High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Fitness Consequences of Northward Dispersal as Possible Adaptation to Climate Change,Using Experimental Translocation of a Migratory Passerine

Climate change leads to rapid, differential changes in phenology across trophic levels, often resulting in temporal mismatches between predators and their prey. If a species cannot easily adjust its timing, it can adapt by choosing a new breeding location with a later phenology of its prey. In this study, we experimentally investigated whether long-distance dispersal to northern breeding grounds with a later phenology could be a feasible process to restore the match between timing of breeding and peak food abundance and thus improve reproductive success. Here, we report the successful translocation of pied flycatchers (Ficedula hypoleuca) to natural breeding sites 560 km to the Northeast. We expected translocated birds to have a fitness advantage with respect to environmental phenology, but to potentially pay costs through the lack of other locally adapted traits. Translocated individuals started egg laying 11 days earlier than northern control birds, which were translocated only within the northern site. The number of fledglings produced was somewhat lower in translocated birds, compared to northern controls, and fledglings were in lower body condition. Translocated individuals were performing not significantly different to control birds that remained at the original southern site. The lack of advantage of the translocated individuals most likely resulted from the exceptionally cold spring in which the experiment was carried out. Our results, however, suggest that pied flycatchers can successfully introduce their early breeding phenotype after dispersing to more northern areas, and thus that adaptation through dispersal is a viable option for populations that get locally maladapted through climate change.     

Mus in Morocco: a Quaternary sequence of intraspecific evolution

North Africa is an intricate biogeographical region at the crossroads of immigration waves from tropical Africa and Asia. Species confined between various barriers (Atlas Mountains, arid environments such as the Sahara in the south, water masses such as the Mediterranean Sea in the north, and the Atlantic Ocean in the west) were generally forced to adapt locally to environmental changes instead of tracking their habitat by shifting their distribution area. The present study aims at providing first insight into the evolution of the genus Mus, and more specifically of the western Mediterranean species Mus spretus in this area. The study relies on the abundant Late Pleistocene and Middle Holocene fossil assemblage from the El Harhoura 2 cave (Rabat‐Témara, Morocco). This exceptional record was studied using geometric morphometrics applied to first upper and lower molars, constituting the most informative and best preserved fossil remains for such small rodents. Two main issues were addressed. (1) Geometric morphometrics was used to clarify taxonomic status and phylogenetic relationships among fossil and modern species in this area. Morphometric analysis revealed good discrimination of most modern and fossil species but failed to document intermediate forms tracing anagenetic evolution. Not mutually exclusive, the occurrence of complex processes of morphological evolution in this genus such as parallel evolution and the action of stabilizing selection may make it difficult to translate patterns of morphological evolution into phylogenetic conclusions. (2) The record was shown to document a sequence of intraspecific evolution of M. spretus. The morphology of the molars through the fossil record of El Harhoura 2 was surprisingly stable despite extensive modern variation. The limited temporal variation largely failed to correlate to palaeoenvironmental proxies. The mouse fossil record at El Harhoura 2 thus presents an intriguing case of morphological stasis despite extensive environmental changes. This long‐term stability may have been recently perturbed by anthropogenic factors including landscape changes and introduction of various competitors and predators, leading to a size reduction. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 599–621.     

Evolution and diversity of Arsenophonus endosymbionts in aphids

Endosymbiotic bacteria are important drivers of insect evolutionary ecology, acting both as partners that contribute to host adaptation and as subtle parasites that manipulate host reproduction. Among them, the genus Arsenophonus is emerging as one of the most widespread lineages. Its biology is, however, entirely unknown in most cases, and it is therefore unclear how infections spread through insect populations. Here we examine the incidence and evolutionary history of Arsenophonus in aphid populations from 86 species, characterizing the processes that shape their diversity. We identify aphids as harbouring an important diversity of Arsenophonus strains. Present in 7% of the sampled species, incidence was especially high in the Aphis genus with more than 31% of the infected species. Phylogenetic investigations revealed that these Arseno‐phonus strains do not cluster within an aphid‐specific clade but rather exhibit distinct evolutionary origins showing that they undergo repeated horizontal transfers (HT) between distantly related host species. Their diversity pattern strongly suggests that ecological interactions, such as plant mediation and parasitism, are major drivers for Arsenophonus dispersal, dictating global incidence across insect communities. Notably, plants hosting aphids may be important ecological arenas for global exchange of Arsenophonus, serving as reservoirs for HT.     

Accounting for Age Uncertainty in Growth Modeling,the Case Study of Yellowfin Tuna (Thunnus albacares) of the Indian Ocean

Age estimates, typically determined by counting periodic growth increments in calcified structures of vertebrates, are the basis of population dynamics models used for managing exploited or threatened species. In fisheries research, the use of otolith growth rings as an indicator of fish age has increased considerably in recent decades. However, otolith readings include various sources of uncertainty. Current ageing methods, which converts an average count of rings into age, only provide periodic age estimates in which the range of uncertainty is fully ignored. In this study, we describe a hierarchical model for estimating individual ages from repeated otolith readings. The model was developed within a Bayesian framework to explicitly represent the sources of uncertainty associated with age estimation, to allow for individual variations and to include knowledge on parameters from expertise. The performance of the proposed model was examined through simulations, and then it was coupled to a two-stanza somatic growth model to evaluate the impact of the age estimation method on the age composition of commercial fisheries catches. We illustrate our approach using the saggital otoliths of yellowfin tuna of the Indian Ocean collected through large-scale mark-recapture experiments. The simulation performance suggested that the ageing error model was able to estimate the ageing biases and provide accurate age estimates, regardless of the age of the fish. Coupled with the growth model, this approach appeared suitable for modeling the growth of Indian Ocean yellowfin and is consistent with findings of previous studies. The simulations showed that the choice of the ageing method can strongly affect growth estimates with subsequent implications for age-structured data used as inputs for population models. Finally, our modeling approach revealed particularly useful to reflect uncertainty around age estimates into the process of growth estimation and it can be applied to any study relying on age estimation.     

