Mechanistic Investigation of Enzyme Triggered Release from a Xyloglucan Matrix Tablet for Controlled Colonic Drug Delivery.

The objective of this study was to investigate the mechanisms underlying drug release from a controlled colonic release (CCR) tablet formulation based on a xyloglucan polysaccharide matrix and identify the factors that control the rate of release for the purpose of fundamentally substantiating the concept and demonstrating its robustness for colonic drug delivery. Previous work demonstrated in vitro limited release of 5-aminosalicylic acid (5-ASA) and caffeine from these tablets in small intestinal environment and significant acceleration of release by xyloglucanase, an enzyme of the colonic microbiome. Targeted colonic drug delivery was verified in an animal study in vivo.

Definition of the binding specificity of the T7 bacteriophage primase by analysis of a protein binding microarray using a thermodynamic model.

Protein binding microarrays (PBM), SELEX, RNAcompete and chromatin-immunoprecipitation have been intensively used to determine the specificity of nucleic acid binding proteins. While the specificity of proteins with pronounced sequence specificity is straightforward, the determination of the sequence specificity of proteins of modest sequence specificity is more difficult. In this work, an explorative data analysis workflow for nucleic acid binding data was developed that can be used by scientists that want to analyse their binding data.

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy.

Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction.

Efficient colonic drug delivery in domestic pigs employing a tablet formulation with dual control concept.

Efficient and reproducible colonic drug delivery remains elusive. The aim of this study was to demonstrate specific colonic delivery in vivo in domestic pigs with a novel tablet formulation based on a dual release control concept using 5-aminosalicylic acid (5-ASA) and caffeine as drug substances. The developed controlled colonic release (CCR) tablet formulation employs a pH-sensitive coating based on Eudragit® FS 30 D to prevent drug release in the upper gastrointestinal tract, and a xyloglucan-based matrix to inhibit drug release after coating removal in the small intestine and to allow microbiome-triggered drug release by enzymatic action in the colon.

The monomeric archaeal primase from Nanoarchaeum equitans harbours the features of heterodimeric archaeoeukaryotic primases and primes sequence-specifically.

The marine thermophilic archaeon Nanoarchaeum equitans possesses a monomeric primase encompassing the conserved domains of the small catalytic and the large regulatory subunits of archaeoeukaryotic heterodimeric primases in one protein chain. The recombinant protein primes on templates containing a triplet with a central thymidine, thus displaying a pronounced sequence specificity typically observed with bacterial type primases only. The N.

Tablet formulation with dual control concept for efficient colonic drug delivery.

Aim of this study was to develop a tablet formulation for targeted colonic drug release by implementing two control mechanisms: A pH-sensitive coating layer based on Eudragit® FS 30 D to prevent drug release in the upper gastrointestinal tract, combined with a matrix based on plant-derived polysaccharide xyloglucan to inhibit drug release after coating removal in the small intestine and to allow microbiome triggered drug release in the colon. In vitro dissolution tests simulated the passage through the entire gastrointestinal tract with a four-stage protocol, including microbial xyloglucanase addition in physiologically relevant concentrations as microbiome surrogate to the colonic dissolution medium. Matrix erosion was monitored in parallel to drug release by measurement of reducing sugar equivalents resulting from xyloglucan hydrolysis.

Stringent Primer Termination by an Archaeo-Eukaryotic DNA Primase.

Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length.

Stable and selective permeable hydrogel microcapsules for high-throughput cell cultivation and enzymatic analysis.

Background: Miniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates.

Recent advances in understanding bacterial and archaeoeukaryotic primases.

DNA replication in all forms of life relies upon the initiation of synthesis on a single strand template by formation of a short oligonucleotide primer, which is subsequently elongated by DNA polymerases. Two structurally distinct classes of enzymes have evolved to perform this function, namely the bacterial DnaG-type primases and the Archaeal and Eukaryotic primases (AEP). Structural and mechanistic insights have provided a clear understanding of the role of the different domains of these enzymes in the context of the replisome and recent work sheds light upon primase-substrate interactions.

Characterization of the housekeeping sortase from the human pathogen : first investigation of a class F sortase.

