In utero RNAi of the dyslexia-associated gene Kiaa0319 in rats (KIA-) degrades cortical responses to speech sounds and increases trial-by-trial variability in onset latency. We tested the hypothesis that KIA- rats would be impaired at speech sound discrimination. KIA- rats needed twice as much training in quiet conditions to perform at control levels and remained impaired at several speech tasks. Focused training using truncated speech sounds was able to normalize speech discrimination in quiet and background noise conditions. Training also normalized trial-by-trial neural variability and temporal phase locking. Cortical activity from speech trained KIA- rats was sufficient to accurately discriminate between similar consonant sounds. These results provide the first direct evidence that assumed reduced expression of the dyslexia-associated gene KIAA0319 can cause phoneme processing impairments similar to those seen in dyslexia and that intensive behavioral therapy can eliminate these impairments. Dyslexia is the most common developmental language disorder and affects approximately 7% of the population KIAA0319, DYX1C1, DCDC2 and ROBO1) KIAA0319 is the most consistently associated gene and the link between KIAA0319 and dyslexia has been replicated in many independent studies KIAA0319 impair speech evoked cortical activity in utero RNA interference (RNAi) of the rat homolog of this gene (Kiaa0319) is sufficient to impair neural processing of speech sounds by elevating trial-by-trial variability in the timing of neural responses Kiaa0319 RNAi also duplicates corpus callosum abnormalities in dyslexia Kiaa0319 would exhibit impaired learning when trained to discriminate speech sounds.Dyslexia is highly heritable and at least four candidate-dyslexia genes have been identified  on a variety of speech sound discrimination tasks designed to evaluate known deficits in human dyslexics, including problems with speech in noise, rapid speech sounds, and isolated phonemes. We recorded action potentials and local field potentials in primary (A1) and posterior auditory fields (PAF) after training and compared responses with untrained KIA- and control rats.In this study, we used an animal model of speech sound processing to evaluate the role of All transfection protocols were designed to minimize any animal suffering and were approved by the University of Connecticut Institutional Animal Care and Use Committee (Protocol # A12-027). All behavioral, surgical, and physiological procedures were designed to minimize any animal suffering and were approved by the University of Texas at Dallas Institutional Animal Care and Use Committee (Protocol # 99-06). Data presented in this study are available upon request.in utero electroporation targeting lateral regions of neocortex that included the auditory cortex by methods described previously Kiaa0319 which can decrease the Kiaa0319 protein expression in cell culture  to identify the experimental status of each subject . The number of fluorescent cells was counted in a 1 mmKiaa0319 (KIA-) and 10 received scrambled RNAi and served as controls. The behavior tasks we tested are described in detail elsewhere We trained 26 rats to discriminate a target speech sound (/dad/) in 4 different contexts. Of these rats, 16 received RNAi of The isolated speech task consisted of a go-no go paradigm in which rats were trained to press a lever in response to the target sound and to reject each of seven distractors: /dad/ versus /bad/, /gad/, /sad/, /tad/, /dud/, /deed/, /dood/ −1) and multi-unit recordings were acquired at cortical layer 4/5 (∼600–800 µm) using four Parylene-coated tungsten microelectrodes (1–2 MΩ). We used previously recorded multiunit responses from 11 experimentally naïve rats to evaluate the effect of training on neural responses; data from 5 untrained KIA- rats and 6 naïve controls was previously published in Centanni et al. 2013a. Each experimental group  underwent neural recordings once for the purposes of this study. Untrained rats were recorded from as described previously Following the approximately 4 months of training needed to complete all 4 tasks; rats were anesthetized with dilute pentobarbital and mapped. The techniques used for acute recordings are described in detail elsewhere At each site, we presented a tuning curve consisting of 90 frequencies (1–47 kHz) at 16 intensities (0–75 dB SPL) to determine the characteristic frequency (CF) of each site, trains of six broadband noise bursts  to evaluate following ability of A1 neurons, and the speech stimuli used in our behavior tasks Though behavior did not differ between groups, we analyzed tuning curves for each group to see if the training order caused differences in the neural responses. To define A1 and PAF sites, multi-unit recording sites were manually analyzed to select the CF of each site, as well as to obtain bandwidth, latency, peak firing and end of peak response information. A1 sites were defined as having a short onset latency (∼15 ms), narrow bandwidths, and tonotopic organization so that CF increased in a posterior to anterior direction. PAF sites were defined based on their long onset latency (>30 ms), broad bandwidths, and poorly defined tonotopic organization. This method of characterizing auditory fields has been previously validated We trained half of the rats using one task order  and the other half using a second task order . This change in order was to determine if KIA- rats would benefit from specific truncated speech training, as is often used in humans with dyslexia. Control A1 and PAF sites were less variable and more accurate at the consonant classifier task following the second task order compared to control A1 and PAF sites after the first task order , while there was no difference in KIA- sites across training task orders. Since there were only minor differences across groups in the neural responses following training, neural activity from both groups was combined for analysis.i is the time of occurrence of the i'th action potential, and T is the inter-stimulus interval. Perfect synchronization would result in a value of one, whereas no synchronization would result in a value of zero.In response to broad band click trains, normalized spike rate  and vector strength (VS) were calculated. VS quantifies the degree of synchronization between action potentials and repeated sounds. Mean VS is calculated with the formula:sites is each recording site and nbins is each of 40 one-millisecond bins being compared between activity evoked by speech sound X versus speech sound Y. For vowel sounds, the classifier counted the number of action potentials over 400 ms from a single trial and compared the value to the average response to each of the sounds We have previously shown that neural responses in KIA- rats are poor predictors of stimulus identity, while responses in control animals are good predictors of stimulus identity. To test whether training can improve KIA- neural responses, single trial response patterns to each of the isolated speech sounds were compared using a well-documented nearest neighbor classifier Kiaa0319 did not also cause hyperactivity. Significantly shorter response time has been linked to the presence of ADHD in both humans in utero knockdown of Kiaa0319 does not cause significant hyperactivity, impulsivity, motor problems, or difficulty hearing speech sounds.KIA- rats (N = 16) learned to detect a target speech sound as quickly as controls (N = 10). Both groups took approximately 8 days to reach the criterion of 10 training sessions with a d' above 1.5  = 2.155, p = 0.14; Auditory training in humans with dyslexia using truncated speech sounds generalizes to other speech tasks Kiaa0319 knockdown, especially in adverse learning conditions such as speech in noise.We then tested these rats on the other tasks to determine whether training on isolated phonemes would generalize to full length speech sounds. KIA- rats were able to perform the full speech task as well as control rats  than control rats  [31]. AfSince earlier studies of plasticity after dyslexia therapy used EEG or fMRI, we hypothesized that the neural plasticity we observed in the action potential patterns of multi-unit recordings of rats would also be visible in the local field potentials (LFPs). After speech discrimination training, LFPs in control A1 responded faster to the onset of the speech sound /dad/ p<0.01; . Trainedin utero RNAi of the candidate-dyslexia gene Kiaa0319 in rats would cause impaired speech sound discrimination in quiet and in noise. KIA- rats were significantly impaired at discriminating a target speech sound from distractor speech sounds in a variety of contexts and required twice the training time to perform at control levels. Training with truncated speech sounds led to improved performance on tasks using full length sounds in quiet and noisy conditions. KIA- rats who trained on truncated speech discrimination were more accurate on the other speech tasks compared to KIA- rats who had not trained on truncated speech. The neurophysiology data we collected demonstrates that behavioral training improved neural discrimination of consonants and reduced the neural variability in KIA- rats. Improved neural processing generalized to sounds that were not trained. These results provide a potential neural justification for the widespread use of intensive auditory training for dyslexia.The behavioral data we collected confirmed our hypothesis that assumed People with dyslexia have impairments in phoneme identification and manipulation that are correlated with abnormal neural responses. Dyslexics have reduced cortical and thalamic responses to non-speech sounds and speech sounds in passive and active listening conditions KIAA0319 have been associated with dyslexia in at least nine independent studies KIAA0319 eliminate the gene, but they do reduce expression of the protein Kiaa0319 to test the consequences of complete protein elimination, However, the rat model we describe here mimics the functional knockdown seen in human cells by suppressing Kiaa0319 protein expression in the developing brain using RNAi and is likely a more accurate model of the functional consequence of KIAA0319 variants in humans Variants in in utero RNAi of Kiaa0319 have significantly increased membrane resistance and are therefore hyperexcitable compared to neurons transfected with control or rescue sequences Kiaa0319 RNAi leads to impaired behavioral discrimination of speech sound stimuli. In addition to the 1–2% of neurons that were affected by the transfection, other studies have shown the Kiaa0319 targeting shRNA also alters non-transfected cells in cortex Cortical neurons affected by In the current study, we used RNA interference, which is a common technique for knocking down protein expression in the brain Many interventions for dyslexia focus on auditory processing to improve the mapping from phonemes to graphemes Training could improve variability and thus improve speech sound processing through synaptic plasticity mechanisms that alter excitation and/or inhibition. Suppression of candidate-dyslexia genes affects the development of GABAergic neurons in the developing brain There is a considerable amount of debate in the literature regarding the effect of behavioral training on neural responses to auditory stimuli. In the current study, we show that KIA- auditory cortex firing properties do change as a result of behavioral training, while control responses remain stable. Several recent studies have looked at the neural responses to auditory stimuli at various points throughout training. These studies used terminal acute recording techniques and therefore required a separate experimental group for each time point. Nevertheless, these studies clearly document the process of neural responses as they become better predictors of stimulus identity during the course of training ROBO1, DYX1C1, and DCDC2) interfere with auditory processing It is possible that candidate-dyslexia genes could interfere with reading without affecting phoneme processing by interfering with cognitive processes such as working memory or visual attention. It is interesting to note that all four of the best characterized candidate-dyslexia genes  transfected with four different conditions and detected with antibodies against an mRFP epitope tag. Column 1 wells were not transfected and this is the background staining level. Column 2 cultures were transfected with pCAG-Kiaa0319-mRFP and a mutant control shRNA that does not match Kiaa0319 coding sequence. The bright red in column 2 indicates intense expression above background of Kiaa0319-mRFP. Column 3 is the same pCAG-Kiaa0319-mRFP construct transfected in 2 with the addition of the shRNA used to knockdown Kiaa0319 in this study. Column 4 is a similar co transfection with another shRNA vector based on the mir-30 system that contains the same shRNA targeting sequence as the shRNA shown in column 3 experiments.