In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.     

The modulation rate transfer function of a harbour porpoise (Phocoena phocoena)

During echolocation, toothed whales produce ultrasonic clicks at extremely rapid rates and listen for the returning echoes. The auditory brainstem response (ABR) duration was evaluated in terms of latency between single peaks: 5.5 ms (from peak I to VII), 3.4 ms (I–VI), and 1.4 ms (II–IV). In comparison to the killer whale and the bottlenose dolphin, the ABR of the harbour porpoise has shorter intervals between the peaks and consequently a shorter ABR duration. This indicates that the ABR duration and peak latencies are possibly related to the relative size of the auditory structures of the central nervous system and thus to the animal’s size. The ABR to a sinusoidal amplitude modulated stimulus at 125 kHz (sensitivity threshold 63 dB re 1 μPa rms) was evaluated to determine the modulation rate transfer function of a harbour porpoise. The ABR showed distinct envelope following responses up to a modulation rate of 1,900 Hz. The corresponding calculated equivalent rectangular duration of 263 μs indicates a good temporal resolution in the harbour porpoise auditory system similar to the one for the bottlenose dolphin. The results explain how the harbour porpoise can follow clicks and echoes during echolocation with very short inter click intervals.     

T cell responses against microsatellite instability-induced frameshift peptides and influence of regulatory T cells in colorectal cancer

High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.     

A brief pre-exercise nap may alleviate physical performance impairments induced by short-term sustained operations with partial sleep deprivation – A field-based study

ABSTRACT

The purpose of the study was to evaluate the recuperative efficacy of pre-exercise napping on physical capacity after military sustained operations (SUSOPS) with partial sleep deprivation. Before and after a 2-day SUSOPS, 61 cadets completed a battery of questionnaires, and performed a 2-min lunges trial and a 3,000-m running time-trial. After the completion of SUSOPS, subjects were randomized to either a control [without pre-exercise nap (CON); n = 32] or a nap [with a 30-min pre-exercise nap (NAP); n = 29] group. SUSOPS enhanced perceived sleepiness and degraded mood in both groups. Following SUSOPS, the repetitions of lunges, in the CON group, were reduced by ~ 2.3%, albeit the difference was not statistically significant (p = 0.62). In the NAP group, however, the repetitions of lunges were increased by ~ 7.1% (p = 0.01). SUSOPS impaired the 3,000-m running performance in the CON group (~ 2.3%; p = 0.02), but not in the NAP group (0.3%; p = 0.71). Present results indicate, therefore, that a relatively brief pre-exercise nap may mitigate physical performance impairments ensued by short-term SUSOPS.     

Predator faunas past and present: quantifying the influence of waterborne cues in divergent ecotypes of the isopod Asellus aquaticus

Waterborne chemical cues are an important source of information for many aquatic organisms, in particular when assessing the current risk of predation. The ability to use chemical cues to detect and respond to potential predators before an actual encounter can improve prey chances of survival. We investigated predator recognition and the impact of chemical cues on predator avoidance in the freshwater isopod Asellus aquaticus. This isopod has recently colonised a novel habitat and diverged into two distinct ecotypes, which encounter different predator communities. Using laboratory-based choice experiments, we have quantified behavioural responses to chemical cues from predators typical of the two predator communities (larval dragonflies in the ancestral habitat, perch in the newly colonised habitat) in wild-caught and lab-reared Asellus of the two ecotypes. Individuals with prior experience of predators showed strong predator avoidance to cues from both predator types. Both ecotypes showed similar antipredator responses, but sexes differed in terms of threat-sensitive responses with males avoiding areas containing predator cues to a larger extent than females. Overall, chemical cues from fish elicited stronger predator avoidance than cues from larval dragonflies. Our results indicate that in these isopods, prior exposure to predators is needed to develop antipredator behaviour based on waterborne cues. Furthermore, the results emphasise the need to analyse predator avoidance in relation to waterborne cues in a sex-specific context, because of potential differences between males and females in terms of vulnerability and life history strategies.     

The mitochondrial oxoglutarate carrier: from identification to mechanism

The 2-oxoglutarate carrier (OGC) belongs to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. Initially, OGC was characterized by determining substrate specificity, kinetic parameters of transport, inhibitors and molecular probes that form covalent bonds with specific residues. It was shown that OGC specifically transports oxoglutarate and certain carboxylic acids. The substrate specificity combination of OGC is unique, although many of its substrates are also transported by other mitochondrial carriers. The abundant recombinant expression of bovine OGC in Escherichia coli and its ability to functionally reconstitute into proteoliposomes made it possible to deduce the individual contribution of each and every residue of OGC to the transport activity by a complete set of cys-scanning mutants. These studies give experimental support for a substrate binding site constituted by three major contact points on the even-numbered α-helices and identifies other residues as important for transport function through their crucial positions in the structure for conserved interactions and the conformational changes of the carrier during the transport cycle. The results of these investigations have led to utilize OGC as a model protein for understanding the transport mechanism of mitochondrial carriers.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

A Novel Bacterial Enzyme with d-Glucuronyl C5-epimerase Activity

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.     

The Shaft of the Type 1 Fimbriae Regulates an External Force to Match the FimH Catch Bond

Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli to host cells. It has been hypothesized that due to their ability to uncoil under exposure to force, fimbriae can reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work, we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbria varies as a bacterium is affected by a time-dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time-dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ∼120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbria shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments.