419 research outputs found

    ACARORUM CATALOGUS IX. Acariformes, Acaridida, Schizoglyphoidea (Schizoglyphidae), Histiostomatoidea (Histiostomatidae, Guanolichidae), Canestrinioidea (Canestriniidae, Chetochelacaridae, Lophonotacaridae, Heterocoptidae), Hemisarcoptoidea (Chaetodactylidae, Hyadesiidae, Algophagidae, Hemisarcoptidae, Carpoglyphidae, Winterschmidtiidae)

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    The 9th volume of the series Acarorum Catalogus contains lists of mites of 13 families, 225 genera and 1268 species of the superfamilies Schizoglyphoidea, Histiostomatoidea, Canestrinioidea and Hemisarcoptoidea. Most of these mites live on insects or other animals (as parasites, phoretic or commensals), some inhabit rotten plant material, dung or fungi. Mites of the families Chetochelacaridae and Lophonotacaridae are specialised to live with Myriapods (Diplopoda). The peculiar aquatic or intertidal mites of the families Hyadesidae and Algophagidae are also included.Publishe

    Strategies to Improve Antineoplastic Activity of Drugs in Cancer Progression

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    The aim of this Special Issue is to collect reports regarding all the recent strategies, directed at the improvement of antineoplastic activity of drugs in cancer progression, engaging all the expertise needed for the development of new anticancer drugs: medicinal chemistry, pharmacology, molecular biology, and computational and drug delivery studies

    The compatible solutes ectoine and 5-hydroxyectoine: Catabolism and regulatory mechanisms

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    To cope with osmotic stress many microorganisms make use of short, osmotically active, organic compounds, the so-called compatible solutes. Examples for especially effective members of this type of molecules are the tetrahydropyrimidines ectoine and 5-hydroxyectoine. Both molecules are produced by a large number of microorganisms, not only to fend-off osmotic stress, but also for example low and high temperature challenges. The biosynthetic pathway used by these organisms to synthesize ectoines has already been studied intensively and the enzymes used therein are characterized quite well, both biochemically as well as structurally. However, synthesis of ectoines is only half the story. Inevitably, ectoines are frequently released from the producer cells in different environmental settings. Especially in highly competitive habitats like the upper ocean layers some bacteria specialized on a niche like this. The model organism used in this work is such a species. It is the marine bacterium Ruegeria pomeroyi DSS-3 which belongs to the Roseobacter-clade. Roseobacter species are heterotrophic Proteobacteria which can live in symbiosis with phytoplankton as well as turning against them in a bacterial warfare fashion to scavenge valuable nutrients. Ectoines can be imported by R. pomeroyi DSS-3 in a high-affinity fashion and be used as energy as well as carbon- and nitrogen-sources. To achieve this, both ectoines rings are degraded by the hydrolase EutD and deacetylated by the deacetylase EutE. The first hydrolysis products α-ADABA (from ectoine) and hydroxy-α-ADABA (from hydroxyectoine) are deacetylated to DABA and hydroxy-DABA which are in additional biochemical reactions transformed to aspartate to fuel the cell’s central metabolism. The role and functioning of the EutDE enzymes which work in a concerted fashion are a central aspect of this work. Both enzymes could be biochemically and structurally characterized, and the architecture of the metabolic pathway could be illuminated. α-ADABA and hydroxy-α-ADABA are not only central to ectoine catabolism, but also to the regulatory mechanisms associated with it. Both molecules serve as inducers of the central regulatory protein of this pathway, the MocR-/GabR-type regulator protein EnuR. In the framework of this dissertation molecular details could be clarified which enable the EnuR repressor molecule to sense both molecules with high affinity to subsequently derepress the genes for the import and catabolism of ectoines

    Crystallographic Studies of Enzymes (Volume II)

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    In this Special Issue of Crystals, entitled "Crystallographic Studies of Enzymes (Volume II)", eleven research papers on key findings and methodologies of structure, function, and reaction mechanisms of enzymes are presented