(TIFF)Click here for additional data file.Figure S2Rats with in utero RNAi of Kiaa0319 are able to learn a simple lever pressing task. All rats (16 KIA- rats and 10 control rats) were first trained to press a lever, which triggered the presentation of the target sound (/dad/) and a sugar pellet reward. KIA- rats learned this task in the same amount of time as control rats . After learning to press the lever, rats were transitioned to detection in which they were required to press the lever only when the target sound /dad/ was presented. Rats with in utero RNAi of Kiaa0319 were not impaired in their ability to switch from free pressing to waiting for the target sound. Both groups were able to reach the performance criterion (10 sessions with a d'≥1.5) in approximately 5 days . B. KIA- rats responded as quickly as control rats except on the first day of training, when they were significantly faster (p<0.01). C. KIA- rats (thick black line) responded to the target sound (squares) less often than control rats (thin black line) during the second and third days of training (* = p<0.01). The false alarm rate was not significantly different between the two groups (triangles).(TIF)Click here for additional data file.Figure S3Figure For all behavioral training, we used a set of consonant-vowel-consonant (CVC) speech sounds that have been used in many previous studies. All speech sounds were recorded in our lab by a female, native English speaker and were shifted up by an octave to better accommodate the rat hearing range (Kawahara 1997). For the truncated speech task, we used only the first 40 ms of the speech sounds, shown in the bottom panel. This figure was reprinted with permission from Porter et al., 2011.(TIF)Click here for additional data file.Figure S4Training affects basic neural firing properties to tonal stimuli in KIA- and Control rats. We trained rats for 4 months on a variety of speech discrimination tasks , but had no effect on bandwidths in the other fields. C. Extensive behavioral training had no effect on auditory thresholds in any group or field . D. Extensive behavioral training increased the number of tone-evoked action potentials fired in control A1 , but reduced the number of tone-evoked spikes fired in KIA- A1 .on tasks . KIA- A1on tasks . This reon tasks , narroweon tasks , and hadon tasks  in contron tasks . Followi(TIF)Click here for additional data file.Figure S5Extensive behavioral training improves neural encoding of vowel sounds in control and KIA- auditory cortex. After training, trial-by-trial variability in onset latency across sites in KIA- A1 and PAF as well as control PAF were significantly reduced  improvement in this fields' vowel classifier performance. H. A representative site from trained KIA- PAF. There was significant reduction in trial-by-trial variability in this field after training, and there was slight (but not significant) improvement in this fields' vowel classifier performance. reduced . Perform reduced . Followi(TIF)Click here for additional data file.Figure S6An additional 4 weeks of behavior training causes additional plasticity in control rats. The 4 weeks of additional training  and control PAF (p<0.01). No significant changes were seen in either field in KIA- rats (p = 0.72 and p = 0.99 in A1 and PAF respectively). B. Additional training improved the ability of control PAF sites to perform the consonant neural discrimination task (p<0.01), but this training did not improve classifier performance in control A1 (p = 0.29), KIA- A1 (p = 0.16), or KIA- PAF (p = 0.88). C. Additional training did not improve the ability of neural activity in any group or field to perform the neural discrimination task using vowel stimuli. Control A1; p = 0.05, KIA- A1; p = 0.42, control PAF; p = 0.36, KIA- PAF, p = 0.70.(TIF)Click here for additional data file.


Intracranial hypotension (IH) occurs typically spontaneous and is a potentially life-threatening condition characterized by symptoms varying from postural headache to coma, with classical magnetic resonance imaging (MRI) findings.We report two cases of clinically relevant trauma-related IH and review of the literature.One patient with a cerebral trauma presented unilateral mydriasis and coma resolved by the Trendelenburg position (-20°) as urgency intervention. In the second patient, IH was caused by a lesion of the brachial plexus after a motor vehicle accident.A history of mild or moderate trauma in association with prolonged postural or permanent headache may indicate IH. Posttraumatic IH is rare, nevertheless life-threatening in case of misdiagnosis. Intracranial hypotension in a trauma context is rarely described and difficult to diagnose. The change from tipical supine 30° to Trendelenburg position (0–20°) can be a life-saving manoeuver in these patients. Intracranial hypotension (IH) is a rare clinical entity which can be caused by i.e. spontaneous CSF leakage, or following a lumbar puncture  Score of 15, with discrete neurological deficits and a slowed psychomotor activity. Admission cranial magnetic resonance imaging (MRI) showed large bilateral chronic SDHs and a 7 mm midline deviation towards the left side. Two bilateral hollow bolt screws and drainage were performed. In the follow-up two revision surgeries because of acute and subacute SDHs were necessary and the patient on the neurointensive care in a supine position 30°. At that time, the patient was awake and fully recovered but six hours after the last surgery, the patient deteriorated to a GCS 4 with decerebrate posturing of his upper extremities. A computer tomography (CT) scan demonstrated sufficient evacuation of the bilateral SDH without any residual signs of mass effect. Four hours later the patient developed a rightsided mydriasis and a second CT demonstrated no change. A fourth explorative rightsided revision demonstrated no mass effect explaining the mydriasis and finally intracranial hypotension was suspected. After changing to a Trendelenburg position -20°) the unilateral mydriasis resolved. With regards to the rapid neurological deterioration which developed within minutes, epileptic seizures activities by electroencephalogram (EEG) or a stroke were excluded. The MRI demonstrated elongation of the mesencephalon. A subsequent spinal MRI and scintigraphy showed no proof of dura fistula. A myelo-CT demonstrated a subtle leak at the dorsal roof exit zone D1 ; 2) spinal meningeal diverticulum ; 3) improvement of symptoms after epidural blood patching. If criteria A and B not met, a criterion C is defined as the presence of all of the following or at least two of the following if typical orthostatic headaches are present: 1) low opening pressure, 2) spinal meningeal diverticulum, and 3) improvement of symptoms after epidural blood patch  suggestive of IH include diffuse pachymeningeal contrast enhancement, sagging of the brain, and subdural fluid collections  as in case I  can be a life-saving manoeuver in these patients.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


Setting aside overmature planted forests is currently seen as an option for preserving species associated with old-growth forests, such as those with dispersal limitation. Few data exist, however, on the utility of set-aside plantations for this purpose, or the value of this habitat type for biodiversity relative to old-growth semi-natural ecosystems. Here, we evaluate the contribution of forest type relative to habitat characteristics in determining species richness and composition in seven forest blocks, each containing an ancient old-growth stand (> 1000 yrs) paired with a set-aside even-aged planted stand (ca. 180 yrs). We investigated the functionally important yet relatively neglected ectomycorrhizal fungi (EMF), a group for which the importance of forest age has not been assessed in broadleaved forests. We found that forest type was not an important determinant of EMF species richness or composition, demonstrating that set-aside can be an effective option for conserving ancient EMF communities. Species richness of above-ground EMF fruiting bodies was principally related to the basal area of the stand (a correlate of canopy cover) and tree species diversity, whilst richness of below-ground ectomycorrhizae was driven only by tree diversity. Our results suggest that overmature planted forest stands, particularly those that are mixed-woods with high basal area, are an effective means to connect and expand ecological networks of ancient old-growth forests in historically deforested and fragmented landscapes for ectomycorrhizal fungi. •Ectomycorrhizal fungi richness was similar in ancient and overmature planted forest.•Community composition was also similar across the forest types.•Fungal fruit body richness was positively related to basal area and tree diversity.•Below-ground ectomycorrhizae richness was positively related to tree diversity. PlantedConcern about the global decline of old-growth forest and the associated loss of biodiversity has motivated initiatives to increase the area of protected old-growth . Althouga.d.) from more recent, secondary forest stands. Whilst differences in species composition between ancient and more recent forests can be attributed to differences in stand structure and microhabitat composition, many studies emphasise the importance of time per se; evidenced by certain species being absent from young forests despite the presence of suitable habitats  comprise a functional group that forms mutualistic associations with most economically and ecologically important temperate tree species . ApproxiWhilst the relative biodiversity value of ancient old-growth and more recent secondary forest has been established for groups including vascular plants and epiphytic lichens , we stilIn historically deforested regions such as the UK, where the proportion of forest has declined to just 12% of total land area, ~ 80% of this forested area is planted and just 5% is classified as ancient , the set22.1Quercus robur), sessile oak (Quercus petraea) and beech (Fagus sylvatica) with the understorey consisting largely of holly (Ilex aquifolium) . The New Forest lies within the Warm Dry climatic zone , with miifolium) .The plantations under study were on average 180 years old. Plantation ages were considered equal to the number of years since planting . Plantations were established following clear-cutting of partially forested (deer parks) to completely forested grounds. All sites likely underwent the same preparation procedure following clear-cutting, involving the upturning of soil and trench formation for water drainage (see Appendix A2 for a detailed description). The management histories of the ancient old-growth stands under study were elucidated using historic maps, Forestry Commission management plans, pollen records and consultation with local experts. The stand ages refer to the oldest record available indicating that the site was forested (Table A1.1), and since then have not experienced major tree removal.2.2In order to compare EMF communities in overmature planted set-asides