB3, SKIP1, SKIP5-1 and SKIP5-2. A total of 129 samples under drought, heat, cold, salt, ABA and high pH treatment were profiled, and software such as geNORM, NormFinder and BestKeeper were used for reference gene evaluation and selection. Different suitable reference genes were selected under different stresses. Across all 129 samples, GAPDH, EF1α and SKIP5-1 were found to be the most stable reference genes, and EF1α+SKIP5-1 is the most stable reference gene combination. Conversely, TUA, TUB and SKIP1 were not suitable for using as reference genes owing to their great expression variation under some stress conditions. The relative expression levels of CkWRKY1 were detected using the stable and unstable reference genes and their applicability was confirmed. These results provide some stable reference genes and reference gene combinations for qRT-PCR under different stresses in C. korshinskii Kom. for future research work, and indicate that CkWRKY1 plays essential roles in response to stresses in C. korshinskii.