Cuculus canorus. Females laying blue eggs belong to an ancient (∼2.6 Myr) maternal lineage, as evidenced by both mitochondrial and W-linked DNA, but are indistinguishable at nuclear DNA from other common cuckoos. Hence, cuckoo host races with blue eggs are distinguished only by maternally inherited components of the genome, which maintain host-specific adaptation despite interbreeding among males and females reared by different hosts. A mitochondrial phylogeny suggests that blue eggs originated in Asia and then expanded westwards as female cuckoos laying blue eggs interbred with the existing European population, introducing an adaptive trait that expanded the range of potential hosts.Maternal inheritance via the female-specific W chromosome was long ago proposed as a potential solution to the evolutionary enigma of co-existing host-specific races (or ‘gentes') in avian brood parasites. Here we report the first unambiguous evidence for maternal inheritance of egg colouration in the brood-parasitic common cuckoo  The common cuckoo lays its eggs in nests of a variety of species and their eggs mimic the ones of their hosts. Here, the authors show that blue egg colouration in the common cuckoo is maternally inherited, originated in Asia and then expanded to Europe. Cuculus canorus is a generalist obligate parasite, exploiting a variety of passerine species across Eurasia, and it has evolved numerous host-specific races (termed ‘gentes'), each having eggs with colour and pattern mimicking the eggs of their respective host species245Gallus gallus, in which blue egg colour is associated with an autosomal, endogenous retroviral insertion, resulting in a dominant Mendelian traitAdaptations and counter-adaptations in the context of co-evolutionary arms races between parasites and their hosts can drive rapid phenotypic changePhoenicurus phoenicurus, where they are excellent mimics of the host's pale blue eggsC. c. bakeri lays blue eggs in the nest of several host species, and they have evolved polymorphic eggs with different intensities of blue matching a similar egg polymorphism in their parrotbill Paradoxornis spp. hosts1011We tested these alternative hypotheses with an expanded geographic sampling of common cuckoos  and a paHere we find that all female cuckoos laying blue eggs in Europe belong to a highly divergent maternal lineage shared with the Asian subspecies, but are indistinguishable at nuclear loci from other common cuckoos. The blue egg colour is therefore maternally inherited, supporting the hypothesis that the genes controlling egg colour are located on the female-specific W chromosome.COLONYFst range: 0.898–0.995) and W chromosome DNA, but not at autosomal or Z-linked loci  and all non-blue eggs being part of the more common ‘canorus' lineage (n=24) and non-blue (n=9) cuckoo eggs collected from a number of different host species in a single sympatric population were strictly associated with the two different mitochondrial lineages , but autosomal and Z-linked markers bear no evidence that would support splitting the common cuckoo into two species  and molecular mechanisms producing blue eggs remain unknown, our data provide the first unambiguous evidence for the long-standing hypothesis that egg colouration in brood-parasitic cuckoos is maternally inherited. In addition, female cuckoos laying blue eggs in Europe are the matrilineal descendants of an ancient lineage that likely evolved in Asia, providing a remarkable example of the geographic expansion and selective introgression of an adaptive trait into an existing population.Genetic samples from 205 European common cuckoos were collected from 7 different host races originating from 8 countries . GeneticGENEMAPPER v.3.7 (Applied Biosystems) and sequence data were assembled and manually checked in GENEIOUS v.4.7.6 . To ensure consistency, one person (FF) scored all of the microsatellite genotypes. The sequencing of DNA samples originating from Asia and from museum eggshells was outsourced to Ecogenics GmbH, Zürich-Schlieren, Switzerland. Mitochondrial 12S and ND2 gene sequences for selected European samples were also generated by Ecogenics GmbH. For amplification of DNA extracted from eggshell samples, we designed a new pair of primers using Primer3 . Multiple mitochondrial and nuclear genetic loci were sequenced  and 13 mer3 ref.  to ampliCOLONYCOLONY utilizes a full-pedigree likelihood approach, which considers the likelihood of the entire pedigree structure and allows for the simultaneous inference of parentage and sib-ships. Moreover, COLONY allows the user to add information on known relationships among the offsprings to increase the probability of correctly assigning individuals to sib-ships. We therefore included information on geographic locality, mitochondrial haplotype and egg appearance to inform the inference of sibling relationships. Offsprings sampled from distant localities are not likely to share either the same father or mother, whereas offsprings with different mitochondrial haplotypes are highly unlikely to share the same mother. Finally, two offsprings having identical mitochondrial haplotypes, but originating from eggs of different appearance are also unlikely to share the same mother. The appearance of eggs from a given cuckoo female is highly repeatable and can be used to assign eggs to individual females, although different females may produce similar eggsCOLONY, but showed a low probability of sib-ship in the analysis. Therefore, we conservatively removed one of the individuals assigned to each pair of putative siblings with a probability lower than 0.95. Of the 205 offsprings analysed for 13 microsatellite loci , 8 offsprings were therefore excluded from further analyses. This left us with 197 offsprings assigned to 104 different mothers for the population genetic analyses. For the sequence-based genetic markers, we utilized subsets of the main data set, including only one offspring per female for statistical analyses. A few siblings were included in the haplotype network for the myoglobin gene, as full siblings often have different genotypes at autosomal loci.Many cuckoo nestlings were collected within the same local area and could be either full- or half-siblings. Thus, to avoid pseudo-replication of the cuckoo lineages associated with a given host, we used N=104). We used exact tests as implemented in the software ARLEQUIN v. 3.5  and, to serve as an outgroup, three species of Hierococcyx cuckoos (n=2 samples per species); Hierococcyx is the sister genus to CuculusCuculus COI sequences and all other available cuculiform COI sequences from GenBank (www.ncbi.nlm.nih.gov/genbank) and Barcode of Life Data Systems (www.boldsystems.org). Given inconclusive resolution of critical ingroup relationships in the COI tree, we generated mitochondrial 12S ribosomal RNA and ND2 gene sequences for representative samples of each Cuculus mitochondrial lineage and combined these data with the comprehensive cuculiform data set from Sorenson and PayneCuculus lineages and, with three times as much sequence data, more robust estimates of relationships and relative divergence times. For both the COI and 12S/ND2 data sets, we completed analyses both with a broad sampling of Cuculiform taxa and with Hierococcyx only as the outgroup to Cuculus. Results for all analyses, including phylogenetic relationships and relative divergence times were broadly consistent except for the placement and divergence time of C. micropterus, which differed between the COI and 12S/ND2 data sets. For each data set, we used jModelTest2  and the predominantly Old World clade comprising all other cuckoos46Cuculus lineages using substitution models and parameter values as determined by jModelTest  under accession codes LN734262 to LN734648 and LN876762 to LN876780.How to cite this article: Fossøy, F. et al. Ancient origin and maternal inheritance of blue cuckoo eggs. Nat. Commun. 7:10272 doi: 10.1038/ncomms10272 (2016).Supplementary Figures 1-2, Supplementary Tables 1-10 and Supplementary References


Carotid sinus syndrome is the association of carotid sinus hypersensitivity with syncope, unexplained falls and drop attacks in generally older people. We evaluated cardiac sympathetic innervation in this disorder in individuals with carotid sinus syndrome, asymptomatic carotid sinus hypersensitivity and controls without carotid sinus hypersensitivity.Consecutive patients diagnosed with carotid sinus syndrome at a specialist falls and syncope unit were recruited. Asymptomatic carotid sinus hypersensitivity and non-carotid sinus hypersensitivity control participants recruited from a community-dwelling cohort. Cardiac sympathetic innervation was determined using Iodine-123-metaiodobenzylguanidine (123-I-MIBG) scanning. Heart to mediastinal uptake ratio (H:M) were determined for early and late uptake on planar scintigraphy at 20 minutes and 3 hours following intravenous injection of 123-I-MIBG.Forty-two subjects: carotid sinus syndrome (n = 21), asymptomatic carotid sinus hypersensitivity (n = 12) and no carotid sinus hypersensitivity (n = 9) were included. Compared to the non- carotid sinus hypersensitivity control group, the carotid sinus syndrome group had significantly higher early H:M  and late H:M . There was, however, no significant difference in early H:M (p = 0.326) or late H:M (p = 0.351) between the asymptomatic carotid sinus hypersensitivity group and non- carotid sinus hypersensitivity controls.Cardiac sympathetic neuronal activity is increased relative to age-matched controls in individuals with carotid sinus syndrome but not those with asymptomatic carotid sinus hypersensitivity. Blood pressure and heart rate measurements alone may therefore represent an over simplification in the assessment for carotid sinus syndrome and the relative increase in cardiac sympathetic innervation provides additional clues to understanding the mechanisms behind the symptomatic presentation of carotid sinus hypersensitivity. Carotid sinus hypersensitivity (CSH) may be associated with syncope in older people, and has also been implicated in the etiology of unexplained falls and drop attack ,2. AsympWe hypothesize that autonomic function is altered in patients with CSS, and that cardiac sympathetic innervation is enhanced in such individuals. In the study reported here, cardiac meta-iodo-benzylguanidine (MIBG) imaging was used to provide a quantitative measure of cardiac sympathetic innervation. This method is particularly useful in older individuals, as unlike other methods using challenge manoeuvres, it requires minimal patient cooperation. We are aware of no such imaging study in CSS. We, therefore, conducted a case-control study of cardiac MIBG imaging in both patients with CSS, asymptomatic CSH and asymptomatic control subjects without CSH.This study was granted ethical approval by the Newcastle and North Tyneside Local Research and Ethics Committee 2. Written informed consent was obtained from all participants. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.Participants with CSS were recruited from consecutive patients diagnosed with CSH during investigations for syncope, unexplained falls or drop attacks at a specialist syncope facility. Control participants with and without CSH were recruited from an asymptomatic community cohort evaluated with carotid sinus massage in an epidemiological study . Potentisyndrome was diagnosed if such a response coincided with symptoms reproducing the patient’s original presentation in the absence of a competing cause for such symptoms following comprehensive evaluation in accordance with European Society of Cardiology guidelines [Carotid sinus hypersensitivity was defined as at least three seconds of asystole or a reduction in systolic blood pressure of at least 50mmHg in response to carotid sinus massage. Carotid sinus idelines . Carotididelines .The patients and asymptomatic community dwellers with and without CSH who consented to the study were invited to attend the Medical Physics Department on a separate occasion. Two doses of potassium iodate 170mg were administered in the preceding 24 hours for thyroid blockade. Participants were injected with 150 MBq of 123-iodine radiolabelled MIBG (123-I-MIBG) administered through a peripheral vein. Early (20 minutes) and delayed (3 hours) planar thoracic scintigraphic images were obtained in an anterior projection using a large field gamma camera fitted with a low energy high-resolution collimator. Static images were collected for 15 minutes with a 20% window centred on the 159keV, 123-I photopeak./ (H:M)early * 100. In all cases, late H and M uptake values were corrected for 3 hours of radioactive decay. Ten randomly selected cases were reanalyzed by the same observer and an independent observer to test intraobserver and interobserver agreement. Both observers followed a standardised method of analysis and were blinded to the groups of the study.On the early anterior view, an oval shaped region of interest was drawn tightly and semi automatically around the entire heart avoiding as far as possible any confounding activity from adjacent left lung . A smalla priori that CSH is associated with changes in cardiac sympathetic innervation. Stepwise entry was used for the potential confounders of age, sex, co-existing medical conditions and medications. The B coefficient therefore represents the estimated mean differences between groups. A two-tailed p-value of < 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS™ 15.0 software package.Histogram plots and the Komolgorov-Smirnov test were used to confirm that continuous variables were normally distributed. Continuous variables were then expressed as mean with 95% confidence intervals, while categorical data were presented as count with percentages in parentheses. Participant characteristics were compared with analysis of variance for continuous variables and the Chi-squared test for categorical variables. Interobserver and intraobserver reliability were determined with Pearson’s correlation. Comparisons for early H:M ratio, late H:M ratio and washout rate were made between the CSS group versus non-CSH controls and the asymptomatic CSH group versus non-CSH controls using linear regression methods, based on our hypothesis generated Forty-two participants mean age 74 years (range 52 to 90 years) were recruited, 21with CSS, 12 with asymptomatic CSH and 9 asymptomatic controls from the same community cohort who did not have CSH. The characteristics of participants in the above three groups are listed in The intraobserver and interobserver values for the H:M uptake ratios showed good correlation at r = 0.997 and r = 0.994 respectively. Linear regression analyses with stepwise entry for potential confounders revealed that the early H:M uptake ratio in individuals with CSS was significantly higher than non-CSH controls (p = 0.005) . The latOur study was the first to examine cardiac sympathetic innervation through 123-I-MIBG myocardial scintigraphy in both CSS and asymptomatic CSH, and controls without CSH. The CSS group had higher early and late H:M uptake ratios than the non-CSH control group. There were no differences in either ratio between the asymptomatic CSH group and the non-CSH control group. These findings indicate that sympathetic presynaptic neuronal uptake is enhanced in individuals with CSS but not those with asymptomatic CSH. There was, however, no notable difference in %WR which has been considered a marker of sympathetic activity.123-I-MIBG scanning has previously shown higher H:M ratios in head-up tilt positive paediatric patients with vasovagal syncope , though Control subjects in previous studies had included patients with negative CSM or asymptomatic controls with unknown CSH status. CSH has been associated with sternocleidomastoid denervation , increasWe have shown that cardiac MIBG uptake is increased in individuals with CSS compared to matched controls without CSH. Meta-iodo-benzylguanidine is an analogue of norepinephrine which is not metabolized in humans, and therefore excreted unchanged. The heart is normally enriched with sympathetic nerve endings which also have a high affinity for MIBG, and this property can be used to perform quantitative assessments of the integrity of cardiac sympathetic nerves ,24. ThesThe relative increase in MIBG uptake observed in the CSS group over the non-CSH control group was, however, not observed in individuals with asymptomatic CSH. The difference in presynaptic neuronal uptake may not, therefore, necessarily account for the heart rate and blood pressure changes that are recorded, but instead appears to be associated with the clinical symptoms that occur with the measured hemodynamic changes. This raises the possibility, reinforcing previous clinical suspicions, that the current practice of assessing heart rate and systolic blood pressure changes alone represents an over simplistic method of assessing overall physiological responses during clinical investigations to evaluate syncope. In addition, this also indicates that individuals with CSH who develop symptoms of falls or syncope have different autonomic responses to asymptomatic individuals, which suggests that CSS is indeed a pathological condition rather than just a feature of normal aging.The interpretation of the lack of difference in %WR is unclear in the presence of increased early and late H:M uptake. While the washout rate is considered an index of sympathetic drive , washoutWhile the number of participants involved in our study outnumbers all the previous studies evaluating the pathophysiological changes associated with CSH published so far, the actual number remains relatively small, introducing the possibility of false negative results. The clinical applicability of our study is also limited by the potential for overlap between the three groups. Nevertheless, the presence of increased or even preserved cardiac sympathetic innervation in CSS represents an entirely new finding, which at the very least deserves further evaluation. This may well represent the early steps towards the identification of an additional therapeutic target for this increasingly important condition. While treatment in CSS reduces syncope recurrence to 7% at 1 year, at 5 years 26% have recurrence of symptoms regardless of treatment modality , so thisCardiac MIBG uptake was significantly increased in symptomatic individuals with CSH compared to controls without CSH, with no corresponding increase in asymptomatic individuals with CSH and no apparent changes in %WR. The absence of changes in MIBG uptake in asymptomatic individuals with CSH highlights possible inadequacies of heart rate and blood pressure measurements in assessing the overall response to carotid sinus massage. The symptomatic presentation of CSH, carotid sinus syndrome, however, appears to be associated with heightened cardiac sympathetic neuronal activity. This represents an important development in the search for potential therapeutic targets for CSS which is now urgently required.


Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings.Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium  Fitneper se. Unlike compensatory mutations, generally beneficial mutations contribute to an increase in fitness, regardless of the genetic background in which they arise [Mycobacterium tuberculosis [One possible mechanism through which bacteria can overcome the cost of resistance is by acquiring compensatory mutations that reduce or eliminate the fitness costs associated with resistance alleles by recovering the functional defects associated with resistance mutations –9. Alterey arise ,11. In tey arise ,12–14. Crculosis –17, and rculosis .−11 mutations per site per generation under laboratory conditions [−9 per genome per generation. On the other hand, generally beneficial mutations have been estimated to occur at a rate between 1 × 10−8 and 1 × 10−5 per genome per generation under laboratory conditions, which is 10–10 000 times higher than our crude estimate of the rate of compensatory mutation [Although compensatory mutations have been identified in a wide range of systems, the fact that resistant populations can also evolve by generally beneficial mutations that increase fitness, irrespective of antibiotic resistance, is far less frequently mentioned in current discussions of antibiotic resistance evolution. It has been estimated that there are, on average, 12 possible compensatory mutations per deleterious mutation in bacteria , and thenditions , which cmutation –23. ThisHowever, adaptation in asexual populations is driven by a small minority of mutations with relatively large benefits that overcome stochastic drift and competition from rival beneficial mutations owing to clonal interference ,22,24,25Pseudomonas aeruginosa that carry different fitness costs, as well as their rifampicin-sensitive ancestor, to evolve for 300 generations in an antibiotic-free rich medium. Extensive whole-genome sequencing was used to systematically identify compensatory mutations and generally beneficial mutations in evolved endpoint populations.To test the hypothesis that high fitness costs promote evolution by compensatory adaptation, we allowed populations of eight isogenic rifampicin-resistant isolates of the opportunistic pathogen 2.(a)rpoB mutants that carried different fitness costs to evolve in a rifampicin-free culture medium for 300 generations. Fitness varied considerably between the rifampicin-resistant mutants that were used to initiate the selection experiment . Fitness increased over the course of the selection experiment , and the fitness of the evolved populations was very similar to that of the rifampicin-sensitive ancestral strain that the resistant mutants were evolved from. These results clearly demonstrate that selection in the absence of antibiotics can rapidly eliminate the substantial fitness cost that is associated with rifampicin resistance for a range of rpoB mutations.To study adaptation to the cost of rifampicin resistance, we allowed three independently propagated populations of eight different rifampicin-resistant (b)To determine the genetic basis of adaptation in antibiotic-resistant populations, we performed whole-genome sequencing on three randomly selected colonies isolated from each evolved endpoint population . As a control experiment, we also sequenced the genomes of evolved isolates from rifampicin-sensitive ancestral populations that were evolved for the same number of generations . Non-synonymous single-nucleotide polymorphisms (SNPs) and short indel (insertion and deletion) mutations of less than 1 kb in protein-coding regions were the most common forms of mutations that we identified. Silent and intergenic mutations were found at low frequencies, as were large deletions and duplications spanning more than one gene.The mutations that we identified may include beneficial, neutral or mildly deleterious mutations. Parallel evolution provides a hallmark of genes that are under strong positive selection –36, and rpoA, rpoB and rpoC) can compensate for the cost of rifampicin resistance [rpoB mutations represents a reversion mutation that swept to fixation in a population that was initiated by the H531R mutant. The reversion mutation restored the rifampicin-sensitive phenotype, which constitutes an evolutionary reversal of antibiotic resistance. Several lines of evidence suggest that the remaining RNA polymerase mutations are compensatory mutations rather than generally beneficial