and ancient old-growth forest communities, locations were selected that paired forest types in a randomised block design . Seven f2.3We used two methods to obtain representative samples of EMF communities in plots: surveys of above-ground sporocarps (‘mushrooms’), and soil cores for analysis of below-ground EMF on root tips . These were a random selection of the available seven locations. Each plot was divided into a 10 × 10 grid, and every intersection given a coordinate. Sixteen soil cores  were taken at randomly generated coordinates. Samples were stored at 4 °C until root tip analysis could be undertaken, always within 5 days. Cores were soaked in tap water for 15 min and washed through a 500 μm sieve. After manual removal of coarse woody debris and senescent roots, live roots were extracted from the core for five minutes under a dissecting microscope. To minimise observer bias, three of the largest roots were selected, and one living root tip was randomly selected from each of these following 2.3.3Identification of ectomycorrhizae followed the methodology of http://www.geneious.com/. Edited sequences were identified using the BLAST algorithm in GenBank and the UNITE database (http://unite.ut.ee/). The best BLAST identification was reported for each fungal taxon. The UNITE species name was validated only when (i) the similarity between the submitted sequence and the sequence in the database exceeded 97%, and (ii) the UNITE identification was plausible taking into account ecological considerations and known geographical distributions of related species  exchangeable cations, K +, Mg 2+, Ca 2+; (vi) total N; and (vii) total C. See Soil was sampled from all plots during a consistently dry period with minimal spatio-temporal variation in rainfall during May 2014. Five random soil cores  were taken from each 10 m × 10 m fungal assessment plot and were pooled to the stand level. Samples were sent to the Forest Research Soil Analysis Service , for analysis. The following soil variables were measured: (i) pH; (ii) moisture content (loss in fresh mass after 2 day at 105 °C); (iii) organic matter (loss on ignition after 1 day at 450 °C); (iv) water-soluble anions, NO2.52.5.1All analyses were computed in R 3.00 software . Prior tr = 0.80, P < 0.001).The simultaneous effects of environmental variables on EMF richness were quantified using mixed effects models. Two response variables were investigated: sporocarp and ectomycorrhizae richness, the former using a generalized mixed model with Poisson error distribution and logarithmic link function, and the latter using a linear mixed model on log-transformed Chao1 estimated richness. Explanatory variables included: forest type (planted or ancient old-growth), tree species richness, understorey species richness, tree basal area, Soil1, Soil2 and Soil3. Canopy closure was not included due to co-linearity with the more precisely measured basal area . NMDS reflects similarities (or dissimilarities) between assemblages. The Sørensen distance measure was used for species presence–absence data. Two-dimensional solutions were produced with 999 iterations, as the reduction in stress was small beyond the second axis.Species fidelities to each forest type were identified by indicator species analysis ISA;  extendedWe estimated the Chao similarity index to quantify the compositional similarity between EMF communities sampled as sporocarps and ectomycorrhizae following 3Lactarius tabidus , Lactarius quietus , Laccaria amethystina  and Russula ochroleuca . Visual examination of species' ranked abundance distributions suggests that the EMF communities from both forest types followed a Zipf–Mandelbrot distribution, indicating that communities have a few species that are very abundant, and a long tail of rarer species . The ancient old-growth stand at location 2 and the planted stand at location 7 were disregarded from the analysis, after the PCA analysis of soil chemistry revealed unrepresentative soil conditions in extreme outliers .A total of 225 EMF taxa were identified across all plots, with 122 species identified in the sporocarp survey and 136 OTUs characterised using the molecular analysis (Appendix A4). The same four species were most frequently observed in the sporocarp and ectomycorrhizae surveys, in terms of the percentage of plots containing them: 3.1R2 values were higher for models explaining sporocarp richness . The NMDS ordination confirmed these results, finding no separation in species composition according to forest type . A two-dimensional solution described 63% and 66% of the variance in the species composition for sporocarp and ectomycorrhizae datasets, respectively  and Cortinarius violaceus (Near Threatened), were found as sporocarps in ancient old-growth stands at locations 1 and 2, respectively. Due to their incidences within single plots, they had low sensitivities to forest type, and so did not have significant indicator values.Indicator species analysis detected associations of a single species with each forest type from the sporocarp surveys, and of a single species with ancient old-growth forest from the ectomycorrhizae samples . IndicatCompositional similarity between communities sampled as sporocarps and ectomycorrhizae was only moderate with Chao similarity indices of 0.41 for ancient and 0.37 for planted forest .44.1This investigation found similar richness and composition of EMF in set-aside plantation and neighbouring ancient stands. It has been suggested that increasing the proportion of overmature planted forests could offer alternative habitats for species typical of old-growth forests, but this possibility lacks empirical evaluation . Many EMThe EMF communities in the overmature plantations were indistinguishable from those of ancient old-growth forests . Our finEMF communities observed within samples of sporocarps and ectomycorrhizae had only moderate overlap in species composition, in contrast to the complete overlap between forest types. Other studies using both methods often report a poor correspondence between them in terms of species richness and community composition . Such diDifferent variables were important in predicting sporocarp and ectomycorrhizae species richness patterns, with sporocarp richness driven by basal area as a proxy for canopy closure and tree diversity, and the richness of ectomycorrhizae driven by tree diversity only . It is p4.2Cenococcum geophilum. However, the molecular sequencing of ectomycorrhizae is expensive and time-consuming resulting in relatively small volumes of soil being screened.We sampled both sporocarps and ectomycorrhizae in our attempt to acquire an accurate unbiased representation of the EMF communities within our plots. Each technique has limitations. Sporocarp production is sensitive to a host of environmental factors, and consequently sporadic annual fruiting patterns necessitate long term monitoring . In mesiLactarius and Russula; or sporadic fruiting, in which species do not produce sporocarps every year (Pseudotsuga menziesii) in Canada, and found old-growth stands contained many species of fungi that infrequently produce sporocarps. With sporocarp surveys disadvantaged by the inherent sporadicity of EMF fruiting and ectomycorrhizae surveys limited by the small volume of soil sampled, either sampling method may have omitted the inclusion of rarer EMF species that fruit infrequently. Indeed, two species of conservation concern were recorded only once, both in ancient old-growth stands. It is possible that further sampling may reveal more rare species, particularly within ancient stands: Sporocarp surveys are essential for detecting those rare species that form conspicuous sporocarps . It is tery year . Smith eA study of EMF communities in forests across the UK in the 1990s  surveyedIt is possible that the ancient old-growth forests of the New Forest National Park comprise a depauperate subset of the EMF community that was once supported in the past. Indeed, potential threats to EMF including nitrogen deposition, selective tree felling and soil compaction by high herbivore densities , might h4.3The setting aside of overmature planted forest is an effective means of conserving EMF communities associated with ancient old-growth forests, given temporal continuity of the order of a century. Whilst this is the first study to investigate the importance of ecological continuity for EMF in overmature broadleaved plantations, similar studies have been done for other taxonomic groups; Our study has identified influences of habitat variables that suggest opportunities for enhancing EMF diversity in planted forest. EMF distribution and composition have been shown to be influenced by the relative proportions of host tree species, indicating a degree of host preference or specificity for some EMF species or genera . In thisBasal area correlated positively with sporocarp richness . AlthougBiology and Biotechnology Research Council (grant no. BB/H531935/1) and British Mycological Society  provided funding to RS, NERC (NE/K006339/1) provided support to MIB and SVDL, and an EU Marie Curie IEF fellowship (FP7-PEOPLE-2009-IEF - 253036 - MYCOIND) provided support to LMS.The


Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity ‘tag’ sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations.We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms.These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally ‘active’ and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the ‘seed bank’ microbial community. Environmental DNA (eDNA) analyses have demonstrated that diversity records are missing significant data regarding protists (reviewed in: -3). ParaRana sphenocephala in the USA in 2003 )Using 454 pyro-sequencing, we investigated the diversity of Perkinsea in a selection of European marine samples, sequencing the V4 region of the SSU rDNA  using boThe taxonomic affiliation of these sequences was checked using phylogenetic analyses of a SSU rDNA dataset comprising representative alveolate groups  moderate topology support (>0.6/60%/60%) and 2) possession of two sequences from separate samples. Using this approach we identified 38 phylogenetic clusters labeled as cluster 1-38 on Figures Perkinsus spp. have highly similar SSU rDNA sequences . If theet al. in 2010, marine-freshwater transitions are likely to have occurred during the diversification of the Perkinsea )erkinsea . Our phy244 (92%) of the V4 sequences classified as Perkinsea were recovered from sediment. Moreover, 27% of the total sequencing effort were sampled from RNA derived cDNA libraries Figure  suggestiIt has previously been suggested that Syndiniales, including Marine Alveolata group I and II, predominate as parasitic protists in marine waters  while itThese results are based on 454 methods targeting a broad spectrum of eukaryotes followed by bioinformatics extraction of Perkinsea-like sequences. Such approaches can lead to partial detection of target groups, dependant on level of sequence saturation achieved and comprehensiveness of the primers selected. In reality achieving single gene-marker primers that allow comprehensive sampling combined with sample saturation is experimentally difficult, unless a narrow group is targeted. As such, future work should incorporate a multiple -group specific- primer approach in order to improve sampling of Perkinsea diversity and map their environmental distribution. A major challenge of future work is to elucidate the ecological roles of this diversity of Perkinsea putative parasites revealed by eDNA surveys.http://biomarks.scrol.fr): offshore Oslo , Naples , Blanes near Barcelona  and Roscoff . Each station was sampled over three depths  using the same sampling protocol (as described in [3) were frozen and stored at −80°C for molecular analysis.Four European coastal stations were sampled Figure A as partribed in  - Figureribed in . BrieflyFor water column samples, DNA and RNA were extracted simultaneously using the NucleoSpin RNA L kit . For sediment