Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX_4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.     

Monitoring of cell therapy and assessment of cardiac function using magnetic resonance imaging in a mouse model of myocardial infarction

We have developed a mouse severe combined immunodeficient (SCID) model of myocardial infarction based on permanent coronary artery occlusion that allows long-term functional analysis of engrafted human embryonic stem cell-derived cardiomyocytes, genetically marked with green fluorescent protein (GFP), in the mouse heart. We describe methods for delivery of dissociated cardiomyocytes to the left ventricle that minimize scar formation and visualization and validation of the identity of the engrafted cells using the GFP emission spectrum, and histological techniques compatible with GFP epifluorescence, for monitoring phenotypic changes in the grafts in vivo. In addition, we describe how magnetic resonance imaging can be adapted for use in mice to monitor cardiac function non-invasively and repeatedly. The model can be adapted to include multiple control or other cell populations. The procedure for a cohort of six mice can be completed in a maximum of 13 weeks, depending on follow-up, with 30 h of hands-on time.     

Transvenous vagus nerve stimulation does not modulate the innate immune response during experimental human endotoxemia: a randomized controlled study

IntroductionVagus nerve stimulation (VNS) exerts beneficial anti-inflammatory effects in various animal models of inflammation, including collagen-induced arthritis, and is implicated in representing a novel therapy for rheumatoid arthritis. However, evidence of anti-inflammatory effects of VNS in humans is very scarce. Transvenous VNS (tVNS) is a newly developed and less invasive method to stimulate the vagus nerve. In the present study, we determined whether tVNS is a feasible and safe procedure and investigated its putative anti-inflammatory effects during experimental human endotoxemia.MethodsWe performed a randomized double-blind sham-controlled study in healthy male volunteers. A stimulation catheter was inserted in the left internal jugular vein at spinal level C5–C7, adjacent to the vagus nerve. In the tVNS group (n = 10), stimulation was continuously performed for 30 minutes (0–10 V, 1 ms, 20 Hz), starting 10 minutes before intravenous administration of 2 ng kg⁻¹Escherichia coli lipopolysaccharide (LPS). Sham-instrumented subjects (n = 10) received no electrical stimulation.ResultsNo serious adverse events occurred throughout the study. In the tVNS group, stimulation of the vagus nerve was achieved as indicated by laryngeal vibration. Endotoxemia resulted in fever, flu-like symptoms, and hemodynamic changes that were unaffected by tVNS. Furthermore, plasma levels of inflammatory cytokines increased sharply during endotoxemia, but responses were similar between groups. Finally, cytokine production by leukocytes stimulated with LPS ex vivo, as well as neutrophil phagocytosis capacity, were not influenced by tVNS.ConclusionstVNS is feasible and safe, but does not modulate the innate immune response in humans in vivo during experimental human endotoxemia.

Trial registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01944228","term_id":"NCT01944228"}}NCT01944228. Registered 12 September 2013.     

Signatures of selection in the three‐spined stickleback along a small‐scale brackish water – freshwater transition zone

Local adaptation is often obvious when gene flow is impeded, such as observed at large spatial scales and across strong ecological contrasts. However, it becomes less certain at small scales such as between adjacent populations or across weak ecological contrasts, when gene flow is strong. While studies on genomic adaptation tend to focus on the former, less is known about the genomic targets of natural selection in the latter situation. In this study, we investigate genomic adaptation in populations of the three‐spined stickleback Gasterosteus aculeatus L. across a small‐scale ecological transition with salinities ranging from brackish to fresh. Adaptation to salinity has been repeatedly demonstrated in this species. A genome scan based on 87 microsatellite markers revealed only few signatures of selection, likely owing to the constraints that homogenizing gene flow puts on adaptive divergence. However, the detected loci appear repeatedly as targets of selection in similar studies of genomic adaptation in the three‐spined stickleback. We conclude that the signature of genomic selection in the face of strong gene flow is weak, yet detectable. We argue that the range of studies of genomic divergence should be extended to include more systems characterized by limited geographical and ecological isolation, which is often a realistic setting in nature.     

Polycystic kidney disease: The complexity of planar cell polarity and signaling during tissue regeneration and cyst formation

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited systemic disease with intrarenal cystogenesis as its primary characteristic. A variety of mouse models provided information on the requirement of loss of balanced polycystin levels for initiation of cyst formation, the role of proliferation in cystogenesis and the signaling pathways involved in cyst growth and expansion. Here we will review the involvement of different signaling pathways during renal development, renal epithelial regeneration and cyst formation in ADPKD, focusing on planar cell polarity (PCP) and oriented cell division (OCD). This will be discussed in context of the hypothesis that aberrant PCP signaling causes cyst formation. In addition, the role of the Hippo pathway, which was recently found to be involved in cyst growth and tissue regeneration, and well-known for regulating organ size control, will be reviewed. The fact that Hippo signaling is linked to PCP signaling makes the Hippo pathway a novel cascade in cystogenesis. The newly gained understanding of the complex signaling network involved in cystogenesis and disease progression, not only necessitates refining of the current hypothesis regarding initiation of cystogenesis, but also has implications for therapeutic intervention strategies. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta

ABSTRACT: BACKGROUND: Electrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments. RESULTS: Intracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644). CONCLUSION: The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.     