mutations. First, the intragenic second-site mutations in rpoB found in this study  are known to recover the reductions in transcriptional efficiency and fitness costs associated with the original rpoB mutations [rpoB at an amino acid level. An additional mutation in the same codon as the original rpoB mutations H531R and H531Y can result in a common amino acid substitution H531C, which was observed in two populations founded by H531R and one population founded by H531Y . H531C can offset the fitness cost of the original rpoB mutations without altering the rifampicin resistance phenotype [Because compensatory mutations interact epistatically with resistance mutations to recover fitness ,31,37, wsistance ,7,16,26.henotype .rpoB mutation . Almost all isolates from populations that were founded by very costly resistance mutants, such as H531R and H531Y, carry compensatory mutations, whereas no compensatory mutations were found in populations that were founded by low-cost resistance mutations such as S536F.To test the hypothesis that low fitness costs constrain compensatory adaptation, we tested for a negative correlation between the frequencies of RNA polymerase mutations and the initial cost associated with each mutation . In supp(c)lasR, a key transcriptional regulator gene involved in quorum-sensing [lasR. There was also a high diversity of mutations in lasR, which include 26 independently evolved SNPs and short indel mutations, as well as four independent large deletions (more than 2 kb) affecting lasR and adjacent genes. These mutations are thought to disrupt LasR function and result in quorum-sensing deficiency [rpoB mutant Q518R, is known to disrupt LasR functions and quorum-sensing-regulated phenotypic traits [lasR led to increased fitness in rpoB mutants . In agreement with this, numerous studies have shown that P. aeruginosa populations adapt to novel environments through the loss of LasR function [Although we found evidence for compensatory adaptation when the cost of resistance was large, the dominant genetic mechanism of adaptation in antibiotic-resistant populations was mutations in -sensing ,40. Abouficiency –45. For c traits . Mutatiofunction –46.lasR mutations were also detected in the evolved control populations , confirming that lasR mutations were not compensatory. More specifically, three independent lasR mutations we observed are known to compromise LasR function by eliminating the start codon (M1I), deleting the lasR promoter region [lasR mutations (33%) was lower compared with that of resistant populations (71%), so it remains to be understood why such discrepancies exist between the resistant and control populations.r region  or prever region . However(d)lasR mutations during the first 24 days of the experiments (b–d). Successful lasR mutants then acquired compensatory mutations in RNA polymerase genes, which began to increase to detectable frequency by the end of the experiment. In the H531Y-A population (a), initial adaptation was dominated by the spread of mutations in the gacA gene. GacS/GacA modulates the expression of more than 500 genes of the RsmA regulon and is indirectly involved in the regulation of quorum-sensing [rpoB gene also began to increase to detectable frequency by day 30.To better understand the conflict between compensatory adaptation and general adaptation, we studied the dynamics of adaptation by sequencing a time series of isolates from a subset of our selection lines. We focused on populations in which both generally beneficial mutations and compensatory mutations were identified . The co-occurrence of generally beneficial mutations and compensatory mutations implies that these populations are likely to provide direct insights into the outcome of competition between these two classes of beneficial mutations. Based on the sequencing results, we constructed the evolutionary history for these four populations . The keyeriments b–d. Succpulation a, initia-sensing –51. SubslasR mutations, suggesting that any mutation which disrupts LasR production and the quorum-sensing phenotype is beneficial under these experimental conditions. By contrast, we found a relatively small number of compensatory mutations in RNA polymerase, which supports the idea that compensatory mutations are rare [rpoB (3/7) was much higher than that in lasR (1/30) among the resistant populations. lasR mutations also confer large fitness benefits. For example, three double mutants that carry single lasR mutations in addition to their original rpoB mutations, showed an increased fitness of between 15.6% and 32.3% . This suggests that the fitness benefit associated with lasR mutations was likely to be comparable to, or greater than, the benefit associated with compensatory mutations, except when the cost of resistance was very large.Several factors are likely to have favoured this repeatable evolutionary dynamic of general adaptation followed by compensatory adaptation. We detected a vast diversity of are rare . This aswspA and wspF from the wsp operon (n = 9), dipA (n = 12), as well as morA (n = 2). The absence of wspA/wspF, dipA and morA mutations in the resistant populations raised the possibility that the fitness benefit conferred by these mutations could be contingent on the absence of rpoB mutations (sign epistasis) [rpoB mutations with dipA or morA mutations and found that dipA and morA mutations were beneficial in the presence of rpoB mutations [Surprisingly, we found that the control populations often carried mutations in genes that were not mutated in resistant populations , includiistasis) . To testutations , implyin3.P. aeruginosa. Although rifampicin-resistant populations quickly evolved to overcome their initial fitness cost, compensatory mutations were fixed in populations founded only by highly costly resistant mutants. Importantly, many studies of evolution in antibiotic-resistant populations tend to focus on evolution in populations carrying very costly resistance mutations [The idea that compensatory adaptation eliminates the cost of resistance and allows resistance alleles to effectively persist in bacterial populations has been extensively studied in evolutionary models of antibiotic resistance ,13,55–57utations ,28–32. OlasR, versus the higher frequency of repeated nucleotide substitutions in rpoB, which was involved in compensatory adaptation. Second, generally beneficial mutations can have large effects on fitness that surpass those associated with compensatory mutations . We argue that compensatory adaptation tends to occur when the cost of resistance is large, because compensatory mutations provide very large fitness benefits in populations carrying costly resistance mutations.Although compensatory adaptation provides a more direct solution to the cost of resistance, two constraints are likely to restrict compensatory evolution. First, generally beneficial mutations are more common than compensatory mutations. While compensatory mutations usually occur in the same protein or pathway as antibiotic resistance mutations do ,52, geneM. tuberculosis [Given that the spread of compensatory mutations can be delayed by the spread of generally beneficial mutations owing to clonal interference, selection for compensatory adaptation will be relatively ineffective under conditions where selection at other sites in the genome is strong. A number of important selective forces are likely to exert strong and recurrent selective pressure on populations of bacterial pathogens, such as antibiotic use ,59, bactrculosis ,15,16, crculosis .Whole-genome sequencing is now being used extensively to study the population biology of bacterial pathogens, and it is often thought that mutations which are found in antibiotic-resistant isolates are compensatory mutations ,59,65–674.(a)rpoB mutants used in the selection experiment were evolved from the rifampicin-sensitive PAO1::mini-Tn7-pLAC-lux ancestral strain using a fluctuation test [rpoB mutant strain were randomly selected for propagating three independent populations (denoted A–C) during the selection experiment. About 1 ml of M9KB liquid culture medium [The eight rifampicin-resistant ion test . All strion test  and incue medium  was inoc(b)LAC-lux ancestral strain and a GFP-tagged derivative strain of PAO1 were diluted 1 : 10 in M9KB medium and regrown to early exponential phase at 30°C with continuous shaking (225 r.p.m.). Each non-fluorescent strain was mixed with the GFP-tagged strain, diluted 200-fold in M9KB medium and incubated in Nunc 96-well microplates  at 30°C overnight for approximately 24 h with continuous shaking. The proportion of fluorescent and non-fluorescent cells in each co-culture was determined using an Accuri C6 flow cytometer  before and after the incubation. The competitive fitness of each non-fluorescent strain was calculated as the ratio of population doublings of each endpoint population relative to the GFP-tagged control strain it was competing against [The competitive fitness of the endpoint populations of each of the propagated populations was quantified relative to the rifampicin-sensitive ancestral strain as previously described . Briefly against . The rellasR mutations are beneficial under the same conditions of the selection experiment, the competitive fitness of three evolved rifampicin-resistant isolates that carried different lasR mutations and the initial rpoB mutant strains was determined relative to the rifampicin-sensitive ancestral strain using a modified version of the competition assays, in which all strains were regrown to exponential phase and co-cultured overnight with the GFP-tagged control strain in 5 ml Falcon tubes (BD Biosciences) using exactly the same growth conditions as those under which the selection experiment was carried out.To demonstrate that (c)rpoB mutants and the endpoint populations of the selection experiment were streaked on M9KB agar plates and incubated overnight at 30°C. On the next day, three colonies of each population were randomly selected to inoculate M9KB liquid medium incubated at 30°C with shaking overnight. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit  according to the manufacturer's protocol. The 12 initial isolates of the rifampicin-sensitive ancestral strain and the three randomly selected colonies from the 12 endpoint populations, which were evolved from these initial isolates during the control experiment, were prepared for whole-genome sequencing using the same procedures.The initial rpoB mutant populations, six random isolates from the 18th and 24th days of the selection experiment were selected for whole-genome sequencing from four selected rpoB mutant populations  using the same procedures as described above. Three additional random isolates from the four endpoint populations were whole-genome sequenced to obtain a total of six isolates for each of the three time points .To search for evidence of clonal interference in rpoB mutant isolates were found to have the same genetic background, with the exception of the specified mutations in rpoB. We discarded mutations that were already present in all the initial isolates with respect to the P. aeruginosa PAO1 reference genome, analysing only those mutations that accumulated throughout the selection experiment.Paired-end whole-genome sequencing with read length of 100 bp was performed using the HiSeq 2000 sequencing system . The average coverage was 44.1× (median coverage: 43.5×). Variants were identified using an experimentally validated in-house pipeline ,73. The (d)All statistical analyses were performed using JMP software v. 11 . Unless otherwise stated, all statistical tests are two-tailed, and the level of significance is 0.05. The degrees of freedom are reported as subscripts next to the test statistics.