samples, DNA and RNA were isolated using the PowerMax Soil DNA Isolation kit and the PowerSoil total RNA Isolation kit . DNA and RNA quality were confirmed using gel electrophoresis (1.5% agarose gels) and quantified using a NanoDrop ND-1000 Spectrophotometer. To avoid contamination by DNA in the RNA extractions, DNAse from the TurboDNA kit  was used to remove traces of DNA. Extracted RNA (100 ng) was reverse transcribed into cDNA using random primers and the Superscript III RT kit  following the protocol outlined by the manufacturer.http://www.genoscope.cns.fr), France.Universal eukaryotic primers TAReuk454FWD1 (5′-CCAGCASCYGCGGTAATTCC-3′) and TAReukREV3 (5′-ACTTTCGTTCTTGATYRA-3′) were used to sample the V4 region (~380 bp) of the SSU rDNA  using po2[2 database, in terms of global alignment identity. This process identified 271 sequences tentatively classified as Perkinsea  under accession numbers PRJEB5698. We have included the six putative chimeras in the submission so these can be checked historically, these six sequences have the reference numbers 37–0005, 54–0139, 134–0005, 138–0140, 206–0147 and 268–0287.All existing Perkinsea SSU rDNA sequences  plus a selection of 31 published sequences that encompass all the other major Alveolata lineages were recovered from the NCBI non-redundant nucleotide database and assembled into a reference dataset of 67 sequences  a dataset encompassing the complete SSU rDNA sequence alignment and including a wide selection of Alveolata lineages  and 2) a second dataset restricted to an alignment mask of the V4 region and focusing only on the Perkinsea phylotypes including sequences from Bråte et al. . Prior et al. to deterWe then conducted Bayesian analyses using MrBayes v3.2.1 . For botFor both datasets, support for the tree topology was evaluated by the bootstrap method and using the Bayesian posterior probabilities (PP) from the MrBayes runs . BootstrThe authors declare that they have no competing interests.For the BioMarKs project, SR carried out the molecular work and SA the global bioinformatics analyses. CdV and TAR are PI’s on the BioMarKs project. AC and CB constructed the sequence alignment. AC and TAR analysed the data and wrote the manuscript. All authors read and approved the final manuscript.Bayesian phylogeny of Alveolata SSU sequences based on the analysis of 98 sequences of 1470 bp and 265 partial sequences from BioMarks V4 sequencing project (~278 bp in length). Posterior probability values and Maximum Likelihood bootstrap values were added at each node (pp/ML bootstrap support). Support values are summarised by black circles on the branch when they are equal to or higher than 0.90/80% and white circle when bootstrap values are between 0.6/60% and 0.9/80. Three ciliates sequences were used as the outgroup. Taxon names are consistent with Bråte et al. 2010. MA corresponds to Marine Alveolates. Arrow identifies the monophyletic Perkinsea clade. Complex clusters of 454 sequences have been reduced to representative triangles, see Figures Click here for fileDetails of published sequences of 18S rDNA used in phylogenetic analysis.Click here for filePublished environmental sequences of 18S rDNA belonging to Perkinsea (Alveolata) used in phylogenetic analysis. Sequences sampled from marine environments are highlighted in grey.Click here for fileCorrespondence between newly described clusters from the present study and the previous studies .Click here for fileDescription of the V4 sequences ID labeling. Each sequence has been labeled by a unique number was given in Figures Click here for fileBayesian model comparison for method selection in phylogenetic inference.Click here for file


These changes in community composition and activity rates were strongly correlated, which suggests the effect of environment on function could be mediated, at least in part, by microbial community composition. Based on their distribution across treatments, each phylotype was categorized as having a salinity preference  and phylogenetic analyses revealed a significant influence of evolutionary history on these groupings. This finding was corroborated by examining the salinity preferences of high-level taxonomic groups. For instance, we found that the majority of α- and γ-proteobacteria in these wetland soils preferred saltwater, while many β-proteobacteria prefer freshwater. Overall, our results indicate the effect of salinity on bacterial community composition results from phylogenetically-clustered salinity preferences.Salinity is a major driver of bacterial community composition across the globe. Despite growing recognition that different bacterial species are present or active at different salinities, the mechanisms by which salinity structures community composition remain unclear. We tested the hypothesis that these patterns reflect ecological coherence in the salinity preferences of phylogenetic groups using a reciprocal transplant experiment of fresh- and saltwater wetland soils. The salinity of both the origin and host environments affected community composition ( Understanding if and how the evolutionary history of a species relates to its ecology is a fundamental question for biologists. For macroorganisms, phylogeny is often an ecologically meaningful way to classify organisms, as closely related taxa frequently have similar ecological characteristics e.g.,  and funcSalinity has been proposed to be a major driver of phylogenetic bacterial community composition across the planet , and num16S rRNA gene sequences) and activity .The current study was designed to: (i) test whether salinity preferences exhibit phylogenetic conservation, and (ii) characterize the response of wetland microbial community composition and function to changes in salinity. To address these goals, we performed a reciprocal transplant experiment of fresh- and saltwater wetland soils and monitored phylogenetic community structure  were randomly collected. Each core was transferred into a chamber made of PVC pipe  with PVC caps secured on the top and bottom. Caps had 8.25-cm diameter holes drilled in them, and these openings were covered with two layers of 0.5-mm Nitex mesh  secured with Amazing Goop . After soil cores were placed in the chambers, the caps were sealed to the PVC cylinder using Amazing Goop. The next morning, chambers were returned the marsh for in situ incubation. Each chamber was gently inserted into one of the holes created the day before (when cores were collected); location assignments within each plot were random. The reciprocal transplant generated four treatments with five replicates of each combination of “soil origin” and “host site” to yield: Fresh–Fresh , Fresh–Salt, Salt–Salt, and Salt–Fresh. Chambers were incubated for 40 days from June–August 2013. When chambers were retrieved, each was sealed in an air-tight plastic bag and immediately returned to the laboratory for analysis.This research was conducted in two tidal wetlands (Cumberland and Taskinas Creek marshes) on the Pamunkey River, a major tributary of the Chesapeake Bay in Virginia (USA). The two marshes are close enough in proximity that weather, land-use, and underlying lithology do not differ, but a strong salinity gradient means that Cumberland Marsh  is freshwater (ppt <0.5) while Taskinas Marsh  is brackish (4–15 ppt). When this study was conducted, the dominant vegetation at Cumberland marsh was g, 15 min), filtered using a 0.45 μm pore-size mixed cellulose ester syringe filter, and stored at -20°C until it could be analyzed for sulfate concentration via ion chromatography .Soil salinity (ppt) and pH were measured using a SG78 SevenGo Duo pro probe . Soil moisture (%) was determined gravimetrically (100°C for 72 h) and organic matter  was measured as mass loss on ignition (500°C for 4 h). Total carbon and nitrogen contents were determined using a Perkin Elmer CHNS/O Analyzer  following grinding and acidification of samples using 10% hydrochloric acid; C:N was calculated by mass. Porewater was extracted from 50-ml soil samples by centrifugation  and stored at -20°C. DNA purity and concentration were analyzed using a Nanodrop ND-1000 . All DNA extracts and PCR products were verified using agarose gel (1.5%) electrophoresis and ethidium bromide staining.16S rRNA and methanogen mcrA gene abundances were assessed using quantitative PCR (qPCR) as described in 16S rRNA gene , and the mlas and mcrA-rev primers were used to target the methyl coenzyme-M reductase encoding the mcrA functional gene . A third qPCR was employed to quantify the abundance of the dsrA gene using the primers dsrA_290F and dsrA_660R designed by Desulfovibrio desulfuricans  was used to establish the standard curve . For all three genes, qPCR assays were performed using SsoAdvanced SYBR Green qPCR Supermix  and a BioRad CFX 96 Real-Time System. Data were analyzed using Bio-Rad CFX Manager Version 2.1. Results were reported as the log(10) of the number of gene copies per g of OM after averaging three technical replicates per sample.Bacterial 16S rRNA gene using the primers and protocol described by 2, and 4 ng DNA template . Thermal cycling conditions were: 95°C for 5 min, 20 cycles of 60 s at 95°C, 60 s at 50°C, 60 s at 72°C, followed by 72°C for 7 min . Triplicate PCR products were pooled and purified using AMPure XP  prior to sequencing on the Illumina Hiseq (at VCU’s Nucleic Acid Research Facility) using 2 × 300 bp read chemistry. Quality filtered reads  were received as pairs of demultiplexed fastq files; the length of the locus was within the high quality region of read one, so the second read was not used. Sequence and accompanying metadata are available on the MG-RAST server , 4-MUB β-D-cellobioside (CBH), 4-MUB- β-D-xylopyranoside (BX), L-leucine-7-AMC (LAP), and 4-MUB-sulfate (AS) and followed incubations of either 1  or 4 h  at 30°C with gentle agitation. POX and PR activities was measured colorimetrically via the oxidation of l-DOPA (in the presence of H2O2 for PR) after a 30 min incubation using the methods of Within one week following sample collection, soils were assayed for extracellular enzyme activity (EEA) using the fluorimetric and colormetric microplate assays described in 2 and CH4 was measured using 48-h anaerobic slurry assays (after -1 h-1) were calculated using linear regression; median correlation coefficients were 0.87 for CO2 and 0.92 for CH4.Production of COys after  employin16S rRNA, mcrA, dsrA) required a log(10) transformation prior to statistical analysis. Effects of host and origin environments on environmental and functional parameters as well as microbial gene abundances were assessed using two-way analysis of variance  with Tukey’s HSD for post hoc comparisons. Similarly, the effect of origin and host environment on the relative abundance of putative sulfate reducing δ-proteobacteria  using 97% sequence identity in Qiime 1.7 using default parameters  in the different salinity environments. Consequently, the “no preference” category may contain organisms that favor one salinity over the other but can survive in both environments. We also recognize that the OTUs we classified as “absent” could still be present in the community, only their abundance is below our detection limit. Overall, our characterization using presence/absence criterion is a conservative approach designed to identify strong salinity preferences. After each phylotype was assigned a salinity preference, the standardized effect size of phylogenetic diversity (SES-PD) and the nearest taxon index (NTI) were calculated for the salinity preference groups and examined to see if these indices reflected more or less phylogenetic relatedness than would be expected by chance using the ses.pd and ses.mntd functions, respectively, in the picante package . None of these parameters changed significantly upon transplant, except that the pH of the soil from the saltwater site decreased. As expected, the host environment regulated the soil porewater salinity and sulfate concentrations, wherein soils hosted in the