    Elucidating the Structure and Regulatory Interactions of the HOTAIR Non-Coding RNA and the Bacterial RNase P. Holoenzyme

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    RNA structures and RNA-protein interactions are studied as potential drug targets, biomarkers in cancer, and can be administered as vaccines. The cancer associated HOTAIR (HOX transcript antisense RNA) exists in higher vertebrates and interacts with chromatin remodeling enzymes. We examined the thermodynamic folding properties and structural propensity of the exonic regions of HOTAIR using biophysical methods and NMR spectroscopy. Different exons of HOTAIR contain variable degrees of structural heterogeneity. We identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing confirm conserved base pair interactions within helix 10 of exon 4 of the human HOTAIR long non-coding RNA (lncRNA). Unlike HOTAIR, the ribonuclease P (RNase P) exists in bacteria, archaea and eukarya. RNase P is a universal RNA-protein endonuclease that catalyzes 5â€Č precursor-tRNA (ptRNA) processing. Protein concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima to understand its oligomerization properties. The identification of a monomeric P protein conformer from NMR relaxation data and chemical shift information provided new insight into the conformational dynamics of the P protein. Taken together, local structural changes of the P protein and the 5â€Č leader RNA facilitate optimal substrate alignment and catalytic activation of the RNase P holoenzyme. As RNase P is an essential enzyme in life, knowledge of the structural differences between pathogenic bacterial and human RNase P may help in the development of new antibiotic therapeutics that target RNase P. The enzyme activity of Mycobacterium tuberculosis RNase P was examined through 32P radioactivity assays, and multidimensional 2D/3D NMR spectroscopy was implemented to study the solution structure of the M. tuberculosis RNase P protein. A comparative analysis of the pathogenic and non-pathogenic RNase P proteins brings important structural insight into the development of antibiotics that target tuberculosis RNase P

    The causes of retinal dystrophy and the development of more comprehensive screening approach

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    Inherited retinal diseases (IRDs) are a group of genetically and phenotypically heterogenous disorders caused by variants in around 280 genes. Additional loci have also been localised to chromosomal regions, though the causative genes remain unknown. Recent improvements in screening technologies have increased the detection of pathogenic variants in IRD. This thesis describes the use of next generation sequencing (second (short-read) and third (long-read) generation sequencing) to find missing or hard to find pathogenic variants in IRD patients. The first results chapter describes use of whole exome sequencing to screen 24 individuals with syndromic and non-syndromic IRDs. This identified pathogenic variants in known genes in eight cases; CDHR1 (c.1527T>G, p.Y509*), RHO (c.284T>C, p.L95P), PRPF31 (c.797delC, p.S266*), CNGA3 (c.1088T>C, p.L363P), BBS10 (c.728-731delAAGA, p. K243Ifs*15), USH2A (c.252T>G, p.C84W), ABCA4 (c.2588G>C, p.G863A and c.6089G>A, p.R2030Q), and SLC25A46 (c.670A>G, p.T224A). In addition, several candidate variants were highlighted for further investigation. In the second results chapter, seven patients with late onset macular dystrophy and one with age related macular degeneration were found to carry the same heterozygous ~126 kb deletion encompassing CRX, TPRX1 and SULT2A1. This phenotype has already been documented in patients with heterozygous variants in the gene encoding retinal transcription factor CRX, while there is no known functional or phenotypic link with variants in TPRX1 or SULT2A1. This therefore confirms that CRX haploinsufficiency is pathogenic, a finding that had previously been debated in the ophthalmic literature. The deletion was characterized using a PCR assay followed by cloning and Sanger sequencing or direct Sanger sequencing. Haplotype analysis was done by microsatellite genotyping. The third results chapter describes use of SMRT PacBio and nanopore long-read sequencing to screen the hard-to-sequence mutation hotspot RPGR-ORF15. Both approaches were effective in reading throughout ORF15 and allowed sequencing indexed pooled samples, and 218 IRD patients were screened, detecting known and new variants. Nanopore sequencing on the smaller Flongle flowcell allowed low-cost optimisation, but pores rapidly blocked, probably due to ORF15 secondary structures. Repeated DNase I washes reopened the pores but required use of the more expensive MinION flowcells. Ultimately, the PacBio sequencer proved simpler to use, cheaper, and more scalable