Recent studies suggest that alpine and arctic organisms may have distinctly different phylogeographic histories from temperate or tropical taxa, with recent range contraction into interglacial refugia as opposed to post-glacial expansion out of refugia. We use a combination of phylogeographic inference, demographic reconstructions, and hierarchical Approximate Bayesian Computation to test for phylodemographic concordance among five species of alpine-adapted small mammals in eastern Beringia. These species  vary in specificity to alpine and boreal-tundra habitat but share commonalities  that might result in concordant responses to Pleistocene glaciations. All five species contain a similar phylogeographic disjunction separating eastern and Beringian lineages, which we show to be the result of simultaneous divergence. Genetic diversity is similar within each haplogroup for each species, and there is no support for a post-Pleistocene population expansion in eastern lineages relative to those from Beringia. Bayesian skyline plots for four of the five species do not support Pleistocene population contraction. Brown Lemmings show evidence of late Quaternary demographic expansion without subsequent population decline. The Wrangell-St. Elias region of eastern Alaska appears to be an important zone of recent secondary contact for nearctic alpine mammals. Despite differences in natural history and ecology, similar phylogeographic histories are supported for all species, suggesting that these, and likely other, alpine- and arctic-adapted taxa are already experiencing population and/or range declines that are likely to synergistically accelerate in the face of rapid climate change. Climate change may therefore be acting as a double-edged sword that erodes genetic diversity within populations but promotes divergence and the generation of biodiversity. Pleistocene glacial cycles have dramatically shaped the spatial distribution of genetic variation in many species . These mDuring previous glacial intervals the Laurentide and Cordilleran ice sheets blanketed much of North America, isolating plant and animal species in multiple refugia . North oIn addition to the Beringian refugium, several cryptic northern refugia appear to have played an important role in the recolonization of previously glaciated regions for some cold-tolerant species ,25. ThesPhylogeographic studies suggest that the majority of the recently colonizing species in eastern Beringia (present-day Alaska) arrived via North America ,21. Pattet al. ) fell into two major phylogeographic groups: a well-supported Beringian clade (hereafter B) currently found in Alaska and northeastern Russia and a Canadian Arctic/eastern Alaska clade to the east (E). Taking into consideration fossil records, they concluded that the eastern lineage was previously isolated south of the Laurentide ice sheet , identified three distinct mtDNA lineages that may have diversified in Beringia . In. Inet alBeringia ,34, suggBeringia ). Phylogd, 1901)  and Colln, 1893)  are more /E[t]) should approach zero. For this dataset the mode of the distribution was close to zero . Analyses with differing priors on tau or theta confirmed the robustness of this pattern, with the mode of the distribution for tau centered on 1. When the N-SE+B split in Arctic Ground Squirrels was used instead of the E-W split, simultaneous divergence was even more strongly supported . The ratio of expected divergence times was similarly close to zero . While the modes of these distributions are similar, analyses including the N-EE-B split in Arctic Ground Squirrels resulted a slightly longer tail on parameter distributions, encompassing greater variation in the range of Ψ, Ω, and the expected time to divergence.Despite differences in divergence depth between eastern and Beringian phylogroups , hABC anOur results support simultaneous intraspecific divergence between central Beringian (B) and eastern (E) populations. Hoary Marmots and Brown Lemmings contained approximately equal genetic diversity and equivalent expansion signals (or lack thereof in Hoary Marmots) between Beringian and eastern phylogroups (B ≅ E). These results refute a history of recent northern expansion in the eastern lineage  and instH and Tajima’s D in Singing Voles. This isolation in multiple microrefugia—small favorable regions of unglaciated habitat such as easternmost Beringia [Pleistocene population centers within Beringia (Alaska) and northwestern Canada likely differed at fine scales by species and over time , resulting in differential genetic diversity but broadly similar phylogeographic patterns  across all five species). Spatial heterogeneity over time may explain differences in ancestral population size among phylogroups, such as the greater genetic diversity in eastern Arctic Ground Squirrels (E>B), which was not predicted under any of the models . PopulatBeringia  or nunatBeringia —would haBeringia  and ArctBeringia . SuitablBeringia .The hypothesis of simultaneous isolation of Beringian and sub-Laurentide populations and subsequent northward expansion of a small colonizing lineage  receivedθ), the degree of genetic divergence between lineages separated by a single event can be highly variable , the fact that a single simultaneous divergence in all five species was strongly supported may seem surprising. However, given sufficient differences in population size and mutation rate between species . In facble (see . This ovble (see ).E+B split for Arctic Ground Squirrels was used. This may reflect simultaneous divergence between all three groups, or it may instead indicate that many coalescent histories can fit the data. A recent multi-locus study of Arctic Ground Squirrels reached a similar conclusion that several hypotheses provide a partial fit to the observed data [It may seem counterintuitive that hABC analyses returned similarly strong support for simultaneous divergence whether the E-B split or the N-Sved data . Given tved data ), arcticved data ,20). If ved data ,82); thiWhile ancestral areas tend to harbor older genotypes, areas with the greatest genetic diversity tend to be regions of secondary contact between previously disjunct groups . The Wrasensu [The cold, arid, windswept “mammoth-steppe” of Beringia during the Pleistocene  is thougsensu ), then nThe demographic consequences of range shifts from the Pleistocene to the present are influenced by the degree of geographic shift as well as population structure. Small habitat patches at the LGM, especially in easternmost Beringia e.g., , may havMarmota olympus) [Ochotona princeps) [For species that have experienced glacial population expansion and interglacial population contraction, it might be more appropriate to consider their current areas of occupation “interglacial refugia” . While tolympus)  and Amerrinceps)  may presIn contrast, there is evidence of demographic expansion without subsequent population decline in Brown Lemmings . The modDifferences in the intraspecific divergence among the taxa in our study also highlight the role that fecundity, vagility, and population fluctuation play in preserving genetic diversity and increasing effective population size . The twoDespite differences in ecology and demography, broadly similar phylogeographic histories are supported for all species, suggesting that these, and likely other, alpine- and arctic-adapted taxa in the region have been shaped by the same historical phenomena. This includes strong support for contemporaneous population divergences across all five species and support for more recent demographic declines in three species. Such declines may be synergistically accelerating due to ongoing rapid climate change. Thus, the habitat affinities that promoted divergence in these species during past episodes of climate change may be leading to the erosion of genetic diversity as populations are confined to increasingly fragmented and isolated alpine habitats.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Involving dTak1 and JNK, this pathway down-regulates the expression of polycomb group of genes to do away with their repressive role on RD genes. Upon genetic inactivation of members of this newly identified pathway, the canonical Dpp signaling fails to trigger RD gene expression beyond a threshold, critical for ectopic photoreceptor differentiation. Moreover, the drop in ectopic RD gene expression and subsequent reduction in ectopic photoreceptor differentiation resulting from inactivation of dTak1 can be rescued by down-regulating the expression of polycomb group of genes. Our results unravel an otherwise unknown role of morphogen in coordinating simultaneous transcriptional activation and de-repression of target genes implicating its importance in cellular plasticity.Understanding the role of morphogen in activating its target genes, otherwise epigenetically repressed, during change in cell fate specification is a very fascinating yet relatively unexplored domain. Our  Cell fate specification and patterning within a tissue, are achieved by positional information provided by the concentration gradients of a group of signaling molecules termed morphogens357Change in cell fate specification, however, poses a unique challenge to morphogen activity. It creates an exceptional situation wherein a morphogen might need to turn on its target genes that were hitherto kept silent in a cell. This demands that apart from transcriptional activation of target genes, epigenetic repression that renders the genes refractory to morphogen signaling needs to be erased. Understanding the mechanism by which both the things are coordinated represents an important aspect of morphogen activity that needs better insight.Drosophila melanogaster. Imaginal discs are sacs of epithelial cells present in larva that serve as anlagen of adult cuticular structures such as wings, legs, antenna and eye. Studies that involved transplanting imaginal disc fragments into the abdomen of adult female flies revealed that even though these regenerating disc fragments when allowed to metamorphose generally differentiate into their corresponding adult structures10antennapedia in the antenna causes antenna to leg transformationvestigial induces ectopic wing like structures in the eyes, legs and antennaeyeless, eyes absent and twin of eyeless in the developing imaginal discs1516One of the best worked out experimental models for understanding the biology of alteration in cell fate specification has been the larval imaginal discs of eyeless expression has been found to be restricted to the cells of developing wing disc that express higher levels of the morphogens Decapentaplegic (Dpp) and Hedgehog (Hh)Intriguingly, the morphogens are known to play an important role during cell fate alteration in imaginal discs. It has been observed that ectopic expression of the morphogen Wingless (Wg) in leg imaginal discs can lead to leg to wing conversionDrosophila by ectopic eyeless expression1521Drosophila. While removal of individual members of the RD network inhibits eye formation2324151617eyeless (ey), eyes absent (eya), sine oculis (so) and dachshund (dac) transcriptionally regulate each others expression by feed back loops2627282930312732dac along with so and eya are also dependent on Dpp signaling2831dac, so and eya in wing disc cells where they are not normally expressed. This in turn serves as a wonderful platform to investigate the role played by Dpp, if any, in modulating the epigenetic landscape to facilitate ectopic expression of these RD genes during ectopic eye induction.To address this issue, we employed the well-established method of induction of eyes in larval wing imaginal discs of Drosophila. We show that besides transcriptional activation of RD genes by the stereotype canonical signaling, Dpp involves another pathway to expunge the existing epigenetic repression on these genes. This two-tiered mechanism employed by Dpp in regulating the ectopic expression of RD genes reveals an otherwise unknown phenomenon by which a morphogen can elicit both instructive and permissive roles to regulate the expression of its target genes.The outcome of this study unravels the mechanism by which Dpp modulates the expression of its target RD genes during ectopic eye induction in wing discs of eyeless (ey), the most potent inducer of ectopic eye formationDpp-Gal4 and Ser-Gal4 to drive the expression of UAS-ey. In case of Dpp-Gal4; UAS-ey late third instar larval wing discs, differentiation of ectopic photoreceptors, as evidenced by immunostaining with antibodies against ELAV, was detected all along the A/P boundary 3839Drosophila Tak1 (dTak1) in ectopic eye formation, we knocked down the expression of dTak1 (dTak1 mutants (thickveins (tkv) (Mother against dpp (Mad)   or Mothepp (Mad) , the twoof dTak1  without of dTak1 . Quantitof dTak1 . In consof dTak1  and S1m.eyeless. Results of both RT-PCR and qRT-PCR analyses did not reveal any appreciable change in the levels of dTak1 expression  to investigate whether knocking down promised . Furtherpromised  and subspromised  remainedbasket , when co-expressed with eyeless, dramatically reduced the number of ectopic photoreceptors in late third instar larval wing discs   . Likewisn flies) , one of n flies)  and 3e.  eyeless . Howeverntiation  thereby puckered (puc), a direct downstream target of JNK signaling. In contrast to wild type late third instar larval wing discs where no puc-lacZ expression was observed in the pouch region  of proteins4243Independent studies that involved ChiP-qPCR and Dpp-Gal4 larvae  and polycomb (Pc), as observed in late third instar wing discs of Dpp-Gal4; UAS-ey larvae got significantly restored upon attenuating the activity of dFos  