Population Bottlenecks Strongly Affect the Evolutionary Dynamics of Antibiotic Persistence

Bacterial persistence is a potential cause of antibiotic therapy failure. Antibiotic-tolerant persisters originate from phenotypic differentiation within a susceptible population, occurring with a frequency that can be altered by mutations. Recent studies have proven that persistence is a highly evolvable trait and, consequently, an important evolutionary strategy of bacterial populations to adapt to high-dose antibiotic therapy. Yet, the factors that govern the evolutionary dynamics of persistence are currently poorly understood. Theoretical studies predict far-reaching effects of bottlenecking on the evolutionary adaption of bacterial populations, but these effects have never been investigated in the context of persistence. Bottlenecking events are frequently encountered by infecting pathogens during host-to-host transmission and antibiotic treatment. In this study, we used a combination of experimental evolution and barcoded knockout libraries to examine how population bottlenecking affects the evolutionary dynamics of persistence. In accordance with existing hypotheses, small bottlenecks were found to restrict the adaptive potential of populations and result in more heterogeneous evolutionary outcomes. Evolutionary trajectories followed in small-bottlenecking regimes additionally suggest that the fitness landscape associated with persistence has a rugged topography, with distinct trajectories toward increased persistence that are accessible to evolving populations. Furthermore, sequencing data of evolved populations and knockout libraries after selection reveal various genes that are potentially involved in persistence, including previously known as well as novel targets. Together, our results do not only provide experimental evidence for evolutionary theories, but also contribute to a better understanding of the environmental and genetic factors that guide bacterial adaptation to antibiotic treatment.     

PHYMYCO-DB: A Curated Database for Analyses of Fungal Diversity and Evolution

Background

In environmental sequencing studies, fungi can be identified based on nucleic acid sequences, using either highly variable sequences as species barcodes or conserved sequences containing a high-quality phylogenetic signal. For the latter, identification relies on phylogenetic analyses and the adoption of the phylogenetic species concept.Such analysis requires that the reference sequences are well identified and deposited in public-access databases. However, many entries in the public sequence databases are problematic in terms of quality and reliability and these data require screening to ensure correct phylogenetic interpretation.

Methods and Principal Findings

To facilitate phylogenetic inferences and phylogenetic assignment, we introduce a fungal sequence database. The database PHYMYCO-DB comprises fungal sequences from GenBank that have been filtered to satisfy stringent sequence quality criteria. For the first release, two widely used molecular taxonomic markers were chosen: the nuclear SSU rRNA and EF1-α gene sequences. Following the automatic extraction and filtration, a manual curation is performed to remove problematic sequences while preserving relevant sequences useful for phylogenetic studies. As a result of curation, ∼20% of the automatically filtered sequences have been removed from the database. To demonstrate how PHYMYCO-DB can be employed, we test a set of environmental Chytridiomycota sequences obtained from deep sea samples.

Conclusion

PHYMYCO-DB offers the tools necessary to: (i) extract high quality fungal sequences for each of the 5 fungal phyla, at all taxonomic levels, (ii) extract already performed alignments, to act as ‘reference alignments’, (iii) launch alignments of personal sequences along with stored data. A total of 9120 SSU rRNA and 672 EF1-α high-quality fungal sequences are now available.The PHYMYCO-DB is accessible through the URL http://phymycodb.genouest.org/.     

Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress

Background

Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.

Methods

Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H₂DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.

Results

Tert-BHP increased CM-H₂DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.

Conclusions

Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.     

A novel peptidomics approach to detect markers of Alzheimer's disease in cerebrospinal fluid

Sensitive and specific diagnosis and monitoring of disease progression are of prime importance to develop new therapies for Alzheimer's disease patients. Although the diagnostic accuracy, verified by pathological examination is high, it is currently not possible to diagnose Alzheimer's disease with a high degree of certainty until relatively late in the disease process. Here, we have undertaken a peptidome analysis of postmortem cerebrospinal fluid of neuropathologically confirmed Alzheimer's disease patients and non-demented controls using a combination of methods and technologies. This includes novel sample preparation based on the enrichment of endogenous, proteolytically derived peptides as well as peptides non-covalently bound to abundant proteins. We observed differences in peptide profiles associated with Alzheimer's disease in the endogenous peptide fraction and in the protein-bound peptide fraction. The discriminating peptides in the unbound peptide fraction were identified as VGF nerve growth factor inducible precursor, and complement C4 precursor, whereas the discriminating peptides in the protein-bound fraction were identified as VGF nerve growth factor inducible precursor, and alpha-2-HS-glycoprotein.