Arnica montana stimulates extracellular matrix gene expression in human macrophages differentiated to wound-healing phenotype.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE77382 | GEO | 2016/11/17
SECONDARY ACCESSION(S): PRJNA310196
REPOSITORIES: GEO
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Project description:Arnica m. effects were associated with a purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. Here Arnica m. dilutions were tested using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24 h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c,9c, 15c or Control. None of these treatments affected cell viability. A total of 20 genes were differentially expressed comparing cells treated with Arnica m. 2c with those treated with Control only. Of these, 7 genes were up-regulated and 13 were down-regulated. Functional gene enrichment analysis showed that the most significantly upregulated function concerned 4 genes with a conserved site of EGF-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p <0.01). Protein assay in supernatants confirmed a statistically significant increase of fibronectin production in Arnica m. 2c treated cells (p<0.05). Pooled extracts of cells treated with increasing dilutions of Arnica m. (3c, 5c, 15c) showed up-regulation of the same group of genes although with lower effect size. The down-regulated transcripts derive from mitochondrial genes coding for some components of electron transport chain. These findings provide new insights into the action of Arnica m. in tissue healing and repair, identifying increased fibronectin production by macrophages as a major therapeutic target.
2016-11-17 | GSE77381 | GEO
Project description:Gelsemium sempervirens L. (Gelsemium) is a traditional medicinal plant which at ultra-low doses is employed as anxiolytic and antidepressant, but consistent evidence is lacking and the possible mechanisms involved at the level of nervous system are largely unknown. In this study we have investigated the gene expression of a human neurocyte cell line treated in vitro with increasing dilutions of Gelsemium extract. Starting from a crude extract containing the active principle gelsemine at the concentration of 6.5Ã10â4 M, six centesimal (c) dilutions of Gelsemium were prepared according to the French homeopathic pharmacopoeia: 2c, 3c, 4c, 5c, 9c and 30c. Human neuroblastoma cells SHSY5Y were exposed for 24h to Gelsemium and their transcriptome was compared with control cells treated with the corresponding vehicle solutions. RNA from cells treated with Gelsemium/controls dilutions was isolated and analyzed by microarray. Four biological replicates were tested for each condition. The exposure to the Gelsemium 2c dilution (the highest dose employed, corresponding to 6.5x10-9 M gelsemine) significantly changed the expression of 56 genes, 49 of which were down-regulated and 7 were overexpressed. These genes belong to G-protein coupled protein signalling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Cytotoxicity was excluded by WST-1 viability test. The mean fold change of the Gelsemium-downregulated geneset was negative compared to controls in each treatment condition. Although the effect in general decreased with increasing drug dilutions, cluster analysis allowed to identify some groups with typical profile of changes with different dilutions. Among the 49 genes down regulated by Gelsemium 2c, most showed decreased expression after treatment with all dilutions of Gelsemium and comparison with respective controls (Wilcoxon test) was highly significant: 47 genes were down regulated with 3c (p<0.0001), 42 with 4c (p<0.0001), 38 with 5c (p<0.0001), 30 with 9c (p<0.0001), 27 in 30c (p<0.0001). No significant differences of gene expression due to Gelsemium were found in a randomly chosen geneset of the whole cell trascriptome. In conclusion this study showed that Gelsemium, a medicinal plant used in traditional medicine, modulates several genes possibly involved in neuronal function. High throughput microarray technology allowed to detect a small but statistically significant response to extremely low doses/high dilutions, thus revealing an extreme sensitivity of human genome to this kind of pharmacological regulation. A 54 array study that includes both SHSY5Y and IMR32 human neuroblastoma cells samples to investigate the expression changes that result in the treatment with increasing dilutions of Gelsemium sempervirens plant ethanolic extract compared to controls. Each experiment with SHSY5Y cells contained 12 samples. The neurocyte cultures were treated for 24h with 6 dilutions of the Gelsemium sempervirens extract, namely 2c, 3c, 4c, 5c, 9c, 30c (c means centesimal dilution following homeopathic Pharmacopoeia) and the corresponding 6 dilutions of the vehicle solution (controls). Four biological replicates of the experiment were analyzed. IMR32 cells were exposed to Gelsemium 2c dilution and the corresponding control in triplicate experiments.