or independently knocked down for the expression of ph-p . Even in case of Ser-Gal4, UAS-ey wing discs we observed ectopic photoreceptor differentiation along the A/P boundary specifically in the Dpp expression domain within the dorsal compartment that spans beyond the identified hot spot region. However, the relevance of hot spots become apparent when we analyze the spatial distribution of ectopic photoreceptor differentiation in wing discs upon attenuating the newly identified signaling cascade. The limited number of ectopic photoreceptor differentiation associated with some of these cases indeed trace back to the identified hot spot regions, suggesting that these cells have greater developmental flexibility than other cells of the Dpp expression domain. In this context, it is important to note that as shown in Dpp-Gal4 wing discs also correlate with one of the hot spots identified in the wing disc. Intriguingly, this correlation is not limited to wing discs but can also be seen in leg and haltere discs  following the standard protocol. Details of primary and secondary antibodies used are provided in Fluorescence intensity of expression was quantified in terms of gray value of Dac and Eya expressing area by using Image J. Each experiment was repeated thrice with appropriate controls and at least five images from each experiment were analyzed. Imaging was done with similar parameter settings. pMad activity was quantified in terms of pixel intensities using plot profile function of Image J.We used ImarisX64 and Image J for calculating the area of expressing cells and normalized with respect to total area of disc to account for variability in discs size. The calculated areas in experimental samples were compared to their corresponding controls to determine the ratio. The results shown here are outcome of three independent experiments. Ommatidial number was also calculated by using ImarisX64.dtak1, tkv, dac, kay, eya, so, Pc and php were analyzed by RT-PCR and qRT-PCR on RNA isolated from 50–60 wing discs. In all cases the expression levels were normalized with levels of rp49 expression. Each experiment was repeated thrice with triplicates in each time. List of primers are in Transcript levels of Data are expressed as mean ± standard deviation (SD) of values from at least three independent experiments. Statistical analysis was performed using two-tailed Student’s t-test. P values of <0.01%; <0.001% and <0.0001%, mentioned as *, **, *** respectively were considered as statistically significant.How to cite this article: Aggarwal, P. et al. The morphogen Decapentaplegic employs a two-tier mechanism to activate target retinal determining genes during ectopic eye formation in Drosophila. Sci. Rep.6, 27270; doi: 10.1038/srep27270 (2016).


Multicentric gliomas are well-separated tumors in different locations of the brain, without anatomical continuity between lesions. We report a rare case of multicentric gliomas that occurred in both supra- and infratentorial regions with different histopathology.A 27-year-old man was admitted to our hospital with mild motor weakness of the right leg. Magnetic resonance imaging (MRI) showed a large tumor occupying the left insula, extending to the left basal ganglia, so tumor resection was performed. Histological diagnosis was diffuse astrocytoma. Tumor cells showed sporadic immunoreactivity for p53 and negative immunostaining for epidermal growth factor receptor (EGFR). Postoperative course was uneventful, and adjuvant therapy was not performed. At 7 months after surgery, MRI disclosed a left cerebellar tumor displaying an irregular ring formation on enhancement with gadolinium (Gd) and marked peritumoral edema. MRI studies including T2-weighted imaging demonstrated that this paravermian tumor had no contact with the initial left insular tumor. In addition, MRI studies of the whole neuraxis, cytological examination of the cerebrospinal fluid, and neurological findings demonstrated that no dissemination had occurred through the subarachnoid space or as intracerebral metastases. Therefore, the second surgery was performed. Histological diagnosis was glioblastoma. Immunohistochemistry revealed that most tumor cells were positively stained for both p53 and EGFR but negatively stained for isocitrate dehydrogenase 1 (IDH1).We reported a case of multicentric gliomas occurring in both supra- and infratentorial regions with different histopathology. Immunohistochemical examinations suggest that different genetic pathways may participate in the occurrence of these tumors. Multicentric gliomas are well-recognized entities, but their occurrence is still rare. Among these, gliomas arising from both supra- and infratentorial regions with different histology are very rare –4. We reA 27-year-old man began to experience disorientation and visited his family doctor. Although magnetic resonance imaging (MRI) showed an abnormal mass in the left insula, extending to the left basal ganglia, he was followed without therapy. The tumor gradually increased in size, and he was referred to our department. Neurological examination on admission revealed clear consciousness and no abnormality in cranial nerve functions other than mild weakness of the right leg . MRI on initial admission showed a tumor mass  extending from the left insula to the basal ganglia. The lesion was hypointense on T1-weighted imaging and hyperintense on T2-weighted imaging. The lesion was not enhanced on administration of gadolinium (Gd) Fig. . No otheMRI performed 3 months after surgery did not show any growth of the left insular tumor or any other lesion Fig. . The resThe clinical study of the abovementioned case report was approved by the Ethics Committee for Clinical Research of Ehime University Hospital, and informed consent was obtained from the patient prior to initiating the study.As for the definition of multicentric gliomas, Batzdorf and Malamud described the criteria that multifocal tumors should be distinguished from multicentric tumors . The mulMulticentric gliomas more often affect middle-aged men and show rapid tumor progression, and the prognosis is extremely poor. Many reported cases of multicentric gliomas occur in the supratentorial compartment, and occurrence in both supra- and infratentorial regions is very rare –7. To thThe pathogenesis of multicentric gliomas remains unclear. Many hypotheses have been proposed to explain the multicentricity. Willis et al. suggested that multicentric lesions could result from a two-step process . At firsReis et al. investigated genetic changes to compare primary and secondary glioblastomas and reported that these tumors were independent of each other, with showing a different pattern of genetic mutations . FurtherWe reported a case of multicentric gliomas occurring in the supra- and infratentorial regions with different histopathology. One tumor was diffuse astrocytoma of the left insula, while the other was glioblastoma of the cerebellum. Immunohistochemical analysis suggested that these tumors might have occurred by different pathways of neoplastic transformation in glial cells.EGFR, epidermal growth factor receptor; Gd, gadolinium; IDH1, isocitrate dehydrogenase 1; MRI, magnetic resonance imaging; PTEN, phosphatase and tensin homologue deleted from chromosome 10.


Dear Sir,A 55-day-old male baby underwent an exploratory laparotomy and a radical left nephro-ureterectomy with peri-hilar and para-aortic lymph nodal dissection for a left sided Wilms’ tumor. Postoperative course was smooth until 4thpostoperative day when mild abdominal distension and bilious nasogastric (NG) tube drainage of 95 ml was noticed. An x-ray abdomen was inconclusive while ultrasound (US) abdomen revealed a bowel mass with a target sign at the right pelvic region suggestive of intussusception. A trial of water-soluble contrast enema reduction was given which showed hold up of contrast at the ileum, followed by free passage of contrast into the normal caliber ileum. The intussusception mass was not visualized at subsequent US. The patient’s clinical condition did not improve with NG tube draining 160 ml bilious aspirate following 24 hours and a repeat US showed recurrence of intussusception. It was then decided for laparoscopic exploration. An ileo-ileal intussusception was found which was easily reduced laparoscopically and bowel appeared viable . The postoperative course was uneventful then. The histopathology reported COG Stage I Wilms’ tumor (triphasic) with favorable histology.Surgical procedures involving extensive retroperitoneal dissection and bowel manipulation cause impairment of bowel innervation and peristalsis, as in resection of Wilms’ tumor, neuroblastoma, pull-through for Hirschsprung’s disease, Ladd’s procedure, diaphragmatic hernia repair and fundoplication are reported to have a high incidence of postoperative intussusception (POI).[1-3]The classical triad of intussusception is usually not seen and in the presence of expectant postoperative ileus, the diagnosis can be easily overlooked which can increase morbidity and mortality.[3] Bilious vomiting, prolonged and excessive NG bilious aspirate and abdominal distension are usually the common findings reported. A similar presentation was seen in our case. When a smooth postoperative course is interrupted by deterioration in clinical symptoms and signs, POI should be suspected. In a case of atypical postoperative ileus, radiologic investigations as US should not be delayed; it is highly accurate, supportive diagnostic tool in cases of POI as in primary intussusception.Reduction of intussusception is the mainstay of treatment. In failed non-operative reduction of intussusception, laparoscopy is a safe and effective operative.(5) In the index case, same approach was used. In case of recurrence, re-laparoscopy can be performed for reduction of intussusception rather than laparotomy.Source of Support: NilConflict of Interest: None declared


Traumatic diaphragmatic rupture remains a diagnostic challenge for both radiologists and surgeons. In recent years, multidetector CT has markedly improved the diagnosis of diaphragmatic injury in polytrauma patients. Herein, we describe two cases of subacute presentation of traumatic diaphragmatic rupture from a penetrating rib fracture and subsequent intrathoracic herniation of omental fat, representing the CT “funky fat” sign. Traumatic diaphragmatic rupture (TDR) may result from a penetrating injury or blunt thoracoabdominal trauma and results in communication between the pleural and peritoneal cavities –3. A penThe fractured rib sign was first described by Holland and Quint  and lateNumerous signs indicating TDR have been described in the literature . We presA 59-year-old helmeted man was thrown 20 feet during a motorcycle collision. Because of multiple comminuted left rib fractures with subcutaneous gas on chest radiograph, a left chest tube was placed. Contrast-enhanced trauma MDCT revealed sternal and scapular fractures, left lung contusion, and a small left pneumothorax. In addition, multiple fractures of the 3rd through 10th left ribs were displacement into the chest cavity, with associated chest wall instability. While the 10th-rib fracture abutted the left diaphragm, the diaphragm appeared intact Figures . The patA 64-year-old male restrained driver suffered blunt chest and abdominal trauma following a motor vehicle collision. He sustained a large left lung contusion, tiny left pneumothorax, fractures of the pelvis and lumbar transverse processes, and multiple comminuted ribs fractures. He did not require intubation. Contrast-enhanced trauma MDCT better depicted multiple displaced compound fractures of the left 3rd through the 11th ribs in close proximity to the diaphragm, but the diaphragm appeared intact Figures . In addiA repeat MDCT was performed four days later to evaluate an enlarging left pleural effusion. CT depicted new herniation of omental fat into the left chest, compatible with evolving TDR Figures . SerosanUp to 8% of patients with severe blunt thoracoabdominal trauma develop a traumatic diaphragmatic injury . TDR is It has been reported that the diagnosis of TDR may be missed initially in up to 30% of cases on MDCT , 8. In sSpontaneous closure of TDR has never been reported, and almost all cases should be surgically repaired . Early dIn all cases with significant blunt thoracoabdominal trauma, thorough evaluation is required to exclude TDR, and follow-up evaluation should be considered to assess potential delayed development of TDR . In diffFarboud et al. reported a 77-year-old man who sustained fractures of the left 3rd to 10th ribs and a left diaphragmatic hernia after falling 8 feet from a ladder. Urgent left thoracotomy revealed that the sharp edge of the displaced left 7th-rib fracture had pierced the diaphragm, and omentum had herniated into the chest through the diaphragmatic defect. Following resection of the rib fragment, the omentum was reduced, the diaphragmatic defect was repaired, and the patient recovered uneventfully .In our cases, both patients had the subacute diagnosis of a small TDR from a penetrating rib injury, with herniation of omental fat within two weeks following trauma. Both had the fractured rib sign on initial CT, with rib fracture fragments protruding into the chest cavity in close proximity to the diaphragm. In this setting, radiologists should have a high index of suspicion for TDR. Intrathoracic omental fat, the CT “funky fat” sign, should also alert the radiologist to search for TDR and may be a delayed finding. In our cases, early diagnosis of TDR likely prevented strangulation of omentum and subsequent development of a large hernia sac containing abdominal visceral organ.


