RNAi revised - target mRNA-dependent enhancement of gene silencing

The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy.We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery.     

Specialize or risk disappearance – empirical evidence of anisomerism based on comparative and developmental studies of gnathostome head and limb musculature

William K. Gregory was one of the most influential authors defending the existence of an evolutionary trend in vertebrates from a higher degree of polyisomerism (more polyisomeric or ‘serial’ anatomical structures arranged along any body axis) to cases of anisomerism (specialization or loss of at least some original polyisomeric structures). Anisomerism was the subject of much interest during the 19th and the beginning of the 20th centuries, particularly due to the influence of the Romantic German School and the notion of ‘primitive archetype’ and because it was conceptually linked to other crucial biological issues (e.g. complexity, scala naturae, progress, modularity or phenotypic integration). However, discussions on anisomerism and related issues (e.g. Williston's law) have been almost exclusively based on hard tissues. Here we provide the first detailed empirical test, and discussion, of anisomerism based on quantitative data obtained from phylogenetic and comparative analyses of the head and forelimb muscles of gnathostomes. Our results strongly support the existence of such a trend in both forelimb and head musculature. For instance, the last common ancestor (LCA) of extant tetrapods likely had 38 polyisomeric muscles (PMs) out of a total of 70 forelimb muscles (i.e. 54%), whereas in the LCAs of extant amniotes and of mammals these numbers were 38/73 (52%) and 21/67 (31%), and in humans are 11/59 (19%). Interestingly, the number of PMs that became specialized during the forelimb evolutionary transition from the LCA of extant tetrapods to humans (13) is very similar to the number of PMs that became lost (14), indicating that both specialization and loss contributed equally to the trend towards anisomerism. By contrast, during the evolution of the head musculature from the LCA of gnathostomes to humans a total of 27 PMs were lost whereas only one muscle became specialized. Importantly, the evolutionary trend towards anisomerism is not related to a general trend leading to the presence of fewer muscles in derived taxa, because for instance humans have more head muscles in total, but many less head polyisomeric muscles than early gnathostomes and extant fish such as sharks, and than early tetrapods and amphibians such as salamanders. This is because new muscles have also been acquired during gnathostome evolution (e.g. facial muscles of mammals). Interestingly, many new PMs have also been acquired during head evolution (but subsequently lost during the transitions towards humans), whereas only a few new PMs were acquired during forelimb evolution. Our comparisons and review of the literature indicate that there is also a trend towards anisomerism during development, thus providing a further example of a parallel between ontogeny and phylogeny, e.g. some forelimb PMs (e.g. contrahentes, intermetacarpales) become specialized or lost (re‐absorbed) during human ontogeny and some head PMs (e.g. constrictores branchiales) become lost during salamander ontogeny. This review will inform future discussions on modularity, complexity, body plans, phenotypic integration and macroevolution, which should ideally include soft tissues and the use of new tools (e.g. anatomical networks) in order to provide a broader and more integrative understanding of these relevant subjects.     

Characterisation of Calcium Phosphate Crystals on Calcified Human Aortic Vascular Smooth Muscle Cells and Potential Role of Magnesium

Background

Cardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. Characterisation of the chemical and crystalline structure of VC was mainly led in patients or animal models with CKD. Likewise, Mg²⁺ was found to be protective in living cells although a potential role for Mg²⁺ could not be excluded on crystal formation and precipitation. In this study, the crystal formation and the role of Mg²⁺ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques.

Methodology/Principal Findings

In HAVSMC incubated with increased Ca x Pi medium, only calcium phosphate apatite crystals (CPA) were detected by Micro-Fourier Transform InfraRed spectroscopy (µFTIR) and Field Effect Scanning Electron Microscope (FE — SEM) and Energy Dispersive X-ray spectrometry (EDX) at the cell layer level. Supplementation with Mg²⁺ did not alter the crystal composition or structure. The crystal deposition was preferentially positioned near or directly on cells as pictured by FE — SEM observations and EDX measurements. Large µFTIR maps revealed spots of CPA crystals that were associated to the cellular layout. This qualitative analysis suggests a potential beneficial effect of Mg²⁺ at 5 mM in noticeably reducing the number and intensities of CPA µFTIR spots.

Conclusions/Significance

For the first time in a model of HAVSMC, induced calcification led to the formation of the sole CPA crystals. Our data seems to exclude a physicochemical role of Mg²⁺ in altering the CPA crystal growth, composition or structure. Furthermore, Mg²⁺ beneficial role in attenuating VC should be linked to an active cellular role.     

RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues.     

Safety and Proof-of-Concept Study of Oral QLT091001 in Retinitis Pigmentosa Due to Inherited Deficiencies of Retinal Pigment Epithelial 65 Protein (RPE65) or Lecithin:Retinol Acyltransferase (LRAT)

Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001). Safety and visual outcomes of a once-daily oral dose of 40 mg/m²/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44%) showed a ≥20% increase and 4 of 18 (22%) showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67%) and 5 (28%) of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI) than non-responders (3.5 ± 1.2 μm). This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01014052","term_id":"NCT01014052"}}NCT01014052     

Fertilization affects the establishment ability of species differing in seed mass via direct nutrient addition and indirect competition effects

Fertilization causes species loss and species dominance changes in plant communities worldwide. However, it still remains unclear how fertilization acts upon species functional traits, e.g. seed mass. Seed mass is a key trait of the regeneration strategy of plants, which influences a range of processes during the seedling establishment phase. Fertilization may select upon seed mass, either directly by increased nutrient availability or indirectly by increased competition. Since previous research has mainly analyzed the indirect effects of fertilization, we disentangled the direct and indirect effects to examine how nutrient availability and competition influence the seed mass relationships on four key components during seedling establishment: seedling emergence, time of seedling emergence, seedling survival and seedling growth. We conducted a common garden experiment with 22 dry grassland species with a two‐way full factorial design that simulated additional nutrient supply and increased competition. While we found no evidence that fertilization either directly by additional nutrient supply or indirectly by increased competition alters the relationship between seed mass and (time of) seedling emergence, we revealed that large seed mass is beneficial under nutrient‐poor conditions (seedlings have greater chances of survival, particularly in nutrient‐poor soils) as well as under competition (large‐seeded species produced larger seedlings, which suffered less from competition than small‐seeded species). Based on these findings, we argue that both factors, i.e. nutrient availability and competition intensity, ought to be considered to understand how fertilization influences seedling establishment and species composition with respect to seed mass in natural communities. We propose a simple conceptual model, in which seed mass in natural communities is determined by competition intensity and nutrient availability. Here, we hypothesize that seed mass shows a U‐shaped pattern along gradients of soil fertility, which may explain the contrasting soil fertility‐seed mass relationships found in the recent literature.     

Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.