2015-11-01 | E-GEOD-42236 | biostudies-arrayexpress
Project description:Gelsemium sempervirens L. (Gelsemium) is a traditional medicinal plant which at ultra-low doses is employed as anxiolytic and antidepressant, but consistent evidence is lacking and the possible mechanisms involved at the level of nervous system are largely unknown. In this study we have investigated the gene expression of a human neurocyte cell line treated in vitro with increasing dilutions of Gelsemium extract. Starting from a crude extract containing the active principle gelsemine at the concentration of 6.5×10−4 M, six centesimal (c) dilutions of Gelsemium were prepared according to the French homeopathic pharmacopoeia: 2c, 3c, 4c, 5c, 9c and 30c. Human neuroblastoma cells SHSY5Y were exposed for 24h to Gelsemium and their transcriptome was compared with control cells treated with the corresponding vehicle solutions. RNA from cells treated with Gelsemium/controls dilutions was isolated and analyzed by microarray. Four biological replicates were tested for each condition. The exposure to the Gelsemium 2c dilution (the highest dose employed, corresponding to 6.5x10-9 M gelsemine) significantly changed the expression of 56 genes, 49 of which were down-regulated and 7 were overexpressed. These genes belong to G-protein coupled protein signalling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Cytotoxicity was excluded by WST-1 viability test. The mean fold change of the Gelsemium-downregulated geneset was negative compared to controls in each treatment condition. Although the effect in general decreased with increasing drug dilutions, cluster analysis allowed to identify some groups with typical profile of changes with different dilutions. Among the 49 genes down regulated by Gelsemium 2c, most showed decreased expression after treatment with all dilutions of Gelsemium and comparison with respective controls (Wilcoxon test) was highly significant: 47 genes were down regulated with 3c (p<0.0001), 42 with 4c (p<0.0001), 38 with 5c (p<0.0001), 30 with 9c (p<0.0001), 27 in 30c (p<0.0001). No significant differences of gene expression due to Gelsemium were found in a randomly chosen geneset of the whole cell trascriptome. In conclusion this study showed that Gelsemium, a medicinal plant used in traditional medicine, modulates several genes possibly involved in neuronal function. High throughput microarray technology allowed to detect a small but statistically significant response to extremely low doses/high dilutions, thus revealing an extreme sensitivity of human genome to this kind of pharmacological regulation.
2015-11-01 | GSE42236 | GEO
Project description:We then compared whole transcriptome bulk RNA-seq data of dHeps isolated from CCl4-injured mice and treated with either 5C, 3C, or DMSO. The expression of hepatocyte-enriched TFs and hepatocyte functional genes was upregulated in both the 5C and 3C groups, while mesenchymal markers (Acta2, Mmp3), inflammatory-related genes (Cxcl10, Ecm1, Ccl6, and Cd68), and oxidative stress-related genes (Ncf2 and Ncf1), were down-regulated compared with their expression in the DMSO control group
2023-11-21 | GSE233081 | GEO
Project description:Though important for gene regulation most studies of genome organisation use either fluorescence in situ hybridisation (FISH) or chromosome conformation capture (3C) methods. FISH directly visualises the spatial relationship of sequences, but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde crosslinking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organisation, but this has not been tested extensively. We investigate the murine HoxD locus with 5C and FISH in different developmental and activity states, and in the presence or absence of epigenetic regulators. We identify situations where the two datasets are concordant, but find other conditions where chromatin topographies extrapolated from 5C or FISH data are not compatible. We conclude that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart – influenced by nuclear environment and chromatin composition. We conclude that results obtained at high-resolution with either 3C methods or by FISH alone must be interpreted with caution and that conclusions about genome organisation should be validated by independent methods. 5C oligonucleotides were designed around EcoRI restriction sites following an alternative scheme
2015-01-02 | E-GEOD-61814 | biostudies-arrayexpress