The incidence of stroke has risen over the past decade and will continue to be one of the leading causes of death and disability worldwide. While a large portion of immediate death following stroke is due to cerebral infarction and neurological complications, the most common medical complication in stroke patients is infection. In fact, infections, such as pneumonia and urinary tract infections, greatly worsen the clinical outcome of stroke patients. Recent evidence suggests that the disrupted interplay between the central nervous system and immune system contributes to the development of infection after stroke. The suppression of systemic immunity by the nervous system is thought to protect the brain from further inflammatory insult, yet this comes at the cost of increased susceptibility to infection after stroke. To improve patient outcome, there have been attempts to lessen the stroke-associated bacterial burden through the prophylactic use of broad-spectrum antibiotics. However, preventative antibiotic treatments have been unsuccessful, and therefore have been discouraged. Additionally, with the ever-rising obstacle of antibiotic-resistance, future therapeutic options to reverse immune impairment after stroke by augmentation of host immunity may be a viable alternative option. However, cautionary steps are required to ensure that collateral ischemic damage caused by cerebral inflammation remains minimal. Stroke is a devastating cerebrovascular event that occurs upon interruption of blood flow to areas of the brain, due to blockage (ischemic stroke) or bursting/bleeding (hemorrhagic stroke) of a cerebral blood vessel. This resulting brain damage contributes to long-lasting disabilities and multiple functional impairments. In 2010, the global prevalence of stroke was 33 million, with an incidence rate of 16.9 million ,5. In orStroke is an ischemia/reperfusion injury that is caused when the blood supply to the brain is insufficient. During occlusion, deprivation of oxygen and glucose can induce immediate, localized neuronal cell death at the ischemic core via excitotoxic activity . NormallSubsequent release of damage associated molecular patterns (DAMPS) by dying cells activates resident microglial cells through stimulation of pathogen recognition receptors (PRRs), such as toll-like receptor (TLR) 2 and TLR 4 ,10,11. TUpon resolution of the vascular blockage, re-oxygenation of the brain promotes mitochondrial-mediated production of ROS to cause further injury to cellular components and promote cell death in surrounding neurons, glial cells and blood vessels . The proVarious immune cells have different roles in infarct development after stroke. Macrophages and neutrophils have been found to infiltrate within 24 h following reperfusion . MacrophNeutrophils are also known to rapidly infiltrate ischemic tissue within hours after stroke. While the role of neutrophils is controversial, trends in literature suggest that neutrophils contribute more toward detrimental outcomes in the brain following stroke . PreventDTR (diphtheria toxin receptor) mice did not alter ischemic lesion volume 3 days after stroke to argue against the neuroprotective role of Tregs [Influx of lymphocytes usually occurs after 2–3 days following stroke. Most notably, T cells have been found to contribute to infarct development through production of IL-1β, interferon gamma (IFN-γ) and macrophage inflammatory protein (MIP)-2 ,35. Miceof Tregs . Confounof Tregs . In factof Tregs . As the of Tregs ,29,33. Hof Tregs . In contof Tregs . Natalizof Tregs . The effof Tregs  or benigof Tregs  effect oof Tregs . Similarof Tregs . Promisiof Tregs ,48 and cof Tregs .The excess infiltration of leukocytes can be largely explained by increased permeability of the blood brain barrier (BBB) . NormallThere is no doubt that sequences of highly inflammatory events add to cerebral insult after stroke and leads to detrimental outcomes, however, despite all the neurological damage that occurs after stroke, a major clinical complication that stroke survivors encounter is infection. It has been reported that between 23% and 65% of patients acquire infection after stroke, with pneumonia and urinary tract infections (UTI) being most common ,57. InfeDiagnosis of infection after stroke can be challenging as the criteria of diagnosis are inconsistent between studies. Hallmark signs of infection in clinics, such as fever and inflammation, can be presented by patients as a consequence of neurological damage that disrupts homeostatic regulation of body temperature . On the Identifying whether patients will succumb to infection could allow for early interventions to reduce post-stroke bacterial burden and improve clinical outcome, however it can be difficult to predict. One study identified a range of independent risk factors that could predict pneumonia after stroke with 76% sensitivity and 88% specificity . These fAlthough initial systemic inflammation, characterized by circulating IL-6 and IFN-γ production, may peak within 6 h following stroke , it has Monocytes and macrophages are phagocytes that can engulf pathogens and infected cells to alert and prime the rest of the immune system . SplenicAnother indicator of immune suppression is the macroscopic shrinking of immune organs, shown to occur within 12 h following stroke in mice . Most noH1) type response to a humoral-mediated anti-inflammatory T-helper 2 (TH2) type response is suggested to occur to protect the brain from further inflammatory damage and promote tissue repair and neuronal regeneration [H2-associated cytokines mediates the immune shift away from a TH1 response and toward a TH2 response after stroke. In particular, IL-10 secretion by monocytes, dendritic cells, and Tregs is greatly increased after stroke in both mice and humans [H1 cells, proliferation of T cells and cytokine responses, TNF-α production by macrophages and cytokine production by monocytes are all inhibited by IL-10 to further emphasize its role in the immune shift toward a TH2 response and immune suppression after stroke [H1 cytokines, were seen 12 h after stroke in mice [Adaptive immune changes can largely predispose to chronic susceptibility to infection . Particuneration ,89. Howed humans ,90, and r stroke . Decreas in mice . In fact in mice . Although immune suppression is apparent following stroke, the mechanism in which this occurs is not well understood. One explanation is that an early, sustained inflammatory response may exhaust the immune system and ultimately lead to immune suppression ,92. AnotCatecholamines signaling through β-adrenergic receptors on immune cells have been shown to reduce TNF-α while promoting IL-10 production. Stimulation of β-adrenergic receptors can also inhibit cytotoxic T-lymphocyte-associated protein (CTLA)-4 expression on T cells . On the Streptococcus pneumoniae was necessary to cause pneumonia and bacteremia in post-stroke mice, whereas sham-surgery mice needed 1000-fold greater amount of bacteria for a similar severity of pneumonia [Pneumonia and UTI’s are the main types of infection after stroke. Pneumonia is seen to be the most common with an incidence rate of up to 57% , while Uneumonia . In a clneumonia . Nonetheneumonia . Howeverneumonia . Stroke-Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae and Psuedomonas aeruginosa [Escherichia coli and Enterobacter cloacae being two of the most frequently detected [Escherichia coli [While infections are common after stroke, the causative agent remains elusive and may be contributed by a number of pathogens. Types of bacteria that have been commonly found in the sputum and urine of stroke patients include ruginosa . Surprisdetected . Indeed,hia coli . Howeverhia coli ,107,111.hia coli . OverallH1-mediated immune response toward MBP [It may appear that we have evolved to react to stroke injury with an apparently maladaptive response (post-stroke infection). During stroke, self-epitopes, which are normally shielded from the systemic immune system by a number of mechanisms, may become exposed to adaptive immunity. This may ‘educate’ the immune system to react to self-antigens in the CNS, and, ultimately, lead to autoimmunity or autoaggression. According to this concept, by globally down-regulating innate and adaptive immunity, stroke-induced immune suppression may help to prevent post-injury autoimmunity. Indeed, brain-specific antigens can be measured in the blood plasma after stroke . At presward MBP  and incrward MBP . Additioward MBP . This maThe obvious strategy and current “gold-standard” treatment to combat infections is the use of antibiotics upon diagnosis of infection . Howevern = 66), it was found that prophylactic treatment with moxifloxacin reduced infections by 24.8% (p = 0.032), though this treatment did not seem to be beneficial using with intention-to-treat (ITT) analysis , which included all recruited patients. ITT analysis may be more viable to evaluate the effectiveness of this treatment as excluding some patients for PP analysis may dramatically impact results due to the small group size in this study (n = 31 in placebo group and n = 35 in treatment group). Overall, the main finding in the PANTHERIS study was that patients did not have improved clinical outcomes or reduced mortality after 6 months despite reduced infection rates and thus discourages prophylactic use of moxifloxacin as a preferential treatment over current treatment guidelines to reduce post-stroke infection.A follow-up study