    Using genome-wide data to model signalling-responsive gene regulatory mechanisms in blood development

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    The control of gene expression driving developmental haematopoiesis crucially depends on distal cis-regulatory elements such as enhancers which directly interact with promoters in the nucleus. However, no global experiments have been conducted which identify the cell type and cell stage-specific activity of enhancers in a chromatin context. It is through these elements that lineage specific transcription factors orchestrate cell fate decisions and direct haematopoietic lineage development emerging from the mesoderm. The roles of transcriptional regulators are beginning to be understood, however, it is still unclear how the myriad of extracellular signals modulate their activity. In this work, we report a global method which enables the identification of thousands tissue-specifically active cisregulatory elements able to stimulate a minimal promoter in cells representing five stages of haematopoietic specification derived from embryonic stem cells. Using serum-free differentiation culture, we demonstrate that our method can identify signalling-responsive enhancer elements and we highlight that it can be adapted to any embryonic stem cell differentiation system generating different cell types. We demonstrate that thousands of cell stage-specific sets of cis-elements are responsive to cytokine signals terminating at signalling-responsive transcription factors. Integrating these data with chromatin accessibility and single cell RNA-Seq data provided important new insights into the regulatory dynamics of the gene regulatory network transitions driving haematopoiesis. Our work identified the cytokine signalling-responsive transcription factors mediating responsiveness of enhancers at each developmental stage. We validated enhancers for Sparc, Pxn, Hspg2, Cdh5, Dlk1 and Mrpl15 as being signalling responsive to VEGF. We found that the cytokine VEGF is a crucial factor that regulates the balance between endothelial and haematopoietic development and our scRNA-seq analysis revealed that in the presence of VEGF Sox17 fails to be downregulated and Runx1 fails to be upregulated in the haemogenic endothelium and progenitor cells. For two Runx1 enhancers (the +23kb and +3.7kb) we studied the transcription factors motifs mediating the responsiveness of the enhancers to VEGF by mutation of these sites. Taken together, our work generated an important novel resource for future studies of haematopoietic differentiation and provides insights into how and where in the genome extrinsic signals program the cell type-specific chromatin landscape driving this process

    Ribosomes as Sensors: How Cells Utilize the Translational Process to Monitor Cellular Conditions

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    The discovery that collisions between ribosomes are the key signaling event that triggers quality control and stress response pathways in eukaryotes has changed our understanding of how cells sense and respond to the environment. Collided eukaryotic ribosomes adopt a unique structure, providing a mechanistic basis for how environmental signals can be sensed by the translational machinery and propagated into wider reprogramming of gene expression. Indeed, the use of ribosomes as a signaling hub is quite apt, as ribosomes make intimate contact with both mRNA and tRNAs. This enables them to monitor the integrity of genetic information coming from the nucleus while simultaneously checking the availability of cellular resources, acting as a sentinel of the central dogma of biology. Here, we discuss the ribosome in the context of the broader translational cycle and how signals arise from disruptions to ribosome function. We also discuss the effects of modifications to the mRNA on the ribosome and the role of the ribosome in the response

    Computational methods for the quantification of RNA transcript abundance and messenger RNA regulation

