RESUMO
BACKGROUND: Inflammatory Bowel Disease (IBD) is a group of chronic idiopathic inflammatory diseases of the gastrointestinal (GI) tract associated with the dysbiosis of gut microbiota. Metabarcoding-based profiling of the gut microbiota of IBD patients is generally based on the stool samples collected from individual patients which rarely represent the mucosa-associated microbiota. The ideal sampling strategy for routine monitoring of the mucosal component of IBD has yet to be determined. METHODS: We hereby compare the microbiota composition of the colonic cleansing fluid (CCF) collected during colonoscopy with stool samples from IBD patients. The relationship between IBD and gut microbiota was revealed through the application of the 16S rRNA amplicon sequencing-based metabarcoding approach. CCF and stool samples were collected from IBD patients with Crohn's disease and ulcerative colitis. RESULTS: The present study shows significant differences in the microbial composition of CCF samples, presumably indicating changes in the mucosal microbiota of IBD patients as compared to the control group. Short-chain fatty acid-producing bacteria under the family Lachnospiraceae, the actinobacterial genus Bifidobacterium, the proteobacterial Sutterella and Raoultella are found to contribute to the microbial dysbiosis of the mucosal flora in IBD patients. CONCLUSIONS: CCF microbiota has the capacity to distinguish IBD patients from healthy controls and, thus, may constitute an alternative analysis strategy for the early diagnosis and disease progression in IBD biomarker research.
Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Microbiota , Humanos , Disbiose/microbiologia , RNA Ribossômico 16S/genética , Doenças Inflamatórias Intestinais/complicações , Microbiota/genética , Colite Ulcerativa/complicações , Mucosa Intestinal/microbiologia , Bactérias/genéticaRESUMO
Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.
Assuntos
Benzimidazóis/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Reprogramação Celular/efeitos dos fármacos , Proteína p300 Associada a E1A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Isoxazóis/farmacologia , Oxazepinas/farmacologia , Piperidinas/farmacologia , Benzimidazóis/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Inibidores Enzimáticos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isoxazóis/química , Estrutura Molecular , Oxazepinas/química , Piperidinas/química , Domínios Proteicos/efeitos dos fármacosRESUMO
Phenylalanine ammonia lyase (PAL) is the first enzyme of the phenylpropanoid pathway, and it is necessary to upregulate flavonoid biosynthesis in most of the plant species. In this study, we have cloned PAL gene from endemic Astragalus chrysochlorus which is a producer of phenolic nicotiflorin (kaempferol-3-O-rutinoside). The cDNA encoding PAL was cloned from A. chrysochlorus using RT-PCR (reverse transcription-polymerase chain reaction) with conserved primer pairs. Amino acid sequence alignments showed that AcPAL (2160 bp, Accession number: KM189182) has more than 95% amino acid identity with their homologues in other Astragalus species. The coding sequence for the protein of AcPAL is 720 amino acids with a calculated molecular weight of 78.53 kDa. Full length AcPAL was cloned and expressed in Escherichia coli. qPCR (quantitative real-time PCR) analysis of the expression of PAL gene of A. chrysochlorus suggested that maximum transcript level was observed in 3 h yeast extract elicited suspension cells. Our findings suggest that AcPAL plays role in early response for yeast extract treatment. The isolation of AcPAL gene could be result in further studies for overproduction of secondary metabolite, nicotiflorin.
Assuntos
Fabaceae/enzimologia , Flavonoides/biossíntese , Fenilalanina Amônia-Liase/genética , Proteínas de Plantas/genética , Sementes/enzimologia , Sequência de Aminoácidos , Técnicas de Cultura de Células , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fabaceae/genética , Flavonoides/isolamento & purificação , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Fenóis/isolamento & purificação , Fenilalanina Amônia-Liase/metabolismo , Células Vegetais/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
In this study, induction of regenerable callus from mature embryos in eight Turkish barley varieties was analysed by using different plant growth regulators (PGRs). Varying concentrations (0.5-4 mg l -1 ) of 2,4-dichlorophenoxyacetic acid (2,4-D) and dicamba (3,6-dichloro-o-anisic acid) were tested for callus induction from mature embryos. Highest percent of callus induction was observed in Bornova 92 variety (98.3%) on MS medium supplemented with 4 mg l -1 dicamba. Calli were transferred to regeneration media with 0.5 mg l -1 dicamba, 0.5 mg l -1 zeatin riboside (ZR) and 2 mg l -1 thidiazuron (TDZ). Low concentrations of dicamba induced multiple shoots during callus regeneration. When the effect of precultivation with 2,4-D or dicamba on the shoot induction were evaluated, lower concentrations (< 4 mg l -1 ) of auxins have been found optimal. On the regeneration medium with 0.5 mg l -1 dicamba, shoots were able to elongate up to 20 cm and shoot numbers were between 1-23 per callus. The use of ZR led to formation of short shoot buds and somatic embryos in 2 weeks period. The effect of TDZ was different from other PGRs by inducing green solid sectors on calli surfaces (Total 51 sectors/20 callus/Akhisar variety). Five plantlets have been grown from these solid cell clumps and transferred to specific media for root formation. As a result, five varieties (Süleyman Bey, Bornova 92, Vamyk Hoca, Kaya and Akhisar) tested in our study showed the potential to produce regenerable callus by using low amounts of dicamba or TDZ. The optimization process starts from culturing embryos to plantlet formation took nearly 4 weeks.