with moxifloxacin, a fourth generation fluoroquinolone, was conducted in the Preventive Antibiotic Therapy in Ischemic Stroke (PANTHERIS) clinical study . With peSimilar results were found for the most recent studies that evaluated the effectiveness of PAT. The preventive antibiotics in stroke study (PASS) used ceftriaxone, a third generation cephalosporin, to determine whether treatment could reduce infection to improve functional outcomes after stroke . PreviouIn the STROKE-INF study, prophylactic use of clarithromycin in combination with either amoxicillin or co-amoxiclav was given within 48 h after stroke for 7 days in dysphagic patients where occurrence of pneumonia and patient functional outcomes at 90 days was assessed ,125. IntPsuedomonas aeruginosa (23.92%), Staphylococcus aureus (15.32%) and Escherichia coli (14.25%). The study found that drug resistance against moxifloxacin, used in the PANTHERIS trial [S. aureus [Escherichia coli and Psuedomonas aeruginosa [Lastly, another possible reason that antibiotic treatment is not effective is the unsuitable use of particular antibiotics due to antibiotic resistant bacteria. One study took sputum samples from patients with stroke-associated pneumonia, cultured for bacteria and tested their drug sensitivity against a range common antibiotics including penicillin, tetracycline, ceftriaxone and moxifloxacin . The preIS trial , was 52.. aureus . Strikin. aureus , was 100ruginosa  which maruginosa . HoweverH2 response after stroke to leave the host susceptible to infections. Targeting these pathways to prevent the immune shift following stroke may offer a viable alternative to antibiotics to reduce infections. Catecholamines are produced as a result of cerebral ischemia and can act upon β-adrenoreceptors on immune cells to induce apoptosis and immune suppressive pathways. Therapeutic blocking of the β-adrenergic receptor after stroke has been shown to reduce infections in experimental models of stroke by counteracting the immunosuppressive properties of catecholamines [The impact that stroke has on the immune system is complex and, as discussed, innervation of the sympathetic nervous system and HPA axis can result in an immune shift towards a Tolamines ,97. Introlamines . A recenolamines . In contolamines . Taken tWe recently showed that the stroke impairs iNKT cell function within hours after stroke to mediate immune suppressive pathways and result in increased host susceptibility to infections in an experimental setting . InteresAdditional studies in the mechanisms of brain-immune system axis are required to reveal potential alternative therapeutic options. However, cautionary steps should be taken with immunomodulatory treatments. Suppression of the immune system after stroke is thought to be a brain-protective mechanism as the majority of penumbral damage is governed by inflammatory events ,89. TherDamage to the brain after stroke is now known to be a largely immune mediated event. As a compensatory response to the highly inflamed environment within the skull, the activation of the sympathetic nervous system and hypothalamic-pituitary-adrenal axis results in the production immune suppressive molecules including catecholamines and glucocorticoids, respectively. The brain-immunity axis is a recently identified and complex pathway that induces immune suppression after stroke. This is believed to be a defensive mechanism of the brain with an attempt to lessen further cerebral damage, however rendering the host more susceptible to infections. Infections such as pneumonia and UTI are extremely common in stroke patients and have been associated with poorer functional outcomes and increased mortality. Therefore, a recent focus has been to reduce infection to improve patient health. Much attention has been drawn to preventive antibiotic treatment, though the majority of clinical studies have swayed against prophylactic antibiotic administration due to a failure in reducing pneumonia and improving clinical outcomes. Importantly, recent studies reveal an underlying issue in the inconsistency of pneumonia diagnosis across clinics. Additionally, with the growing emergence of antibiotic-resistant pathogens, alternative therapies to reduce infection are required. Immunomodulation therapies that augment the immune system to combat infections is an attractive avenue, however care is needed to ensure further cerebral injury is avoided. Future studies may want to focus on the precise mechanisms of immune suppression after stroke in order to unveil alternative therapeutic targets to reduce stroke-associated infections.


Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic syndrome. The hallmark of the disease is multiple hamartomatous lesions in multiple organ systems. Common cardiac manifestations of TSC are rhabdomyomas, which are a benign tumor of striated muscle. In some patients with TSC, myocardial fatty foci (MFF) deposition has been described with or without the presence of rhabdomyomas.We present the case of a 24-year-old female with TSC and refractory seizures, who was evaluated with cardiac magnetic resonance (CMR) for an intracardiac right ventricular mass thought to be rhabdomyoma on echocardiography and for multiple areas of myocardial fatty deposition. Myocardial fatty deposition is a common finding in patients at cardiac imaging. In patients with TSC, it is critical that fatty deposits and lipomas are clearly distinguished from rhabdomyoma. CMR is an integral part of characterizing cardiac masses as it has superior soft tissue characterization and a wider field of view compared to echocardiography. A positive correlation has been shown between the number of MFF and the degree of extracardiac tuberous sclerosis (TS) manifestations suggesting that MFF may indicate more severe multiorgan disease in patients with TSC.Cardiac MR is superior to echocardiogram in evaluating and distinguishing intracardiac lipomas and fatty deposits from rhabdomyomas. Published studies have indicated that in patients with TSC, the presence of MFF correlates with the severity of multiorgan disease as was seen in our case. Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic syndrome of variable phenotypes arising due to mutation of cellular proliferation genes TSC1 or TSC2. This leads to deregulation of cellular proliferation protein mammalian target of rapamycin (mTOR), resulting in cellular hyperproliferation -4. A limA 24-year-old female presented to our hospital with refractory seizures. The patient had been diagnosed with TSC at the age of seven months, outside of the United States. Manifestations of the disease in this patient included bilateral renal angiomyolipomas treated with embolization, lymphangioleiomyomatosis (LAM), and subependymal giant cell astrocytoma (SEGA).Screening transthoracic echocardiography (TTE)  demonstrated normal global left ventricular wall motion, contractility, and ejection fraction. An echogenic mass, initially thought to be a rhabdomyoma, was noted within the right ventricle (RV) at the mid septal region Figure . There wA multiplanar contrast-enhanced CMR demonstrated normal cardiac anatomy. At least 10 definitive areas of MFF were identified within the midwall of the interventricular septum, lateral wall, moderator band, and septal leaflet attachment site. The largest midwall deposit was in the lateral wall measuring 8 x 5 mm Figure . The MFFMyocardial fatty deposition is a common finding at cardiac imaging, especially in patients of advanced age. Pathologic conditions with myocardial fat deposition include physiologic fat, healed myocardial infarction (MI), arrhythmogenic right ventricular cardiomyopathy (ARVC), dilated cardiomyopathy, and muscular dystrophy. Distinction of these different entities can be made using the patient’s age, clinical history, ventricular size and function, myocardial thickness, location of fat deposition, and regional wall motion abnormalities . In patiCMR imaging is an integral part of characterizing cardiac masses. CMR has superior soft tissue characterization and a wider field of view compared to echocardiography. Additional anatomic pre-surgical information such as mass mobility, valvular involvement, and possible myocardial and extracardiac extension can be obtained using CMR. Contrast-enhanced CMR imaging is also useful in defining the margins of the tumor, characterizing the mass, and distinguishing from cardiac thrombus.Rhabdomyomas on CMR are usually isointense to myocardium on T1-weighted images although they can be slightly hyperintense. They are hyperintense on T2-weighted images and demonstrate hypoenhancement relative to myocardium on contrast-enhanced images . In contA positive correlation has been shown between the number of MFF and the degree of extracardiac TS manifestations. This suggests that the presence of MFF may indicate more severe multiorgan disease in patients with TSC . Our patWe report a case of myocardial fatty foci and subendocardial lipoma diagnosed using cardiac magnetic resonance imaging in a patient with tuberous sclerosis complex. Similar to previous reports of myocardial fatty foci, our patient had multiple intracardiac fatty lesions in addition to significant multiorgan disease related to tuberous sclerosis complex. We demonstrate that, although difficult to definitively characterize on echocardiography, myocardial fatty foci and intracardiac lipoma can be easily distinguished from rhabdomyoma using cardiac magnetic resonance imaging.