saltwater site had greater solute concentrations than their freshwater counterparts .Soils originating from the freshwater site had lower pH but higher moisture content, OM, and C:N than the soils originating from the saltwater site (16S rRNA) abundance was not significantly different across the treatments (Table 1). In contrast, both the abundance of methanogens (mcrA) and SRB  were affected by the origin of the soil, wherein the freshwater soils had higher methanogen abundance and lower SRB abundance. An interaction effect was observed for methanogens, wherein abundance in soils originating from the saltwater site increased upon transplantation to the freshwater environment. Bacterial abundance was not correlated with that of methanogens  or that of δ-proteobacterial SRB . However, a strong negative relationship was found between the abundance of methanogens and δ-proteobacterial SRB .Bacterial (16S rRNA gene sequences), all soils were dominated by Proteobacteria , Chloroflexi, Acidobacteria, and Bacteroidetes . However, soils of freshwater origin had higher relative abundances of β-proteobacteria  and Nitrospirae , while soils originating from the saltwater site had greater relative abundances of γ-proteobacteria . Saltwater control soils  had greater phylotype richness than freshwater control soils (Fresh–Fresh), and transplantation into an alternate salinity increased the phylotype richness of soils originating from both environments . Phylogenetic community structure (assessed as weighted UniFrac distances) was interactively affected by both origin and host environments as determined by two-way PerMANOVA . Though each community shifted when transplanted to the contrasting salinity environment, they remained more closely related to their origin environment controls than their host environment controls .In terms of relative abundance . Soils of freshwater origin had lower relative abundance of SRB  and a greater fraction of Desulfarculaceae, while SRB in soils of saltwater origin were dominated by the Desulfobacteraceae, Desulfobulbaceae, and Desulfuromonadaceae families. Both origin and host environment salinity influenced the relative abundance of the putative FeRB Geobacter ; Geobacter relative abundance was higher in soils originating from and or hosted in the freshwater environment.The relative abundance and composition of putative SRB in the δ-proteobacteria clade differed between soils originating from the freshwater and saltwater environments . These salinity preferences were then compared to phylogeny to assess whether evolutionary history influenced habitat preference . The phylotypes in these categories were more closely related to each other than would be expected by chance as assessed by both the standardized effect size of phylogenetic diversity (SES-PD) and NTI. The strongest influence of phylogeny was seen in the phylotypes that had no salinity preference , followed by the saltwater  and freshwater  preference categories. Phyla varied with respect to their salinity preferences  but were not found in the Salt–Salt treatment. Another 820 phylotypes were categorized as having a preference for saltwater by virtue of the fact that they were found in both soils incubated at the saltwater site , but not the Fresh–Fresh controls. The 307 phylotypes present in both the fresh and saltwater controls were categorized as having no salinity preference (Table 1). Overall, this variation in function was strongly correlated to variation in bacterial community structure .As was observed for community composition, microbial activity appears to be most strongly controlled by the origin of the soil sample (Table 1). Soils of freshwater origin had a greater activity of BG (∼6.5-fold), AS (∼2.9-fold), and LAP (∼2.3-fold). In contrast, soils with a saltwater origin had a greater activity of POX (∼1.3-fold) and PR (∼1.6-fold). The only enzyme activity affected by the host environment was PR, which was ∼30% higher when hosted in the freshwater site.Analysis of EEA revealed no treatment effects for either CBH or BX  than those from the freshwater site. Host environment had a modest but significant effect wherein the soils incubated at the freshwater site had rates of CO2 production that were ∼20% higher than their saltwater counterparts (Table 1). The production of CH4 was interactively regulated by soil origin and host environment wherein rates were very low in all treatments except for the soils originating from, and hosted in, the freshwater environment.Production of COin situ salinity manipulation experiment, and then assessed whether phylogenetic clades of bacteria exhibited ecological coherence with regard to their salinity preferences. We found a tight coupling of microbial community structure and function, and demonstrated that salinity selects for organisms with phylogenetically-clustered salinity preferences. An influence of phylogeny was also detected within organisms that displayed no salinity preference, which suggests a genomic basis for this flexibility. Understanding how salinity changes will affect microbial communities and their associated functions is especially important given that climate change associated sea-level rise will expose many aquatic ecosystems to saltwater intrusion . This is likely due, at least in part, to the colonization of organisms from the new host environment. Interestingly, few phylotypes disappeared from the transplanted soils relative to their origin controls, which could be interpreted as a widespread tolerance to the salinity change. However, a recent study by 16S rRNA gene library . These results are consistent with a scenario wherein organisms from the host environment have begun to colonize the transplanted soil (thus changing community membership) but have yet to establish large populations. This sort of gradual change in community composition is consistent with other salinity manipulations that have found saltwater to have little effect on community composition in the short term [∼1 month (Table 1). These results are consistent with past work showing that environmental history can have lasting effects on microbial community composition  is consistent with other several other wetland studies (mcrA) and SRB (dsrA) abundances across the treatments.When the relative abundance of phylotypes is considered along with these changes in community membership, it becomes clear that the origin of the soil was the dominant influence on microbial community composition , as was the relative abundance of known SRB families . This finding is consistent with past work that has found SRB to be abundant in saline marshes (2).In addition to inhibiting methanogens, saltwater intrusion is expected to influence sulfate and iron-reducing bacteria. In this study, the abundance of δ-proteobacteria SRB was higher in the soils of saltwater origin . This is consistent with pure culture studies that have found high salt concentrations to inhibit the growth of Geobacter species  and a higher than expected preference for freshwater . Our study demonstrates that high levels of taxonomic organization can serve as ecologically meaningful units, and suggests that a more thorough consideration of an organism’s evolutionary history could help resolve several unanswered questions in microbial ecology. In particular, our work highlights the potential use of phylogenetic information for predicting the distribution of bacterial taxa along environmental gradients and for determining how environmental perturbations may shape community composition. As microbial ecologists gather this sort of information for an increasingly large number of environmental variables, we will develop a more robust understanding of microbial biogeography and become better able to predict bacterial community responses to global change.EM and RF designed the research. EM performed the laboratory and data analyses. EM and RF wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adult-onset Still’s disease (AOSD), a rare autoinflammatory disorder, resembles systemic juvenile idiopathic arthritis (SJIA). The superimposable systemic clinical features of AOSD and SJIA suggest both clinical phenotypes represent the same disease continuum with different ages of onset. To further characterize the similarity between AOSD and SJIA at the molecular level, 2 previously identified response gene sets in SJIA were used to investigate how genes that respond to interleukin (IL)-1β inhibition with canakinumab in SJIA patients behave in AOSD patients with active disease prior to IL-1β targeting therapy, relative to healthy subjects.All genes downregulated in SJIA patients following canakinumab treatment were upregulated in most patients with active AOSD prior to canakinumab treatment, relative to healthy subjects. A few patients with milder AOSD had expectedly gene-expression patterns that resembled those in healthy subjects. Comparison of the gene-expression patterns with neutrophil counts showed a correlation between elevated neutrophil numbers and upregulation of canakinumab-responsive genes. Correspondingly, most genes upregulated following canakinumab treatment in patients with SJIA patients were downregulated in the majority of AOSD patients.These results further support the concept of a Still’s disease continuum that includes both a pediatric/juvenile onset (SJIA) and adult onset (AOSD) form. Blood saffy and gcrma R packages. Normalized data were then scaled to a trimmed mean value of 150. The significance of gene-set enrichment was estimated using the ROAST method as implemented in R, [Whole blood samples were collected in PAXgene Blood RNA tubes  and stored at −80 °C. Total RNA was subsequently isolated with the PAXgene Blood RNA Kit (Qiagen). The synthesis of cDNA was performed using the Ovation® RNA Amplification System V2 including the Ribo-SPIA® amplification process according to the instructions of the manufacturer . The amplification process was performed in 3 stages: (1) a 1st-strand cDNA synthesis with oligo(dT) primers and Ovation WB Reagent (NuGEN), (2) a 2nd-strand cDNA synthesis, and (3) a single-primer, linear isothermal amplification  that produced amplified single-stranded biotin-labeled cDNA. The cDNA was hybridized to GeneChip® Human Genome U133 Plus 2.0 Array as specified by the manufacturer . Gene-expression values were stored in CEL files that were used for robust multi-array average normalization with the ed in R,  applyingThe behavior of canakinumab responsive genes in patients with AOSD and healthy subjects is shown in Figs. While relatively new, gene expression allows analysis of hundreds and thousands of different genes at a given time instead of a handful of candidates, and represents the most advanced and comprehensive approach to screening gene activity as well as molecular networks involved in the innate immune system. Nonetheless, SJIA and AOSD are rare autoinflammatory conditions; therefore, large prospective and longitudinal trials are difficult to generate which explains the relatively small sample size of this analysis. Disease duration and severity, prior treatment with other biologics and concomitant corticosteroid exposure during the trial may have influenced the SJIA findings. With regard to the AOSD baseline analysis it should be noted that future analysis needs to confirm the hypothesized treatment response and downregulation of canakinumab responsive genes.In conclusion, the results of this gene-expression analysis are consistent with and further support the concept of a Still’s disease continuum that includes both a pediatric/juvenile onset (SJIA) and adult onset (AOSD). These findings support further clinical evaluation of anti − IL-1β therapy in patients with AOSD given that AOSD appears to be an IL-1–driven condition similar to SJIA.Study NCT02204293 (Canakinumab for Treatment of Adult Onset Still’s Disease) was approved by the Paul-Ehrlich-Institut (PEI) Germany  on April 3, 2012 and the Ethic Committee Landesamt für Gesundheit und Soziales (LAGeSo) .


