Attenuation of cGAS‐STING signaling is mediated by a p62/SQSTM1‐dependent autophagy pathway activated by TBK1

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING. Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62‐deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy‐associated vesicles. Thus, DNA sensing induces the cGAS‐STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.     

MobiSeq: De novo SNP discovery in model and non‐model species through sequencing the flanking region of transposable elements

In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome‐scale studies to characterize both model and non‐model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome‐wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site‐associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms—enabling the exploration of diverse evolutionary and conservation questions.     

A Legume Genetic Framework Controls Infection of Nodules by Symbiotic and Endophytic Bacteria

Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule.     

Expression of genes for cytokines and cytokine-related functions in leukocytes infected with Herpes simplex virus: comparison between resistant and susceptible mouse strains

Cytokines and chemokines play an important role in the first line of defence against viral infections. Moreover, these groups of proteins also contribute significantly to regulation of the acquired immune response. Therefore, knowledge of the expression of cytokines, chemokines and factors involved in their action may provide information about the immune reaction responsible for elimination of viral infections and for immune-mediated pathology. Using cDNA arrays, we have evaluated the expression of cytokines and genes related to cytokine function in resting murine peritoneal cells and in inflammatory macrophages infected with Herpes simplex virus (HSV)-1 and -2. To allow comparison, the experiments were performed using both the resistant mouse strain C57BL/6 and the susceptible strain BALB/c. The work identified a group of genes that is differentially expressed during HSV infection of cells from the two strains. Another group of genes was affected by HSV-1 but not HSV-2 infection and vice versa. Further analysis of these genes may provide new information about host defense against viral infections and could also lead to identification of the molecular basis for the pathological differences between infections with HSV-1 and -2.     

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with (beta)arr2. However, when the wild-type receptor was coexpressed with both (beta)arr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level.     

Randomised Controlled Trials May Underestimate Drug Effects: Balanced Placebo Trial Design

Background

It is an inherent assumption in randomised controlled trials that the drug effect can be estimated by subtracting the response during placebo from the response during active drug treatment.

Objective

To test the assumption of additivity. The primary hypothesis was that the total treatment effect is smaller than the sum of the drug effect and the placebo effect. The secondary hypothesis was that non-additivity was most pronounced in participants with large placebo effects.

Methods

We used a within-subject randomised blinded balanced placebo design and included 48 healthy volunteers (50% males), mean (SD) age 23.4 (6.2) years. Experimental pain was induced by injections of hypertonic saline into the masseter muscle. Participants received four injections with hypertonic saline along with lidocaine or matching placebo in randomised order: A: received hypertonic saline/told hypertonic saline; B: received hypertonic saline+lidocaine/told hypertonic saline; C: received hypertonic saline+placebo/told hypertonic saline+pain killer; D: received hypertonic saline+lidocaine/told hypertonic saline+pain killer. The primary outcome measure was the area under the curve (AUC, mm²) of pain intensity during injections.

Results

There was a significant difference between the sum of the drug effect and the placebo effect (mean AUC 6279 mm² (95% CI, 4936–7622)) and the total treatment effect (mean AUC 5455 mm² (95% CI, 4585–6324)) (P = 0.049). This difference was larger for participants with large versus small placebo effects (P = 0.015), and the difference correlated significantly with the size of the placebo effect (r = 0.65, P = 0.006).

Conclusion

Although this study examined placebo effects and not the whole placebo response as in randomised controlled trials, it does suggest that the additivity assumption may be incorrect, and that the estimated drug effects in randomised controlled trials may be underestimated, particularly in studies reporting large placebo responses. The implications for randomised controlled trials and systematic reviews need to be discussed.     

Genetic association analysis of human longevity in cohort studies of elderly subjects: an example of the PON1 gene in the Danish 1905 birth cohort

Although the case-control or the cross-sectional design has been popular in genetic association studies of human longevity, such a design is prone to false positive results due to sampling bias and a potential secular trend in gene-environment interactions. To avoid these problems, the cohort or follow-up study design has been recommended. With the observed individual survival information, the Cox regression model has been used for single-locus data analysis. In this article, we present a novel survival analysis model that combines population survival with individual genotype and phenotype information in assessing the genetic association with human longevity in cohort studies. By monitoring the changes in the observed genotype frequencies over the follow-up period in a birth cohort, we are able to assess the effects of the genotypes and/or haplotypes on individual survival. With the estimated parameters, genotype- and/or haplotype-specific survival and hazard functions can be calculated without any parametric assumption on the survival distribution. In addition, our model estimates haplotype frequencies in a birth cohort over the follow-up time, which is not observable in the multilocus genotype data. A computer simulation study was conducted to specifically assess the performance and power of our haplotype-based approach for given risk and frequency parameters under different sample sizes. Application of our method to paraoxonase 1 genotype data detected a haplotype that significantly reduces carriers' hazard of death and thus reveals and stresses the important role of genetic variation in maintaining human survival at advanced ages.     

Body composition analysis by DEXA by using dynamically changing samarium filtration

Gotfredsen, Anders, Lene Bæksgaard, and Jannik Hilsted.Body composition analysis by DEXA by using dynamically changing samarium filtration. J. Appl. Physiol.82(4): 1200-1209, 1997.Dual-energy X-ray absorptiometry (DEXA)has a high accuracy for body composition analysis but is influenced bybeam hardening and other error sources in the extremes of measurement.To compensate for beam hardening, the Norland XR-36 introduces adynamically changing samarium filtration system, which depends on thecurrent-absorber thickness. With this system we found a good agreement(r = 0.99) between reference andmeasured amounts of tissue or fat percentages in a plastic phantom andin smaller (~0.5-4 kg) and larger (~5-20 kg) piles oftissue (ox muscle and lard). Scans of six healthy volunteers coveredwith combinations of beef and lard (~5-15 kg) showed a goodagreement (r = 0.99) between referenceand DEXA values of added soft tissue mass and fat percentage. Weconclude that the DEXA method (and, in particular, the Norland XR-36using dynamic filtration) has a high accuracy for body compositionanalysis. It has a potential for gaining status as a reference methodin the future and may presently be used as a supplement to thetraditional methods for body composition analysis.

     

Gene flow and mating patterns in individuals of wych elm (Ulmus glabra) in forest and open land after the influence of Dutch elm disease

Dutch elm disease has severely reduced the number of large trees of U. glabra in Denmark. Consequently, the distance between large trees has increased and the overall density of the species has decreased. Patches of small trees with stem diameters up to 10 cm are, however, relatively frequent. With four microsatellites we studied potential differences in genetic diversity, mating patterns and pollen flow in trees of U. glabra that occur either in a continuous forest (Suserup Forest) or isolated in the open land. We found no indications of selfing in forest or open land but indications of biparental inbreeding in offspring of isolated trees. Estimates of effective pollen donors (N _ep) and minimum number of pollen donors (N _p) were alike in forest and open land (N _ep of 31 and 34 and N _p of 4 to >6 and 3 to >6, respectively). The number of alleles was also very similar. With indirect methods we found that average pollen dispersal was 104 m under forest conditions. The average distance between the isolated trees and their potential pollen donors was further, thus suggesting that effective pollen in the open land on average moves further than in a dense forest. Finally, 28% of small trees (diameters up to 10 cm) produced fruits. Reproduction at a young age may be a key stone in the survival of U. glabra as the vectors of the disease prefer older trees.