Measurement of drug concentration and bacterial contamination after diluting morphine for intrathecal administration: an experimental study

Background: Low concentrations of morphine are required for safe dosing for intrathecal injections. Sometimes, manual dilution of morphine is performed to achieve these low concentrations, but risks dilution errors and bacterial contamination. The primary goal was to compare the concentrations of morphine and bupivacaine between four groups of syringes. The secondary goal was to investigate the difference in contamination rate between these groups. Methods: Twenty-five experienced anesthesia providers were asked to prepare a mixture of bupivacaine 2.0 mg/ml and morphine 60 μg/ml using 3 different methods as clean and precise as possible. The fourth method used was the aspiration of ampoules prepared by the pharmacy. The concentrations of morphine and bupivacaine were measured by High-Pressure Liquid Chromatography (HPLC). The medication was cultured for bacterial contamination. Results: Group 1 (median 60 μg/ml; 95% CI: 59–110 μg/ml) yielded 3 outliers above 180 μg/ml morphine concentration. Group 2 (76 μg/ml; 95% CI: 72–80 μg/ml) and 3 (69 μg/ml; 95% CI: 66–71 μg/ml) were consistently higher than the target concentration of 60 μg. The group “pharmacy” was precise and accurate (59 μg/ml; 95% CI: 59–59 μg/ml). Group 2 and “pharmacy” had one contaminated sample with a spore-forming aerobic gram-positive rod. Conclusion: Manually diluted morphine is at risk for deviating concentrations, which could lead to increased sideeffects. Medication produced by the hospital pharmacy was highly accurate. Furthermore, even when precautions are undertaken, contamination of the medication is a serious risk and appeared to be unrelated to the dilution process

Plant ‘evo-devo’ goes genomic: from candidate genes to regulatory networks

Plant development gives rise to a staggering complexity of morphological structures with different shapes, colors, and functions. Understanding the evolution of control mechanisms that underlie developmental processes provides insights into causes of morphological diversity and, therefore, is of great interest to biologists. New genomic resources and techniques enable biologists to assess for the first time the evolution of developmental regulatory networks at a global scale. Here, we address the question of how comparative regulatory genomics can be used to reveal the evolutionary dynamics of control networks linked to morphological evolution in plants

Plant ‘evo-devo’ goes genomic: from candidate genes to regulatory networks

Plant development gives rise to a staggering complexity of morphological structures with different shapes, colors, and functions. Understanding the evolution of control mechanisms that underlie developmental processes provides insights into causes of morphological diversity and, therefore, is of great interest to biologists. New genomic resources and techniques enable biologists to assess for the first time the evolution of developmental regulatory networks at a global scale. Here, we address the question of how comparative regulatory genomics can be used to reveal the evolutionary dynamics of control networks linked to morphological evolution in plants

Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [ChIP-Seq]

Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the importance of natural variation in plant developmental programs, there is a need to understand the molecular basis of this variation at the level of developmental gene regulation. However, until now, the evolutionary turnover and dynamics of TF binding sites among plant species has not yet experimentally determined. Here, we performed comparative ChIP-seq studies of the MADS-box TF SEPALLATA3 (SEP3) in inflorescences of two Arabidopsis species: A. thaliana and A. lyrata. Comparative RNA-seq analysis shows that the loss/gain of BSs is often followed by a change in gene expression. Chromatin was crosslinked and isolated from wildtype A. lyrata inflorescences up to floral stage 10/11. The immunoprecipitation of protein-DNA complexes was performed using a peptide SEP3 antibody that was raised against A. thaliana SEP3, and is also able to recognize the homolog from A. lyrata due to protein similarity. As a control, de-crosslinked 'input-DNA' was used. SEP3 ChIP-seq on Arabidopsis thaliana can be found in the accessions GSM364939 and GSM364941. Re-analyzed peak positions for these samples are included as a supplementary file on the series level ([email protected])

Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [ChIP-Seq]

Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the importance of natural variation in plant developmental programs, there is a need to understand the molecular basis of this variation at the level of developmental gene regulation. However, until now, the evolutionary turnover and dynamics of TF binding sites among plant species has not yet experimentally determined. Here, we performed comparative ChIP-seq studies of the MADS-box TF SEPALLATA3 (SEP3) in inflorescences of two Arabidopsis species: A. thaliana and A. lyrata. Comparative RNA-seq analysis shows that the loss/gain of BSs is often followed by a change in gene expression. Chromatin was crosslinked and isolated from wildtype A. lyrata inflorescences up to floral stage 10/11. The immunoprecipitation of protein-DNA complexes was performed using a peptide SEP3 antibody that was raised against A. thaliana SEP3, and is also able to recognize the homolog from A. lyrata due to protein similarity. As a control, de-crosslinked 'input-DNA' was used. SEP3 ChIP-seq on Arabidopsis thaliana can be found in the accessions GSM364939 and GSM364941. Re-analyzed peak positions for these samples are included as a supplementary file on the series level ([email protected])

Comparative analysis of binding patterns of MADS-domain proteins in Arabidopsis thaliana