To assess the treatment progression during the 24 months following a formal diagnosis of chronic obstructive pulmonary disease (COPD) in the UK primary care setting.2-agonists (LABAs), long-acting muscarinic antagonists (LAMAs), inhaled corticosteroids (ICSs), and respective combinations. At each 3-month interval, discontinuation, switching, addition, and stepping down patterns were analyzed cumulatively for the first 12 months and over the 24-month of follow-up.A retrospective cohort of newly diagnosed COPD patients was identified in the Clinical Practice Research Datalink (CPRD) from 1/1/2008 until 31/12/2009. Maintenance therapy prescribed within the first 3 months of diagnosis and in the subsequent 3-month intervals for 24 months were analyzed. Treatment classes included long-acting βth-6th month interval were included in the analysis. At diagnosis (0–3 months), the most frequently prescribed maintenance therapy was LABA+ICS (43%), followed by LAMA (24%) and LABA+LAMA+ICS (23%). Nearly half the patients  starting on a monobronchodilator had additions to their treatment in 24 months. Compared to other medications, patients starting on a LAMA were most likely to escalate to triple therapy in 24 months. Nearly one-fourth of the patients prescribed triple therapy at baseline stepped down to LABA+ICS (25%) or LAMA (31%) within 24 months.A total of 3199 patients with at least one prescription of a maintenance therapy at baseline and during 4Disease progression is evident over the 24 months after COPD diagnosis, as more patients were prescribed additional maintenance therapy in the 24-month period compared to baseline. The changes in therapy suggest that it is difficult to achieve a consistently improved COPD disease state. Chronic obstructive pulmonary disease (COPD) is “a preventable and treatable disease characterized by airflow limitation that is not fully reversible” 2-agonists (SABAs) are used on an as-needed basis for symptomatic relief in early stages. As COPD is a progressive condition, daily maintenance drugs are usually needed. GOLD and NICE guidelines suggest that bronchodilators such as β2-agonists and antimuscarinics form the mainstay of therapy 1) following bronchodilator therapy can be small; however, the accompanying larger change in lung volume contributes toward reduction in perceived breathlessness 2-agonists (LABAs) or long-acting muscarinic antagonists (LAMAs) are also used to reduce the number and severity of exacerbations 1 percent predicted. For patients with FEV1 ≥ 50%, a LAMA or a LABA is recommended as the initial choice. If the FEV1 is <50%, the treatment choice is between a LAMA or LABA+ICS. Patients starting on LAMA or LABA+ICS would then progress to triple therapy (LABA+LAMA+ICS) if exacerbations or breathlessness persist Once COPD is diagnosed and its severity is established, pharmacological treatment aims to reduce symptoms and exacerbations Numerous studies report less than optimal compliance with guidelines in patients with COPD In this retrospective cohort study, we evaluated treatment patterns of patients with COPD identified from the primary care setting using the Clinical Practice Research Datalink (CPRD), which is the National Health Service (NHS) observational data and interventional research service 1/FVC < 0.7; FVC, forced vital capacity) at COPD index date/cohort entry date . Patients were labeled as cases of newly diagnosed COPD only if they had no prior diagnosis of COPD ever in their records. Further, these patients were required to have at least a 24-month follow-up history, unless death occurred during the follow-up period. Subjects with an occurrence of an event code of a medical condition incompatible with COPD diagnosis at any time in their history were excluded. These events included lung or bronchial developmental anomalies, degenerative processes , bronchiectasis, pulmonary resection, or other significant respiratory disorders that can interfere with clinical COPD diagnosis or substantially change the natural history of the disease.Patients aged ≥40 years with the first diagnosis of COPD and at least 12 months of history preceding the diagnosis were included. The diagnosis was confirmed by spirometry , smoking status, available Medical Research Council (MRC) dyspnea scale score, and prior diagnosed and/or treated comorbid conditions (according to the Charlson index) In order to assess the progression through individual maintenance therapy, patients prescribed one of the following six index therapies at baseline and at each 3-month interval were included: LABA, LAMA, LABA+LAMA, LABA+ICS, LAMA+ICS, and LABA+LAMA+ICS. ICS as a standalone treatment was excluded, as ICS is not licensed as monotherapy for COPD. Patients were categorized into a baseline treatment class based on their first prescription in the 0- to 3-month interval. Patients were considered to continue on this index therapy if they were prescribed the same therapy in subsequent 3 monthly time intervals. Patterns of first change in treatment  were analyzed at each time interval and cumulatively in the first (1–12 months) and second year (0–24 months). Patients with any change in therapy were excluded in subsequent intervals, thereby restricting the analysis to patients continuing on index therapy, while recording their first change.In addition, the time to first prescription of triple therapy by index therapy among all newly diagnosed patients was evaluated using a Kaplan-Meier curve. Exploratory analysis was performed using Cox proportional hazards models by excluding asthma and adjusting for factors associated with progression to triple therapy .1 < 50% (35% vs 27%) and MRC score ≥ 3 (26% vs 21%).Among the cohort of 7881 newly diagnosed patients with COPD, 3925 (50%) were prescribed maintenance therapy during the baseline period (0–3 months after diagnosis). Of the 3925 patients, 80% (n = 3199) were prescribed a maintenance therapy at baseline and during the 4–6 month interval and were included in this analysis. These patients had a mean (SD) age of 67 (11) years. The cohort consisted of 47% women, 15% former smokers, and 36% current smokers. Additionally, 97% and 66% of patients had an available stage of lung function and MRC dyspnea scale score, respectively . The demThe frequency distribution of each type of prescribed therapy following a new diagnosis of COPD is shown in Approximately one-quarter of the cohort was not prescribed their initial therapy again in the first 4–6 months following diagnosis; half of the cohort was not prescribed their initial therapy again after 12 months; and approximately three-quarters of the cohort was not prescribed their initial therapy again after 2 years .Patients on LABA+ICS combination demonstrated highest continuation, with 60% at interval 4 (10–12 months), and 35% at interval 8 (22–24 months) continuing on their index therapy. Patients on LABA and LAMA monotherapies had comparable continuation with 42% and 44% patients on the index treatment at interval 4 and 21% and 22% on index treatment at interval 8, respectively. The proportion of patients remaining on their initial therapy at the end of 24 months varied between initial medications prescribed. The proportions ranged from 8% for LAMA+ICS to 35% for LABA+ICS and about 25% for most other therapies . There wFew patients were prescribed another class of maintenance therapy within 24 months of diagnosis. Similar to additions, a majority of switches occurred within the 12 months of diagnosis . A smallPatients prescribed more than one maintenance medication in combination were included in the stepped down therapy analysis. Among patients starting on dual therapies, majority of step downs occurred in patients starting on LAMA+ICS therapy with 44% stepping down to LAMA monotherapy within first 12 months. Several patients starting on LABA+LAMA combination also stepped down to a LABA (13%) or a LAMA (17%) monotherapy within 12 months. Majority of patients prescribed triple therapy at baseline stepped down to LABA+ICS (19%) or LAMA (23%) during the 24-month follow-up .Results from the Kaplan-Meier survival analysis showed that the proportion of patients that progressed to triple therapy varied depending on the initial medication prescribed . At 1 yeIn this study, we evaluated the treatment paradigm evolving over 24 months after the first diagnosis of COPD in the UK general practice. Maximum variability in treatment was observed within the first 6 months, with approximately 15% to 49% of patients changing their initial therapy. Beyond this period, patients gradually stabilized on their respective treatments, with limited changes over the next 18 months of the follow-up. Majority of changes beyond 6 months included addition of treatments with limited switches. In addition, the index treatment was discontinued for most patients within the 24 months of diagnosis irrespective of the treatment class. This gradual yet significant change in therapy may indicate the difficulty in achieving a stable disease state in a progressive disease such as COPD. Thus, this observed change from initial therapy, with addition or switching, may suggest suboptimal disease control.The results of this study are consistent with other studies that examined treatment patterns in COPD. Overall retention of treatment was low across all medications, although differences were observed among different treatment classes. In a study using the Ontario Drug Benefit Program database, Cramer et al demonstrated that 15–63% of patients continued on their index drug for more than six months The Cramer et al study found that patients stayed on tiotropium treatment significantly longer than other daily medications. In contrast, the treatment class with maximum retention in our study was LABA+ICS. The patients in the current study could represent COPD patients with concomitant yet undiagnosed asthma as 44% of the patients prescribed LABA+ICS in the total cohort had at least one asthma diagnosis in their record. These patients may also have had exacerbations in their previous history and received benefit from the ICS.Guidelines suggest adding an ICS as anti-inflammatory agents to reduce the rate of exacerbation in patients with severe disease Many patients starting on a long-acting bronchodilator (LAMA or LABA) or an ICS combination that includes a long-acting bronchodilator received a prescription for triple therapy by the end of the 2-year study period. This finding was observed with two different analyses: when patients were analyzed from their first maintenance therapy until first change in prescription, and when patients were followed up throughout the study period. Thus, the results suggest that COPD treatment evolution is progressive and patients probably need more than one maintenance medication to achieve symptom relief and/or prevent exacerbations.Stepping down therapy may be reflective of prescriptions of multiple inhalers used intermittently. Among triple therapy initiators who stepped down during the follow-up period, majority of patients stepped down to a LAMA or an LABA+ICS combination. This study only addressed first change in therapy and perhaps these patients may have been prescribed triple therapy again at a later date.Study limitations included the fact that GPRD only captures diseases diagnosed in the primary care setting. Also, there may be a possibility of misclassification of drug exposure because the GPRD provides information on prescribed rather than dispensed medications. However, audits of electronic medical records in the UK have shown a relatively high concordance of dispensing to prescription (99.7% of prescriptions tracked were recorded by the electronic patient record during a month) Challenges in optimally managing the condition were evident over the 24 months after COPD diagnosis, as more patients compared to baseline were prescribed additional maintenance therapy in the 24-month period. A high proportion of patients were switched or discontinued from their index treatment within 24 the months of diagnosis. Patients starting on LAMA or LABA+ICS therapy were most likely to change to triple therapy. Thus, the results suggest that COPD treatment evolution is progressive and patients likely need more than one maintenance medication to provide symptom relief and/or prevent exacerbations. The changes in therapy observed in this real world cohort suggest that it is difficult to achieve a consistently improved COPD disease state over two years and the unmet need remains.