Project description:Amartya Sanyal mailto:amartya.sanyal@umassmed.edu (Wet Lab), Bryan R. Lajoie mailto:bryan.lajoie@umassmed.edu, Gaurav Jain mailto:gaurav.jain@umassmed.edu (Dry Lab), Job Dekker mailto:job.dekker@umassmed.edu (Principal Investigator) This track contains chromatin interaction data generated using the 5C (Chromatin Conformation Capture Carbon Copy) method by the ENCODE group (Dekker Lab) located at the University of Massachusetts, Worcester, MA. This track shows the significant looping interactions between transcriptional start sites (TSS) and distal regulatory elements in the context of the 44 ENCODE pilot regions spanning 1% of the human genome. Although the DNA is a linear sequence, the chromatin, which is packed and organized inside the nucleus, does not function linearly. This is most clearly illustrated by the fact that genes are often regulated by elements that are located hundreds of kilobases away in the linear genome. Imaging techniques have shown that regulatory elements can act over large genomic distances by engaging in direct physical interactions with target genes, resulting in the formation of chromatin loops. Based on these observations, we have envisaged that the spatial organization of the genome resembles a three-dimensional network that is driven by physical associations between genes and regulatory elements, both in cis (within the same chromosome) and in trans (between different chromosomes) (Dekker, 2006). Apart from imaging technology which is labor intensive and low-throughput, long-range chromatin looping interactions can be detected using the Chromosome Conformation Capture (3C) technology (Dekker et al., 2002). The 3C method employs formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. Cells are subsequently lysed and chromatin is digested with a restriction enzyme of choice. The digested fragments are then ligated under dilute conditions to facilitate intramolecular ligation. The result is a genome-wide interaction library of ligation products corresponding to all possible chromatin interactions. Specific ligation products can then be detected by PCR using specific primer pairs. The 5C method was developed to dramatically increase 3C throughput (Dostie et al., 2006; Dostie and Dekker, 2007). The 5C method greatly increases the scale of chromatin interaction detection by replacing the PCR detection step of 3C with ligation-mediated amplification (LMA). LMA is advantageous due to a much higher level of multiplexing by using thousands of primers in a single reaction to detect millions of chromatin interactions (ligation junctions) in parallel. The LMA step effectively "copies" 3C ligation products into much smaller 5C ligation products that precisely correspond to ligation junctions formed during the 3C procedure. The products of the multiplexed LMA reaction constitute the 5C library. The composition of the 5C library is determined using high-throughput DNA sequencing. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf The aim of the pilot study was to generate a "connectivity map" between transcription start sites (TSS) and distal regulatory elements within the 44 ENCODE PILOT regions. In the current scheme, 5C primers were designed for all HindIII restriction fragments. Reverse primers were designed on fragments containing the TSS of annotated genes. Forward primers were designed on all other fragments. This design allowed for the interrogation of all TSS with all other restriction fragments, thus generating an interaction map between TSS and regulatory elements. For gene desert ENCODE pilot regions (for example ENr313), an altered scheme of forward and reverse primers was designed. Primers were selected for relative uniqueness using a custom 15-mer frequency table and BLAST. A custom hexamer barcode was added to each primer to ensure the sequence was unique relative to the primer pool being used. Primers were also selected for the appropriate melting temperature and GC-content and a universal tail sequence for amplification. The 44 ENCODE regions were analyzed in two groups using two separate 5C primer pools. The first group (ENm) contained the manually-picked ENCODE regions, ENm001-014 and ENr313. The second group (ENr) contained the 30 randomly-picked ENCODE regions. The two 5C primer pools were made by pooling 5C primers for interrogating long-range interactions in the two groups of ENCODE regions. The primer pool for the ENm group contained a total of 3,150 primers (476 reverse 5C primers and 2674 forward 5C primers). This primer pool allowed interrogation of a total of 1,272,824 interactions. Of these, 83,427 interactions were between fragments that were both located in the same ENCODE region. This primer pool for the ENr group contained a total of 3,152 primers (505 reverse 5C primers and 2647 forward 5C primers). This primer pool allowed interrogation of a total of 1,336,735 interactions. Of these, 34,859 interactions were between fragments that were both located in the same ENCODE region. In total, 981 reverse primers and 5,321 forward primers were designed (corresponding to ~77.1% (6,302/8,174) of all HindIII fragments in the 44 ENCODE regions). Currently, data for two biological replicates have been generated for ENCODE Tier I (GM12878 and K562), Tier II (HeLa-S3), and H1 human embryonic stem cells (H1-hESC), spanning 14 ENCODE manual regions along with one random region (ENr313) as well as 30 random regions separately using high-throughput paired-end sequencing in the Illumina GA2 platform. The looping interactions, which are detected in both the biological replicates, are considered significant.