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    Experiments investigating the regulation of RNA transcripts have been revolutionised by technology developed over the last 40 years. The data acquired from these experiments have revealed novel regulatory mechanisms for the localisation, degradation and modification of RNA transcripts. However, the volume and complexity of the data sets have led to an unprecedented reliance on statistical software. Inadequate analysis of data sets is contributing to the ongoing crisis around reproducing conclusions from published research. Rigorous implementation of statistical analysis software can continue to uncover novel regulatory mechanisms, but closed, obscure, and incorrect analyses will propagate the reproducibility crisis to unassailable new heights. The objective of this research project is to develop open-source software and implement reproducible analyses to enable further exploration of regulatory mechanisms acting on RNA transcripts. This thesis focuses on the analysis of transcriptomics data sets, predominately from the model organism Saccharomyces cerevisiae. This first project discusses the standardisation of the analysis of qPCR data. The chapter compares the R package tidyqpcr, developed by the author, to other current software available. This case highlights how quality software supported by comprehensive documentation can improve the quality of an entire experimental assay. The next chapter showcases how the implementation of quality analysis can detect subtle interactions between regulatory motifs. The design of several reporter constructs using insights from published data sets shows how even short regulatory motifs can be affected by their overall context. The final results chapter outlines the development of a statistical software package to rigorously analyse noisy transcriptomic data from RNA-Seq assays exploring RNA localisation. The statistical software package uses a Bayesian hierarchical model of fractionation-based assays to overcome common biases in RNA-Seq data sets. In summary, this thesis presents and implements two examples of research software that improve the reproducibility and quality of conclusions from data acquired from common experimental assays in molecular biology. The thesis also outlines how to implement open-source development practices and create inclusive documentation in an academic setting. Software developed within this framework is then used to elucidate subtle ways that cells regulate their transcriptome

    Mechanisms of Regulatory Adaptation in the Evolving Genome

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    The development from a single cell into a complex organism requires the precise control of gene expression in space and time. To achieve this, the activity of genes is governed by large regulatory chromatin landscapes that when disrupted can cause gene mis-regulation and disease. However, at the same time, the successful modification of these landscapes is thought to be a major driver of phenotypic innovation during evolution. Given the vulnerability of these landscapes in disease settings, it remains largely unknown how their integrity is maintained when novel genes are “safely” incorporated during evolution, which is addressed in this work. Specifically, here, multiple mechanisms are dissected that adapted the Fat1 regulatory landscape to maintain its integrity while simultaneously incorporating a novel gene, Zfp42, during evolution. First, comparative evolutionary genomics was used to reconstruct the history of the locus (section 1). Second, the three-dimensional chromatin configuration of the locus was examined in relationship to the gene activities using genomics-technologies (HiC, DamID) combined with super resolution microscopy and in silico modeling (section 2). Finally, the mechanisms that adapted the landscape in ESCs (section 3) and embryonic limbs (section 4) for the emergence of Zfp42 were investigated using genome engineering and genomics. Two tissue-specific mechanisms were identified that enabled the independent activities of Zfp42 and Fat1 despite sharing the same regulatory chromatin landscape: In ESCs, the landscape physically restructures and isolates the genes together with their regulatory information, from one another, thereby allowing their independent regulation. Surprisingly, this restructuring is not driven by the most recognized chromatin structuring force, loop extrusion, but rather by the underlying epigenetic state of chromatin. A different mechanism operates in embryonic mouse limbs where both genes are exposed to the same regulatory information driving Fat1 activation, but surprisingly not Zfp42. The inactivity of Zfp42 cannot be explained by nuclear envelopment attachment nor by enhancer-promoter specificity. Instead, Zfp42 is kept inactive by a highly context-dependent silencing mechanism driven by DNA methylation. As such, Zfp42 is ectopically active and responsive to the surrounding regulatory information when DNA methylation is removed or when the gene is slightly repositioned within its domain. Combined, we find that 3D-restructuring and context-dependent silencing adapted the Fat1 landscape to integrate Zfp42. More generally, this demonstrates that even single regulatory landscapes harbor an enormous regulatory complexity and, thus can accommodate multiple independently regulated genes. We believe that this has significant consequences for human genetics where similar genomic alterations do not drive disease in patients. This is possible, because additional, yet still unknown, mechanisms control how regulatory information is used in the genome
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