Lynx lynx throughout north-eastern Europe using microsatellite, mitochondrial DNA control region and Y chromosome-linked markers. Using SAMOVA we found analogous patterns of genetic structure based on both mtDNA and microsatellites, which coincided with a relatively little evidence for male-biased dispersal. No polymorphism for the cytochrome b and ATP6 mtDNA genes and Y chromosome-linked markers were found. Lynx inhabiting a large area encompassing Finland, the Baltic countries and western Russia formed a single genetic unit, while some marginal populations were clearly divergent from others. The existence of a migration corridor was suggested to correspond with distribution of continuous forest cover. The lowest variability (in both markers) was found in lynx from Norway and Białowieża Primeval Forest (BPF), which coincided with a recent demographic bottleneck (Norway) or high habitat fragmentation (BPF). The Carpathian population, being monomorphic for the control region, showed relatively high microsatellite diversity, suggesting the effect of a past bottleneck  on its present genetic composition. Genetic structuring for the mtDNA control region was best explained by latitude and snow cover depth. Microsatellite structuring correlated with the lynx's main prey, especially the proportion of red deer (Cervus elaphus) in its diet. Eurasian lynx are capable of maintaining panmictic populations across eastern Europe unless they are severely limited by habitat continuity or a reduction in numbers. Different correlations of mtDNA and microsatellite population divergence patterns with climatic and ecological factors may suggest separate selective pressures acting on males and females in this solitary carnivore.Due to their high mobility, large terrestrial predators are potentially capable of maintaining high connectivity, and therefore low genetic differentiation among populations. However, previous molecular studies have provided contradictory findings in relation to this. To elucidate patterns of genetic structure in large carnivores, we studied the genetic variability of the Eurasian lynx,  Various studies on population genetics in large carnivores show diverse patterns of genetic structure. Some research emphasizes that, due to their high mobility, carnivore populations are maintained through long distance dispersal, which contributes to high gene flow across large spaces and low differentiation among populations Some of these discrepancies may result from using markers of different heritability, e.g. microsatellites and mitochondrial DNA (mtDNA) – the markers most frequently used in population genetic studies. For instance, a microsatellite-based study on the genetic structure of Scandinavian brown bears Lynx lynx), two studies Lepus timidus)  or domestic reindeer (Rangifer tarandus) (Norway) and the south-western populations feeding on Cervids From the research concerning the population genetic structure of the Eurasian lynx  and used three types of genetic markers: maternally inherited mtDNA, Y-chromosome markers and nuclear microsatellites. Our primary goal was to understand the relative role of factors affecting the genetic diversity in different populations. We predicted that: 1) lynx are capable of maintaining high rates of gene exchange providing that habitat is continuous; 2) genetic variability and distinctiveness of contemporary lynx populations in Eastern Europe are affected by both human activity  and past climatic events ; 3) genetic differentiation between lynx populations is correlated with climatic factors and/or 4) it is correlated with the main prey type of lynx. This knowledge will help in interpreting the importance of current and historical processes in shaping the distribution and genetic structure of the lynx population, as a model species of large, mobile carnivores.The license for lynx live-trapping and blood sampling was obtained from the National Ethics Committee for Animal Experiments (no: DB/KKE/PL – 110/2001) and the Local Ethics Committee for Animal Experiments at the Medical University of Białystok (no: 52/2007). No animals were harmed during live-trapping and handling. An effort was made to minimize the time animals spent immobilized in the traps. All traps were equipped with radio- or GSM-alarm systems that allowed us to remotely control trapping and release the captured animals from the traps within 15 minutes to 1 hour.We analysed samples of Eurasian lynx from northern, central and eastern Europe bordered by Norway, the Carpathian Mountains and Russia (Kirov Republic) . We arbiAltogether, 332 lynx individuals were sampled between 1992 and 2011. Sample sizes per population are given in 2O , 7 µl primer mix (containing 2pmol of each primer). Aliquots of 8 µl of this mix was then added to 2 µl of isolated DNA template. The thermocycling profile was as follows: 95°C for 15 min, followed by 30–45 cycles of denaturation at 94°C for 30 s, annealing at 57°C (Panel 1 and 3) or 56.5°C (Panel 2) for 90 s, extension at 72°C for 60 s and final extension at 60°C for 30 min. The PCR products were mixed with 10 µl ultragrade formamide and 0.2 µl GeneScan 500-LIZ size standard , denatured at 95°C for 5 min, immediately cooled and then separated using a four-capillary ABI 3130 Genetic Analyser . Allele sizes and genotypes were scored using GeneMapper 4.0 software . PCRs were repeated twice to test the consistency of the results for a subset (5%) of randomly chosen individuals. A total of 54 hair samples from Lithuania and Belarus were analyzed. Hair samples which gave low amplification rate and displaying contamination within hair traps were excluded. Microsatellite panels in the remaining hair samples (20) were amplified between two and four times until they gave reliable genotypes, e.g. heterozygotes at least three times out of four. Samples with inconsistent genotypes were discarded from the analysis. Possible genotyping errors due to stuttering, short allele dominance and null alleles were tested by MICRO-CHECKER 2.2.3. Genomic DNA was extracted using the Genomic Mini Kit  according to the manufacturer's protocol and was stored at −20°C until used. The amplification of 13 microsatellite markers (twelve nuclear and one Y-linked) was carried out in three multiplex panels . The above-mentioned microsatellite loci were developed for domestic cats G GAA TTT AAC CAT GAC C -3′ and LcytbR: 5′-GAC TCT TCA TTT GAG GAG ACG. The entire 681 bp of the ATP6 gene (LATP_F: 5′-TCC AGA ACC TAA ATC CAC AAC C-3′ and LATP_R: 5′-GCA TGA GTT TGG TGG GTC ATT A-3′) was amplified in 8 lynxes from Norway, Finland, Latvia and Poland. All laboratory procedures and quality test controls for cr mtDNA followed DBY4, which had an annealing temperature of 48°C. We obtained clear PCR products for DBY4 (185 bp), DBY7 (275 bp), DBY8 (115 bp), SMCY7 (625 bp) and SMCY17 (125 bp) but no clear products were obtained for UBE1Y6 and UTY11. The five Y-chromosome markers that gave clear PCR products were sequenced in 43 males from Russia, Finland, Estonia, Latvia, Poland, Romania and Belarus according to A portion (613 bp) of the mtDNA control region (cr mtDNA) was amplified in 122 lynxes using the primer pair LGL283 and ISM015 A), allelic richness (AR), gene diversity (Hs) and inbreeding coefficient (FIS) in the populations. Differences among groups of samples in AR, Hs, expected heterozygosity (He), relatedness coefficient (r), and FST were tested using a permutation procedure (10000 iterations) in FSTAT. The same software was used to tests for linkage disequilibrium among 12 microsatellite loci within each population and for the total sample (13200 permutations). Hardy–Weinberg equilibrium probabilities were calculated using GENEPOP 4.0 π), number of segregating sites (s) and mean pairwise difference were calculated using ARLEQUIN v 3.1 FST (for microsatellites) and ΦST (for mtDNA) values for the 10 populations were estimated using ARLEQUIN and their significance was tested using 1000 permutations corrected for multiple tests by Bonferroni correction. For comparative purposes, we have also calculated pairwise DESTST/(1- FST) http://ibdws.sdsu.edu/~ibdws/For the autosomal microsatellite loci, FSTAT 2.9.3 http://www.goldensoftware.com/demo-downloads). Contour lines of the first axis of the PCA were interpolated and superimposed onto a geographic map of the study area. By interpolating the PC1 scores for each individual lynx throughout the study area, we tried to identify regions where genetic dissimilarity between individuals was considerably higher or lower than would be expected from the IBD effect alone. Such geographic areas may represent dispersal barriers or migration corridors, respectively.Principal coordinate analysis (PCA) was performed based on a sample-wise matrix of genetic distance for microsatellite and mtDNA data in GenAlEx v. 6.0 ΦST and FST). To explore patterns of genetic divergence in more detail, we applied the spatial AMOVA procedure using SAMOVA ver. 1.0 CTΦ values (among group variance) and minimized ΦSC values (among populations within group variance). The significance of Φ-statistics was tested using 10000 permutations for K = 2 to K = 9 partitions of the sampling sites. The pRST - RST tests between identified groups were performed in SPAGEDI Analysis of molecular variance  replicates after the burn-in period. The program was run 5 times for each number of clusters (K) between 1 and 10 to verify the consistency of estimates across runs. To determine most likely number of genetic clusters we evaluated the probability logarithm of the data (lnP(D); Contemporary migration rates (within the past one to three generations) between lynx populations were evaluated using a Bayesian approach implemented in BayesAss ver. 1.3 −7 substitutions/site/year with SD  = 2.28×10−7 substitutions We applied Approximate Bayesian computation , average snow cover depth (SCD) in January www.worldclimate.com), To identify correlations between genetic distance between lynx populations and abiotic climatic, as well as biotic factors (lynx basic prey), we performed multivariate multiple regression analysis using DISTLM ver. 5 We obtained control region mtDNA sequences for 332 lynx samples. Sixteen 613 bp haplotypes of the cr mtDNA were observed, of which four  were previously unreported . All newATP6 gene among 8 lynxes from different populations (GenBank accession no.: KM000083).The cytochrome b gene (1094 bp) showed no polymorphism among 12 individuals from different populations (GenBank accession no.: KM000081). Likewise, no polymorphism was found among 681 bp of the entire No sequence polymorphism was found in the five Y-linked chromosome markers , among 43 male lynx from seven sampling locations  and the Y-linked microsatellite locus 278g21-4 possessed only a single allele (146 bp long) in all males studied.R and He) were lowest for the Norwegian and BPF populations, and largest for lynx from Russia  than for the remaining lynx populations, possibly due to bottlenecks. Interestingly, the Carpathian population did not show any signs of reduced levels of polymorphism at the microsatellite loci; however it exhibited a positive and significant FIS value, as did the population from Belarus  linkage disequilibria between loci within any of the populations studied, as well as the frequencies of null alleles low . Measures of genetic diversity for the microsatellite loci  and the majority of comparisons were significant , with an x-intercept at 498 km and 586 km for females and males, respectively . For fem2 = 0.028, p = 0.28) nor for microsatellite loci  among our sampling locations  but not for the cr mtDNA   pattern for mtDNA (rocations . When we = 0.08) .