A report on the Plant and Animal Genome XXII meeting held in San Diego, California, USA, January 11–15, 2014. The Plant and Animal Genome XXII meeting was held in the warm and sunny coastal city of San Diego, California, on 11–15 January this year, during a time when many other US cities were plagued with snowstorms and extreme cold weather. More than 2,800 attendees from 62 countries joined the meeting. There were eight plenary talks and many workshops and posters covering topics ranging from genomics and functional genomics to epigenomics and systems biology. Tremendous progress in our understanding of the structure, evolution and regulation of plant and animal genomes has been made in recent years. Revolutionary high-throughput sequencing techniques have enabled complete sequencing of many large and complex genomes containing extensive transposable elements (TEs) and repeats, and have also helped in the identification of many hidden regulators, such as small RNAs, especially in non-model species. Here, I highlight some of the exciting presentations on epigenomics and small RNAs.Whole genome sequencing analysis has revealed that TEs constitute more than half of the genomes of many animals and crop plants. For example, more than 80 % of the maize genome and 60 % of our human genome is composed of TEs. Many studies and presentations support the notion that proliferation of TEs is one of the major driving forces for genome expansion. Nathan Springer  described the complex interplay between TEs and genes within the maize genome. He showed that although DNA methylation and a repressive histone methylation marker, H3K9me2, are present in almost all TE regions in maize, DNA methylation patterns and their ability to spread to surrounding regions vary among different transposon families. It was also shown that many genes with TEs in their introns are still actively transcribed, suggesting that maize transcription machinery can tolerate blocks of heterochromatin.de novo DNA methylation and histone modifications. Nathaniel Street  presented the genome assembly and small RNA profiles of Norway spruce (Picea abies), a tree with a similar number of genes as the model plant Arabidopsis, but which has a genome 100 times larger and with many more TEs. The large expansion of the genome is due to the slow and steady accumulation of a diverse set of long terminal repeat (LTR) retrotransposons. The 24-nucleotide class of siRNAs is much less abundant in gymnosperms than in angiosperms, which may partially explain the mass proliferation of TEs in gymnosperms. By comparative analysis with related species, it was found that the distribution of small RNA classes was highly diverse and tissue-specific. In animals, PIWI proteins and PIWI-interacting small RNAs (piRNAs) are key regulators in suppressing TE proliferation in germ cells. Michael Vandewege  discussed the interplay between piRNAs and TEs among Laurasiatherian mammals. Small RNAs from the testes of dog, cat, vesper bat and horse were profiled and it was shown that piRNAs play an essential role in suppressing the expression of active retrotransposons in mature testes in dog, cat and horse, whereas the DNA transposons in the vesper bat, which lacks a RNA intermediate, are not targeted by piRNAs. This demonstrates the complex dynamics between TEs and genomic defenses in mammalian genomes.Although TE transposition and replication are beneficial for adaptive evolution in many eukaryotic organisms, uncontrolled transposition activities can be detrimental and even fatal. Therefore, TEs are usually under tight epigenetic control via DNA methylation or histone modifications. In plants, the 24-nucleotide heterochromatic small interfering RNAs (siRNAs) are mainly responsible for silencing TEs by guiding mPing, in rice. In a few rice landraces, mPing has been amplified to more than 750 copies during recent domestication. She further illustrated that one strategy for successful rapid amplification is that mPing has a preference for inserting into AT-rich regions and thus minimizes exon insertions because rice protein coding regions tend to be GC-rich. This study has provided an excellent example that links TEs with genome restructuring and regulation.Despite robust genome integrity maintenance, certain TEs can still circumvent the epigenetic surveillance and amplify rapidly in the genome without killing its host or being silenced. Sue Wessler  gave a plenary talk on one such successful TE - a miniature inverted repeat transposable element (MITE), FLOWERING LOCUS C (FLC) locus, the antisense transcripts COOLAIR are alternatively spliced and polyadenylated, which recruit distinct chromatin-modifying complexes for different FLC chromatin states. FLC is one of the best-studied gene loci that are under precise control via multiple layers of regulation at the epigenetic level.Caroline Dean  gave a plenary talk on co-transcriptional coupling mechanisms that link chromatin dynamics with antisense transcripts for quantitative gene regulation. At the flowering repressor Arabidopsis, soybean and Medicago truncatula) and monocots , and discovered a novel class of 24-nucleotide phasiRNAs from monocots that are generated by a monocot-specific Dicer-like (DCL) protein. This class of phasiRNAs, emerging with germinal cell maturation and meiosis, shares characteristic features with animal piRNAs. Doris Wagner  introduced the function of ATP-dependent SWI/SNF chromatin remodeling proteins, SPLAYED and BRAHMA, during lateral organ formation in Arabidopsis. Meanwhile, the animal epigenetics workshop covered topics ranging from genomic imprinting and transcriptome analysis to public resources, such as the livestock epigenetics database (epiDB).The plant and animal epigenome workshops were held separately during the conference. Rob Martienssen  and Eric Lyons  introduced the Epigenomics of Plants International Consortium (EPIC), a joint effort to support the community working on plant epigenomics and epigenetics. In plants, epigenetic control is associated with the RNA-directed DNA methylation (RdDM) pathway. Plant-specific RNA polymerase IV (Pol IV) is a central player that initiates siRNA biogenesis in the RdDM pathway. Julie Law  presented work on the characterization of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a protein that targets Pol IV to chromatin. She found that SHH1 was able to recognize both H3K9 methylation and an unmodified H3K4 residue, which connects DNA methylation and H3K9 methylation within the context of the RdDM pathway. Blake Meyers  discussed phased secondary siRNAs (phasiRNAs) in plants. He compared phasiRNAs and their generation loci between dicots  have organized informative ‘Small RNA’ workshops at this meeting for the last eight years. This year, six speakers from both animal and plant fields presented their findings. In addition, small RNAs have become an indispensable part of genomics studies and appeared in many genomics and epigenomics presentations.Small RNAs are short non-coding regulatory RNAs that direct gene silencing in a sequence-dependent manner. It has been 20 years since the first microRNA (miRNA) was discovered in let-7, in cardiovascular differentiation using whole genome miRNA screening, revealing a remarkable conservation of function from amphibians to mammals. Robert Sullivan  reported findings on extracellular microvesicle-associated miRNAs in the male reproductive tract. Distinct miRNA repertories were found in the epididymal intraluminal compartment that are likely to be involved in intercellular communication through the epididymis via extracellular microvesicles. Beth Thompson  introduced the function of DCL1 in maize development. Pamela Green gave a talk on the genome-wide analysis of Brachypodium miRNAs in the Brachypodium Genomics workshop. Conserved and novel miRNAs were identified and their potential targets were predicted and confirmed by parallel analysis of RNA ends.Several talks addressed the function of small RNAs in various cellular processes. For example, Alexandre Colas  presented findings on the function of several miRNAs, including Botrytis cinerea, can be delivered into host plant cells to silence host immunity genes by hijacking host RNA interference (RNAi) machinery to facilitate its infection. This finding represents a naturally occurring cross-kingdom RNAi, which serves as an advanced virulence mechanism for aggressive eukaryotic pathogens. Barbara Baker  described analyses of several plant endogenous small RNAs that regulate host immunity. The tobacco N gene that confers resistance to tobacco mosaic virus is regulated by both promoter-targeted transposon MITE-associated siRNAs and coding-region-targeted miRNAs. They also identified a new class of miRNAs derived from long inverted repeats, one of which targets Cf9, a tomato gene that confers resistance to fungal pathogen Cladosporium fulvum.In addition to their role in animal and plant development, small RNAs also play essential roles in regulating host-pathogen interactions. I  presented our work on pathogen small RNAs that regulate host immunity in the ‘Small RNAs’ and ‘Host-Microbe Interactions’ workshops. A group of small RNAs from an aggressive fungal pathogen, cis-regulatory feature through small RNA processing machinery in Arabidopsis. By performing RNA-seq on double-stranded RNA and single-stranded RNA pools, a negative correlation between mRNA abundance and structure complexity was identified. Thus, in addition to long double-stranded RNAs or miRNA precursors, highly structured mRNAs can also potentially serve as substrates of DCL proteins and give rise to small RNAs with a wide size range, unveiling a new regulatory function of DCLs on highly structured mRNAs.Brian Gregory  showed that the specific secondary structure of mRNAs can act as a potent As the premier and largest ag-genomics meeting in the world, this year’s Plant and Animal Genome Conference was a great success, and there are many more exciting and stimulating talks that I unfortunately do not have space to discuss here. Even if you were overwhelmed by the wealth of information presented at the meeting, you would have certainly been inspired. It was very satisfying to see that epigenetics and small RNAs have become an important part of many genomics studies, especially in complex non-model systems, which indicates their essential roles in regulating genes and genomes.DCL: Dicer-like; miRNA: microRNA; MITE: Miniature inverted repeat transposable element; phasiRNA: phased secondary siRNA; piRNA: PIWI-interacting small RNA; Pol IV: Polymerase IV; RdDM: RNA-directed DNA methylation; RNAi: RNA interference; SHH1: SAWADEE HOMEODOMAIN HOMOLOG 1; siRNA: small interfering RNA; TE: Transposable element.The author declares that she has no competing interests.


Different strategies of neurostimulation have been developed as treatment tools for migraine. Among them, vagus nerve stimulation (VNS) can be performed both invasively and non-invasively. Recently, “Gammacore” has been approved as a non-pharmacological and non-invasive tool for headache, and a recent study demonstrated its efficacy in 22% of patients with acute migraine attacks.Although the pathophysiology of migraine is not yet fully understood, many studies have shown a role of sterile inflammation of cerebral vessels and of the change in diameter of the intracranial arteries. Blood flow velocities and vasomotor reactivity (VMR) in patients suffering from migraine without aura in the intercritical phase were found either increased or normal compared to non-migraineurs healthy controls, 3. SincWe enrolled 20 patients aged between 18 and 65 years, suffering from chronic migraine and 20 healthy non-migraineur subjects matched for demographic characteristics. None of them assumed vaso-reactive drugs for at least 30 days before registration.Subjects enrolled underwent registration of blood flow velocity in the middle cerebral artery bilaterally through transcranial Doppler (TCD), in the morning and fasting from food and caffeine. The monitoring was performed at baseline, during and after apnea lasting 30 seconds. After the recording, the subjects underwent external stimulation of the vagus nerve with “Gammacore” device for 90 seconds, and 20 minutes after we once again registered VMR through apnea test. VMR was calculated by means of Breath holding index according to the following formula: VMR/apnNo statistically significant differences emerged comparing VMR before and after VNS in our population, irrespective of groups. No patient suffered from adverse event during or after VNS.Non-invasive VNS resulted safe and did not seem to influence VMR, neither in migraineurs nor in healthy volunteers. However, the small sample of our study population does not allow to draw definitive conclusions, hence the study will be further continued to extend sample size.Written informed consent to publication was obtained from the patient(s).