The intrinsically disordered CARDs‐Helicase linker in RIG‐I is a molecular gate for RNA proofreading

The innate immune receptor RIG‐I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG‐I''s C‐terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non‐specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG‐I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs‐Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD‐chosen RNAs to bind the helicase domain, while at the same time blocking non‐specific RNAs. These findings also indicate that CHL could represent a novel target for RIG‐I‐based therapeutics.     

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.     

Testing the precision and sensitivity of density estimates obtained with a camera‐trap method revealed limitations and opportunities

The use of camera traps in ecology helps affordably address questions about the distribution and density of cryptic and mobile species. The random encounter model (REM) is a camera‐trap method that has been developed to estimate population densities using unmarked individuals. However, few studies have evaluated its reliability in the field, especially considering that this method relies on parameters obtained from collared animals (i.e., average speed, in km/h), which can be difficult to acquire at low cost and effort. Our objectives were to (1) assess the reliability of this camera‐trap method and (2) evaluate the influence of parameters coming from different populations on density estimates. We estimated a reference density of black bears (Ursus americanus) in Forillon National Park (Québec, Canada) using a spatial capture–recapture estimator based on hair‐snag stations. We calculated average speed using telemetry data acquired from four different bear populations located outside our study area and estimated densities using the REM. The reference density, determined with a Bayesian spatial capture–recapture model, was 2.87 individuals/10km² [95% CI: 2.41–3.45], which was slightly lower (although not significatively different) than the different densities estimated using REM (ranging from 4.06–5.38 bears/10km² depending on the average speed value used). Average speed values obtained from different populations had minor impacts on REM estimates when the difference in average speed between populations was low. Bias in speed values for slow‐moving species had more influence on REM density estimates than for fast‐moving species. We pointed out that a potential overestimation of density occurs when average speed is underestimated, that is, using GPS telemetry locations with large fix‐rate intervals. Our study suggests that REM could be an affordable alternative to conventional spatial capture–recapture, but highlights the need for further research to control for potential bias associated with speed values determined using GPS telemetry data.     

Small‐scale genetic structure and mating patterns in an extensive sessile oak forest (Quercus petraea (Matt.) Liebl.)

Oaks (Quercus) are major components of temperate forest ecosystems in the Northern Hemisphere where they form intermediate or climax communities. Sessile oak (Quercus petraea) forests represent the climax vegetation in eastern Germany and western Poland. Here, sessile oak forms pure stands or occurs intermixed with Scots Pine (Pinus sylvestris). A large body of research is available on gene flow, reproduction dynamics, and genetic structure in fragmented landscapes and mixed populations. At the same time, our knowledge regarding large, contiguous, and monospecific populations is considerably less well developed. Our study is an attempt to further develop our understanding of the reproduction ecology of sessile oak as an ecologically and economically important forest tree by analyzing mating patterns and genetic structure within adult trees and seedlings originating from one or two reproduction events in an extensive, naturally regenerating sessile oak forest. We detected positive spatial genetic structure up to 30 meters between adult trees and up to 40 meters between seedlings. Seed dispersal distances averaged 8.4 meters. Pollen dispersal distances averaged 22.6 meters. In both cases, the largest proportion of the dispersal occurred over short distances. Dispersal over longer distances was more common for pollen but also appeared regularly for seeds. The reproductive success of individual trees was highly skewed. Only 41 percent of all adult trees produced any offspring while the majority did not participate in reproduction. Among those trees that contributed to the analyzed seedling sample, 80 percent contributed 1–3 gametes. Only 20 percent of all parent trees contributed four or more gametes. However, these relatively few most fertile trees contributed 51 percent of all gametes within the seedling sample. Vitality and growth differed significantly between reproducing and nonreproducing adult trees with reproducing trees being more vital and vigorous than nonreproducing individuals. Our study demonstrates that extensive, apparently homogenous oak forests are far from uniform on the genetic level. On the contrary, they form highly complex mosaics of remarkably small local neighborhoods. This counterbalances the levelling effect of long‐distance dispersal and may increase the species’ adaptive potential. Incorporating these dynamics in the management, conservation, and restoration of oak forests can support the conservation of forest genetic diversity and assist those forests in coping with environmental change.     

Synthesis of heteroatom-substituted analogues of stearic acid

We report herein the syntheses of several analogues of stearic acid in which oxygen or sulfur atoms or sulfoxide groups have replaced the methylene groups at either position 9 or position 10 of the polymethylene chains. These compounds have been fully characterized by the results of proton and carbon 13 nuclear magnetic resonance, and low and high resolution mass spectral studies.     

Fractionation of subcellular membranes of the secretory pathway from the peroxidase-producing white-rot fungus Phanerochaete chrysosporium

Abstract Mycelia from the basidiomycete Phanerochaete chrysosporium , producing lignin and manganese peroxidases, were homogenized and fractionated on a sucrose gradient. The main subcellular fungal membrane fractions were successfully separated. Lipid composition analyses of the isolated membranes as well as associated marker enzymes distribution gave evidence to similarities with membranes originating from plants. Lignin and manganese peroxidases were investigated by immunodetection in subcellular fractions. Our results show that lignin and manganese peroxidases are mainly associated with Golgi apparatus vesicles and, to a lesser extent, with endoplasmic reticulum and light density vesicles, but not with plasma membranes.