2012-07-18 | E-GEOD-39510 | biostudies-arrayexpress
Project description:Amartya Sanyal mailto:amartya.sanyal@umassmed.edu (Wet Lab), Bryan R. Lajoie mailto:bryan.lajoie@umassmed.edu, Gaurav Jain mailto:gaurav.jain@umassmed.edu (Dry Lab), Job Dekker mailto:job.dekker@umassmed.edu (Principal Investigator) This track contains chromatin interaction data generated using the 5C (Chromatin Conformation Capture Carbon Copy) method by the ENCODE group (Dekker Lab) located at the University of Massachusetts, Worcester, MA. This track shows the significant looping interactions between transcriptional start sites (TSS) and distal regulatory elements in the context of the 44 ENCODE pilot regions spanning 1% of the human genome. Although the DNA is a linear sequence, the chromatin, which is packed and organized inside the nucleus, does not function linearly. This is most clearly illustrated by the fact that genes are often regulated by elements that are located hundreds of kilobases away in the linear genome. Imaging techniques have shown that regulatory elements can act over large genomic distances by engaging in direct physical interactions with target genes, resulting in the formation of chromatin loops. Based on these observations, we have envisaged that the spatial organization of the genome resembles a three-dimensional network that is driven by physical associations between genes and regulatory elements, both in cis (within the same chromosome) and in trans (between different chromosomes) (Dekker, 2006). Apart from imaging technology which is labor intensive and low-throughput, long-range chromatin looping interactions can be detected using the Chromosome Conformation Capture (3C) technology (Dekker et al., 2002). The 3C method employs formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. Cells are subsequently lysed and chromatin is digested with a restriction enzyme of choice. The digested fragments are then ligated under dilute conditions to facilitate intramolecular ligation. The result is a genome-wide interaction library of ligation products corresponding to all possible chromatin interactions. Specific ligation products can then be detected by PCR using specific primer pairs. The 5C method was developed to dramatically increase 3C throughput (Dostie et al., 2006; Dostie and Dekker, 2007). The 5C method greatly increases the scale of chromatin interaction detection by replacing the PCR detection step of 3C with ligation-mediated amplification (LMA). LMA is advantageous due to a much higher level of multiplexing by using thousands of primers in a single reaction to detect millions of chromatin interactions (ligation junctions) in parallel. The LMA step effectively "copies" 3C ligation products into much smaller 5C ligation products that precisely correspond to ligation junctions formed during the 3C procedure. The products of the multiplexed LMA reaction constitute the 5C library. The composition of the 5C library is determined using high-throughput DNA sequencing. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2012-07-19 | GSE39510 | GEO
Project description:Though important for gene regulation most studies of genome organisation use either fluorescence in situ hybridisation (FISH) or chromosome conformation capture (3C) methods. FISH directly visualises the spatial relationship of sequences, but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde crosslinking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organisation, but this has not been tested extensively. We investigate the murine HoxD locus with 5C and FISH in different developmental and activity states, and in the presence or absence of epigenetic regulators. We identify situations where the two datasets are concordant, but find other conditions where chromatin topographies extrapolated from 5C or FISH data are not compatible. We conclude that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart – influenced by nuclear environment and chromatin composition. We conclude that results obtained at high-resolution with either 3C methods or by FISH alone must be interpreted with caution and that conclusions about genome organisation should be validated by independent methods.
2015-01-02 | GSE61814 | GEO
Project description:Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C generates high quality, high resolution maps of looping interactions genome-wide, but is intractable for high-throughput screening of loops across conditions due to the requirement of an enormous number of reads (>6 Billion) per library. Here, we describe 5C-ID, an updated version of Chromosome-Conformation-Capture-Carbon-Copy (5C) with restriction digest and ligation performed in the nucleus (in situ ChromosomeConformation-Capture (3C)) and ligation-mediated amplification performed with a new double alternating design. 5C-ID reduces spatial noise and enables higher resolution 3D genome folding maps than canonical 5C, allowing for a marked improvement in sensitivity and specificity of loop detection. 5C-ID enables the creation of high-resolution, high-coverage maps of chromatin loops in up to a 30 Megabase subset of the genome at a fraction of the cost of Hi-C.
2018-05-07 | GSE114121 | GEO
Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing "in-nucleus ligation" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications.
2015-08-12 | GSE70181 | GEO