Assuntos
Hordeum/fisiologia , Regeneração , Sementes/fisiologia , Proliferação de Células/efeitos dos fármacos , Genótipo , Hordeum/efeitos dos fármacos , Hordeum/embriologia , Reguladores de Crescimento de Plantas/farmacologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Técnicas de Cultura de TecidosRESUMO
In this study it was aimed to investigate inducibility of secondary metabolism in Astragalus chrysochlorus by yeast extract which is known to cause the synthesis of defensive phenolic metabolites. Cell suspension cultures of Astragalus chrysochlorus responded to elicitor treatment (10 gl(-1) yeast extract) by increasing phenylalanine ammonia lyase (PAL E.C. 4.3.1.5) activity the key enzyme of phenylpropanoid pathway and accumulation of phenolic compounds. Yeast extract was added on 13th day in the cultures when cells were at early stage of logarithmic phase. The highest PAL activity (2.71 U microg protein(-1) min(-1)) was measured at 36 hr after addition of yeast extract and increasing of total phenolics accompanied with 221 microg g(-1) value as fresh weight (FW). Total phenolic content reached maximum value, 343.86 microg g(-1) FW, at 48 hr while control's value was 162 microg g(-1) FW. Maximum PAL activity and total phenolic content were 2.88 and 2.12 times higher than the controls of A. chrysochlorus cells, respectively. Our results indicate that there is a positive correlation between induced PAL activity and accumulation of total phenolics. It is considered that early defense response given to environmental stressors such as biotic and abiotic factors by upregulation of phenolic branch of secondary metabolism occurs in A. chrysochlorus with addition of yeast extract.
Assuntos
Astrágalo/metabolismo , Misturas Complexas/farmacologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Leveduras/química , Astrágalo/efeitos dos fármacos , Astrágalo/fisiologia , Células Cultivadas , Fenóis/metabolismoRESUMO
Astragalus species are medicinal plants that are used in the world for years. Some Astragalus species are known for selenium accumulation and tolerance and one of them is Astragalus chrysochlorus, a secondary selenium accumulator. In this study, we employed Illumina deep sequencing technology for the first time to de novo assemble A. chrysochlorus transcriptome and identify the differentially expressed genes after selenate treatment. Totally, 59,656 unigenes were annotated with different databases and 53,960 unigenes were detected in NR database. Transcriptome in A. chrysochlorus is closer to Glycine max than other plant species with 43,1 percentage of similarity. Annotated unigenes were also used for gene ontology enrichment and pathway enrichment analysis. The most significant genes and pathways were ABC transporters, plant pathogen interaction, biosynthesis of secondary metabolites and carbohydrate metabolism. Our results will help to enlighten the selenium accumulation and tolerance mechanisms, respectively in plants.
Assuntos
Astrágalo/metabolismo , RNA de Plantas/genética , Selênio/metabolismo , Transcriptoma , Adaptação Fisiológica/genética , Astrágalo/genética , Astrágalo/fisiologiaRESUMO
In this study it was aimed to investigate inducibility of secondary metabolism in Astragalus chrysochlorus by yeast extract which is known to cause the synthesis of defensive phenolic metabolites. Cell suspension cultures of Astragalus chrysochlorus responded to elicitor treatment (10 gl-1 yeast extract) by increasing phenylalanine ammonia lyase (PAL E.C. 4.3.1.5) activity, the key enzyme of phenylpropanoid pathway and accumulation of phenolic compounds. Yeast extract was added on 13th day in the cultures when cells were at early stage of logarithmic phase. The highest PAL activity (2.71 U μg protein-1 min-1) was measured at 36 hr after addition of yeast extract and increasing of total phenolics accompanied with 221 μg g-1 value as fresh weight (FW). Total phenolic content reached maximum value, 343.86 μg g-1 FW, at 48 hr while control’s value was 162 μg g-1 FW. Maximum PAL activity and total phenolic content were 2.88 and 2.12 times higher than the controls of A. chrysochlorus cells, respectively. Our results indicate that there is a positive correlation between induced PAL activity and accumulation of total phenolics. It is considered that early defense response given to environmental stressors such as biotic and abiotic factors by upregulation of phenolic branch of secondary metabolism occurs in A. chrysochlorus with addition of yeast extract.