Background Correct flower formation requires highly specific temporal and spatial regulation of gene expression. In Arabidopsis thaliana the majority of the master regulators that determine flower organ identity belong to the MADS-domain transcription factor family. The canonical DNA binding motif for this transcription factor family is the CArG-box, which has the consensus CC(A/T)6GG. However, so far, a comprehensive analysis of MADS-domain binding patterns has not yet been performed. Results Eight publicly available ChIP-seq datasets of MADS-domain proteins that regulate the floral transition and flower formation were analyzed. Surprisingly, the preferred DNA binding motif of each protein was a CArG-box with an NAA extension. Furthermore, motifs of other transcription factors were found in the vicinity of binding sites of MADS-domain transcription factors, suggesting that interaction of MADS-domain proteins with other transcription factors is important for target gene regulation. Finally, conservation of CArG-boxes between Arabidopsis ecotypes was assessed to obtain information about their evolutionary importance. CArG-boxes that fully matched the consensus were more conserved than other CArG-boxes, suggesting that the perfect CArG-box is evolutionary more important than other CArG-box variants. Conclusion Our analysis provides detailed insight into MADS-domain protein binding patterns. The results underline the importance of an extended version of the CArG-box and provide a first view on evolutionary conservation of MADS-domain protein binding sites in Arabidopsis ecotypes

Comparative analysis of binding patterns of MADS-domain proteins in Arabidopsis thaliana

Background Correct flower formation requires highly specific temporal and spatial regulation of gene expression. In Arabidopsis thaliana the majority of the master regulators that determine flower organ identity belong to the MADS-domain transcription factor family. The canonical DNA binding motif for this transcription factor family is the CArG-box, which has the consensus CC(A/T)6GG. However, so far, a comprehensive analysis of MADS-domain binding patterns has not yet been performed. Results Eight publicly available ChIP-seq datasets of MADS-domain proteins that regulate the floral transition and flower formation were analyzed. Surprisingly, the preferred DNA binding motif of each protein was a CArG-box with an NAA extension. Furthermore, motifs of other transcription factors were found in the vicinity of binding sites of MADS-domain transcription factors, suggesting that interaction of MADS-domain proteins with other transcription factors is important for target gene regulation. Finally, conservation of CArG-boxes between Arabidopsis ecotypes was assessed to obtain information about their evolutionary importance. CArG-boxes that fully matched the consensus were more conserved than other CArG-boxes, suggesting that the perfect CArG-box is evolutionary more important than other CArG-box variants. Conclusion Our analysis provides detailed insight into MADS-domain protein binding patterns. The results underline the importance of an extended version of the CArG-box and provide a first view on evolutionary conservation of MADS-domain protein binding sites in Arabidopsis ecotypes

Phylogenomic Synteny Network Analysis of MADS-Box Transcription Factor Genes Reveals Lineage-Specific Transpositions, Ancient Tandem Duplications, and Deep Positional Conservation

Conserved genomic context provides critical information for comparative evolutionary analysis. 29 With the increase in numbers of sequenced plant genomes, synteny analysis can provide new 30 insight into gene family evolution. Here, we exploit a network analysis approach to organize and31 interpret massive pairwise syntenic relationships. Specifically, we analyzed synteny networks of 32 the MADS-box transcription factor gene family using fifty-one completed plant genomes. In 33 combination with phylogenetic profiling, several novel evolutionary patterns were inferred and 34 visualized from synteny network clusters. We found lineage-specific clusters that derive from 35 transposition events for the regulators of floral development (APETALA3 and PI) and flowering36 time (FLC) in the Brassicales and for the regulators of root-development (AGL17) in Poales. We 37 also identified two large gene clusters that jointly encompass many key phenotypic regulatory 38 Type II MADS-box gene clades (SEP1, SQUA, TM8, SEP3, FLC, AGL6 and TM3). Gene39 clustering and gene trees support the idea that these genes are derived from an ancient tandem 40 gene duplication that likely predates the radiation of the seed plants and then expanded by 41 subsequent polyploidy events. We also identified angiosperm-wide conservation of synteny of 42 several other less studied clades. Combined, these findings provide new hypotheses for the43 genomic origins, biological conservation and divergence of MADS-box gene family members

Evolution of DNA-Binding Sites of a Floral Master Regulatory Transcription Factor

Flower development is controlled by the action of key regulatory transcription factors of the MADS-domain family. The function of these factors appears to be highly conserved among species based on mutant phenotypes. However, the conservation of their downstream processes is much less well understood, mostly because the evolutionary turnover and variation of their DNA-binding sites (BS) among plant species has not yet been experimentally determined. Here, we performed comparative ChIP-seq experiments of the MADS-domain transcription factor SEPALLATA3 (SEP3) in two closely related Arabidopsis species: A. thaliana and A. lyrata which have very similar floral organ morphology. We found that binding site conservation is associated with DNA sequence conservation, the presence of the CArG-box BS motif and on the relative position of the BS to its potential target gene. Differences in genome size and structure can explain that SEP3 BSs in A. lyrata can be located more distantly to their potential target genes than their counterparts in A. thaliana. In A. lyrata, we identified transposition as a mechanism to generate novel SEP3 binding locations in the genome. Comparative gene expression analysis shows that the loss/gain of BSs is associated with a change in gene expression. In summary, this study investigates the evolutionary dynamics of DNA BSs of a floral key-regulatory transcription factor, and explores factors affecting this phenomenon. <br/