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance.

A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved.

Elucidation of the Recognition Sequence of Sortase B from Bacillus anthracis by Using a Newly Developed Liquid Chromatography-Mass Spectrometry-Based Method.

Sortases are enzymes that are responsible for the attachment of secreted proteins to the cell wall of Gram-positive bacteria. Hereby, the sortases recognize short, five-residue amino acid sequences present in the target proteins and fuse them to the peptidoglycan layer via a transpeptidation reaction, creating a new peptide bond between the C-terminus of the recognition sequence and the cell wall. The transpeptidation activity of sortases is widely used in protein engineering for modification of target proteins.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine.

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions.

Structures to complement the archaeo-eukaryotic primases catalytic cycle description: What's next?

DNA replication is a crucial stage in the transfer of genetic information from parent to daughter cells. This mechanism involves multiple proteins with one key player being the primase. Primases are single-stranded DNA dependent RNA polymerases.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored.

Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1.

We have determined the minimal replicon of the crenarchaeal plasmid pRN1. It consists of 3097 base pairs amounting to 58% of the genome of pRN1. The minimal replicon comprises replication operon orf56/orf904 coding for a transcriptional repressor and the replication protein of pRN1.

Construction of a chimeric thermoacidophilic beta-endoglucanase.

Background: The archeaon Sulfolobus solfataricus P2 encodes a thermoacidophilic cellulase which shows an extreme acid and thermal stability with a pH optimum at 1.8 and a temperature optimum at 80°C. This extraordinary enzyme could be useful for biotechnological exploitation but the expression and purification in expression hosts like E.

Structure and function of the primase domain of the replication protein from the archaeal plasmid pRN1.

The replication protein of the archaeal plasmid pRN1 is a multifunctional enzyme which appears to carry out several steps at the plasmid replication initiation. We recently determined the structure of the minimal primase domain of the replication protein and found out that the primase domain consists of a catalytic primase/polymerase domain and an accessory helix-bundle domain. Structure-guided mutagenesis allowed us to identify amino acids which are important for template binding, dinucleotide formation and a step before primer extension.

The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis.

The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40-370. While the N-terminal part of that minimal region (residues 47-247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248-370) is unknown.

Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius.

Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E.

Structure-based stability analysis of an extremely stable dimeric DNA binding protein from Sulfolobus islandicus.

ORF56 is a small and thermodynamically extremely stable dimeric protein from the archaeon Sulfolobus islandicus. This DNA binding protein is encoded on plasmid pRN1 and possibly controls the copy number of the plasmid. We report the solution NMR structure as well as the crystal structure of ORF56 comprising a ribbon-helix-helix fold.

DNA translocation activity of the multifunctional replication protein ORF904 from the archaeal plasmid pRN1.

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear.

Biofunctionalization of dispense-plotted hydroxyapatite scaffolds with peptides: quantification and cellular response.

Hydroxyapatite (HA) ceramic is a widely used synthetic bone substitute material for the regeneration of bone defects. We manufactured HA scaffolds with adjustable pore sizes and pore geometry by dispense-plotting. In addition, we attached peptides covalently onto the HA surface and are able to simultaneously quantify the amount of covalently attached and adsorbed peptide down to the picomolar range with a novel fluorescence-based detection method.

Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus.

Although Sulfolobus species are among the best studied archaeal micro-organisms, the development and availability of genetic tools has lagged behind. In the present paper, we discuss the latest progress in understanding recombination events of exogenous DNA into the chromosomes of Sulfolobus solfataricus and Sulfolobus acidocaldarius and their application in the construction of targeted-deletion mutant strains.

Molecular biology of the pRN1 plasmid from Sulfolobus islandicus.

The pRN1 plasmid is a rather small multicopy plasmid which was isolated from a Sulfolobus islandicus strain in 1993 by Wolfram Zillig and co-workers. Sequence analysis of the genome sequence suggested that three conserved genes are important for plasmid replication. These genes code for two sequence-specific DNA-binding proteins (ORF56 and ORF80) and for a large multifunctional replication protein (ORF904).