ΦST = 0.570, p<0.001, FST = 0.464, p<0.001). SAMOVA was subsequently used to identify the subdivision that most likely explains the mtDNA structure observed in lynx. The data were best explained assuming four groups of lynx populations: (1) Norway, (2) BPF, (3) Carpathians and (4) the remaining 7 sampling sites .Geographical structuring among lynx cr mtDNA haplotypes in the study area was highly supported by AMOVA results where all sampling sites were treated as a single group . The data were best explained in SAMOVA analysis assuming the same four groups of lynx populations: (1) Norway, (2) BPF, (3) Carpathians and (4) the remaining 7 sampling locations . RST did not differ significantly from pRST for any pairwise between-group comparisons .For the microsatellite data, AMOVA supported the geographical structuring of the populations  is correlated with latitude  but not with longitude. Similarly, the number of days with snow cover, snow cover depth, and the NAO index, as revealed by marginal tests, had significant effect on mtDNA differentiation among sampling locations  were separated into three clearly distinct genetic entities. Based on the different heritability of mtDNA and microsatellites, as well as previous research that suggested different genetic structure in the brown bear with use of the same types of markers et al. RST and pRST values for microsatellite loci suggest genetic drift as the primary reason for observed genetic divergence among the lynx populations at microsatellite nuclear loci.Spatial autocorrelation analysis provided relatively little evidence for male-biased dispersal in the lynx. This is not fully concordant with the available telemetry data, suggesting that similar to other large felids The strong genetic structure in the Eurasian lynx revealed by our analyses corresponds well with our previous study on mtDNA that showed large differentiation among the populations at the westernmost peripheries of its range Our study has also indicated that the genetic structure in the Eurasian lynx may be explained, at least in part, with climatic conditions, particularly the depth of snow cover, which was significantly correlated with mtDNA population differentiation, even when geographic coordinates were included as co-variables in the conditional tests. The lack of a barrier on the synthetic genetic map for mtDNA in northern Scandinavia throughout Finland and Russia coincides well with similar climatic conditions (e.g. high snow cover depth) in all northern lynx populations. Very similar results have recently been reported based on mtDNA for a smaller dataset of lynx samples from the western part of its range The relationships between microsatellite population differentiation and climatic factors were less clear, with the only significant effect of snow conditions found in the sequential test. On the other hand, a significant effect on microsatellite differentiation in the marginal and sequential tests were found for prey, especially the proportion of red deer in the lynx's diet. Although drawing firm conclusions from the latter result can be somewhat speculative, red deer are killed more often by lynx in North-Eastern Poland (BPF and KARPF) than in other areas (Lynx canadensis) The results of multivariate multiple regression, along with the IBD effect may suggest that despite generally concordant patterns of population subdivision, there might be different factors such as climate and prey type affecting mtDNA and the microsatellites genetic structure of lynx population, respectively. The effect of climate may have resulted in the matrilineal clusters of lynx being specifically adapted to hunting prey under local snow conditions. Moreover, as male lynx are more likely to kill larger prey This study revealed differing levels of gene flow within the range of the Eurasian lynx. Low genetic divergence and high rate of migrants identified between the eastern part of European Russia and some western lynx sampling locations suggested high gene flow along the east-westward direction, despite the distance between locations. Several independent analyses of our microsatellite data indicated possible migration corridors situated between Russia (Kirov and Arkhangelsk Oblast), Finland and Belarus. In contrast, little genetic exchange appears to have occurred between neighboring populations situated at the westernmost peripheries of the lynx range, e.g. Norway, the BPF and the Carpathians.The possibility of recent gene flow between lynx inhabiting the far east of European Russia and the westernmost edge of the lynx range (approximately 1100–1300 km) may not be surprising considering the long distances large carnivores (including lynx) are capable of covering during dispersal and migrations et al.Studies documenting long-distance, cross-continental movements in wildlife are rare due to difficulties in collecting representative material The lynx population inhabiting eastern European Russia was characterized by high genetic variation, and therefore one may expect it to constitute a possible source population for these cats in Europe. However, we found present-day gene flow between Russian and Finnish lynx in both directions with migration rates slightly higher from Finland to Russia than in the opposite direction. The pattern of lynx population history revealed with DIYABC analysis of the mtDNA data supports the scenario of a Russian origin for the European lynx populations.The highest variability at both types of markers was found in sampling locations grouped into clusters situated within the core lynx range (Russia) or being directly connected to it through the continuity of the habitat present . Whereas high genetic variability in this cluster can be directly linked with the central location and habitat continuity Among the three most divergent and least variable populations, the Norwegian and Carpathian lynx present different patterns of genetic variability. Whereas the lynx in both areas showed a complete lack of polymorphism in mtDNA, they had different levels of microsatellite variability. As the Norwegian lynx had one of the lowest reported values for allelic richness and heterozygosity, it is clearly concordant with the relatively recent bottleneck experienced by this population during the twentieth century In the case of the BPF population, it is most likely that both its peripheral location and strong isolation from the core range were the main reasons for the low microsatellite and mitochondrial variability. A similar relationship between genetic variability and location relative to the core of the species range was reported for the Canada lynx The Eurasian lynx is capable of maintaining a panmictic population across eastern Europe unless they are severely limited by habitat continuity or a reduction in numbers. The effect of climatic conditions (snow cover) on the genetic divergence between populations with respect to mtDNA, but not nuclear microsatellite loci, may suggest different selective pressures acting on males and females in solitary carnivores. However, further research is needed to identify such selective pressures.S1 FigResults of isolation by distance (IBD) analysis of the Eurasian lynx populations. Two upper panels show IBD for microsatellite data for all ten putative populations (A) and excluding Kirov (Russia) (B). Two lower panels show IBD for mtDNA data for all populations (C) and excluding Kirov (Russia) (D). A significant relationship was only obtained for microsatellite data after excluding the Russian population.(PDF)Click here for additional data file.S2 FigNumber of genetic clusters of the Eurasian lynx population in north-eastern Europe. Evaluation of the most likely number of genetic clusters with the ΔK method (A) and the probability logarithm of the data (lnP(D) (B). ΔK clearly suggests that K = 4 is the most likely number of clusters, while lnP(D|K) shows leveling off from K = 4 and the highest likelihood at K = 8.(PDF)Click here for additional data file.S1 TableHaplotype frequencies of the mtDNA control region in ten populations of Eurasian lynx Lynx lynx.(DOC)Click here for additional data file.S2 TablePairwise differentiation between Eurasian lynx populations.FST values based on 12 microsatellites  and FST values based on the mtDNA control region . Nonsignificant values are given in italics.(DOC)Click here for additional data file.S3 TablePairwise differentiation between Eurasian lynx populations.DEST  and RST  values based on 12 microsatellites.(DOC)Click here for additional data file.S4 TableHabitat type, climate and lynx diet composition of the ten lynx populations studied. * References used for data on lynx diet: Norway (DOC)Click here for additional data file.S1 AppendixList of Eurasian lynx genotypes based on 12 microsatellite loci.(XLS)Click here for additional data file.S2 AppendixProcedures adopted in the Approximate Bayesian Computations (DIYABC 2.0.4 software) and the list of all scenarios tested.(DOC)Click here for additional data file.