Epistatic Interaction between BANK1 and BLK in Rheumatoid Arthritis: Results from a Large Trans-Ethnic Meta-Analysis

Background

BANK1 and BLK belong to the pleiotropic autoimmune genes; recently, epistasis between BANK1 and BLK was detected in systemic lupus erythematosus. Although BLK has been reproducibly identified as a risk factor in rheumatoid arthritis (RA), reports are conflicting about the contribution of BANK1 to RA susceptibility. To ascertain the real impact of BANK1 on RA genetic susceptibility, we performed a large meta-analysis including our original data and tested for an epistatic interaction between BANK1 and BLK in RA susceptibility.

Patients and Methods

We investigated data for 1,915 RA patients and 1,915 ethnically matched healthy controls genotyped for BANK1 rs10516487 and rs3733197 and BLK rs13277113. The association of each SNP and RA was tested by logistic regression. Multivariate analysis was then used with an interaction term to test for an epistatic interaction between the SNPs in the 2 genes.

Results

None of the SNPs tested individually was significantly associated with RA in the genotyped samples. However, we detected an epistatic interaction between BANK1 rs3733197 and BLK rs13277113 (P_interaction = 0.037). In individuals carrying the BLK rs13277113 GG genotype, presence of the BANK1 rs3733197 G allele increased the risk of RA (odds ratio 1.21 [95% confidence interval 1.04–1.41], P = 0.015. Combining our results with those of all other studies in a large trans-ethnic meta-analysis revealed an association of the BANK1 rs3733197 G allele and RA (1.11 [1.02–1.21], P = 0.012).

Conclusion

This study confirms BANK1 as an RA susceptibility gene and for the first time provides evidence for epistasis between BANK1 and BLK in RA. Our results illustrate the concept of pleiotropic epistatic interaction, suggesting that BANK1 and BLK might play a role in RA pathogenesis.     

Behavioral and functional strategies during tool use tasks in bonobos

Different primate species have developed extensive capacities for grasping and manipulating objects. However, the manual abilities of primates remain poorly known from a dynamic point of view. The aim of the present study was to quantify the functional and behavioral strategies used by captive bonobos (Pan paniscus) during tool use tasks. The study was conducted on eight captive bonobos which we observed during two tool use tasks: food extraction from a large piece of wood and food recovery from a maze. We focused on grasping postures, in‐hand movements, the sequences of grasp postures used that have not been studied in bonobos, and the kind of tools selected. Bonobos used a great variety of grasping postures during both tool use tasks. They were capable of in‐hand movement, demonstrated complex sequences of contacts, and showed more dynamic manipulation during the maze task than during the extraction task. They arrived on the location of the task with the tool already modified and used different kinds of tools according to the task. We also observed individual manual strategies. Bonobos were thus able to develop in‐hand movements similar to humans and chimpanzees, demonstrated dynamic manipulation, and they responded to task constraints by selecting and modifying tools appropriately, usually before they started the tasks. These results show the necessity to quantify object manipulation in different species to better understand their real manual specificities, which is essential to reconstruct the evolution of primate manual abilities.     

Immune gene variability influences roe deer natal dispersal

Dispersal is a key life‐history trait governing the response of individuals, populations and species to changing environmental conditions. In the context of global change, it is therefore essential to better understand the respective role of condition‐, phenotype‐ and genetic‐dependent drivers of dispersal behaviour. Although the importance of immune function and pathogen infestation in determining patterns of dispersal is increasingly recognised, no study to our knowledge has yet investigated the influence of immune gene variability on dispersal behaviour. Here, we filled this knowledge gap by assessing whether individual heterozygosity at five immune gene loci (one from the Major histocompatibility complex and four from encoding Toll‐like receptors) influences roe deer natal dispersal. We found that dispersal propensity was affected by immune gene diversity, suggesting potential pathogen‐mediated selection through over‐dominance. However, the direction of this effect differed between high and low quality individuals, suggesting that dispersal propensity is driven by two different mechanisms. In support of the condition‐dependent dispersal hypothesis, dispersal propensity increased with increasing body mass and, among high quality individuals only (standardized body mass > 18 kg), with increasing immune gene diversity. However, among poor quality individuals, we observed the opposite pattern such that dispersal propensity was higher for individuals with lower immune gene diversity. We suggest that these poor quality individuals expressed an emergency dispersal tactic in an attempt to escape a heavily infested environment associated with poor fitness prospects. Our results have potentially important consequences in terms of population genetics and demography, as well as host–pathogen evolution.