Infections related to stroke-induced immunodepression are an important complication causing a high rate of death in patients. Several experimental studies in mouse stroke models have described this process but it has never been tested in other species such as rats.Our study focused on the appearance of secondary systemic and pulmonary infections in ischemic rats, comparing with sham and naive animals. For that purpose, male Wistar rats were subjected to embolic middle cerebral artery occlusion (eMCAO) or to transient MCAO (tMCAO) inserting a nylon filament. Forty-eight hours after ischemia, blood and lung samples were evaluated.In eMCAO set, ischemic rats showed a significant decrease in blood-peripheral lymphocytes  together with an increase in polymorphonuclears (PMNs) , while no change in monocytes was observed. The increase in PMNs counts was positively correlated with worse neurological outcome 48 hours after eMCAO . However, sham animals showed similar changes in peripheral leukocytes as those seen in ischemic rats . Analysis of bacteriological lung growth showed clear differences between naive  and both sham  and ischemic  groups. Additionally, naive animals presented non-pathological lung histology, while both sham and ischemic showed congestion, edema or hemorrhage. Concordant results were found in the second set of animals submitted to a tMCAO.Inflammatory and infection changes in Wistar rats subjected to MCAO models may be attributed not only to the brain ischemic injury but to the surgical aggression and/or anaesthetic stress. Consequently, we suggest that stroke-induced immunodepression in ischemic experimental models should be interpreted with caution in further experimental and translational studies, at least in rat stroke models that entail cervicotomy and cranial trepanation. Stroke not only produces local brain injury but also causes a systemic inflammatory response followed by an immunodepression process which predisposes patients to infections. This immunodepression state is thought to be induced through a sympathetic nervous system over-action which contributes to an increase of catecholamines and glucocortocoids Streptococcus peumoniae is intranasally applied, only mice with stroke shift to severe infection Experimental studies with different animal models have been crucial in understanding several aspects related to the physiopathology of the infection during the acute phase of stroke. Some studies in mouse models of cerebral ischemia (suture MCAO model) have demonstrated that nervous system damage results in spontaneous bacterial infections because of a deterioration of cell-mediated immune responses All procedures were approved by the Animal Ethics Committee of the Vall d'Hebron Research Institute (02/09 and 58/13 CEEA) and were conducted in compliance with the Spanish legislation and in accordance with the Directives of the European Union. In all experiments, male Wistar rats  were used. Rats were kept in a climate-controlled environment on a 12-hour light/12-hour dark cycle. Food and water were available ad libitum during all experimental process and all efforts were made to minimize suffering.Infarction in the territory of the middle cerebral artery (MCA) was induced by embolic occlusion (embolic MCAO) as previously described A total of 59 rats were used for this study. Nine of them were naive rats, 13 were sham and 37 were submitted to embolic MCAO. Eleven of the ischemic animals were excluded after applying the following criteria: inappropriate occlusion of the MCA after embolization (n = 6); spontaneous reperfusion within the next ten minutes after occlusion (n = 3) or sudden death during the surgery process (n = 2). Blood samples were drawn through transcardiac puncture 48 hours after embolic MCAO or after sham surgery. Afterwards, rats were transcardially perfused with sterile PBS and lungs were carefully removed in aseptic conditions for posterior evaluation. From the total of 26 rats submitted to embolic MCAO and included in the study, 10 of them died before the experimental protocol finished (eight during the first 24 hours and two of them between 24 and 48 hours after the occlusion). No animals died in the sham group.In order to ratify our findings, experiments were repeated in a new set of rats submitted to a transient MCAO model using an intraluminal filament as described previously From the total of 10 rats submitted to tMCAO, two of them were excluded due to bad reperfusion and another one died before the experimental protocol finished. No animals died in the sham group.Infarct volume was measured using 2,3,5- triphenyltetrazolium chloride  staining as described Rats were assessed using a 9-point neurological deficit scale, as previously described + and GrM-PE+; monocytes CD11b-FITC+ and GrM-PE- and lymphocytes CD11b-FITC- and GrM-PE-. Data were analyzed with FCS Express version 3  and expressed as percentage of total leukocytes.Blood-leukocyte populations  and lymphocytes) were evaluated by flow cytometry technique. With this aim, 500 µl of blood were incubated with Ammonium-chlorid-kaliumhydrogencarbonat (ACK) buffer  to lysate erythrocytes and later the pellet was rinsed with Hanks' balanced salt solution - Hepes . The same process (lysis-cleanliness-centrifugation) was repeated thrice. Cells were resuspended in 100 µl of FACS buffer, blocked with 0.25 µg purified mouse anti-Rat CD32  on ice for 10 minutes and stained with 0.4 µg anti-Granulocyte Marker-PE (GrM-PE) (eBioscience) and 1 µg anti-Rat CD11b- FITC (eBioscience) antibodies for 20 minutes on ice. A specific antibody to detect cell viability, To-Pro-3 (eBioscience) was used to assess samples preservation. A total of ten-thousand events per sample were acquired using FacScalibur . To discriminate leukocyte types, the following populations were defined: PMNs were CD11b-FITCLungs were carefully removed and separated into right and left lung under sterile conditions. Left lung was immersed in formaldehyde 4%  for 48 hours, embedded in paraffin wax and sectioned with a microtome in 4 µm thick sections. Hematoxilyn & Eosin (HE) stain was carried out to perform lung tissue histological evaluation by an investigator blinded for study group.Lung imprints were performed pressing 4 times on blood-agar plates' surface with a piece of sterile right lung. Blood was withdrawn by cardiac puncture in sterile conditions and lungs were collected, minced and homogenized also under sterile conditions. One-hundred µL of all specimens were serially diluted in sterile PBS up to 200 µL of final solution, which was plated onto blood agar plates . After 24 and 48 hours of incubation at 37°C, agar plates were analyzed for growth colonies. Results of lung imprints were expressed as the number of observed colonies. Results of both blood and lung homogenate were expressed as a logarithmic function (log10).For the measurement of blood CXCL-1 cytokine, a simple ELISA kit was purchased from R&D systems and used according to the manufacturer's protocol.p value<0.05 was considered statistically significant at a 95% confidence level.Data were analyzed using GraphPad Prism_v5 software. Statistical significance for intergroup differences was tested by Student's t-test and ANOVA followed by Bonferroni post hoc test for parametric data. For non-parametric data, Mann Whitney and Kruskal-Wallis test followed by Dunn's Multiple Comparison test was performed. Correlations between parameters were tested by Pearson (parametric data) or Spearman tests (non- parametric data). For parametric data, bars represent mean± SD and for non- parametric data, box plots represent median (Interquartil range). A Infarct volumes of a subgroup of rats submitted to an eMCAO were calculated and the mean was 40.62±10.09% (n = 6), whereas no infarct lesion was observed in sham animals in any case (n = 13). Mean neurological score at 48 hours was 4.43±2.9 for ischemic animals and 0.28±0.82 for sham animals.As shown in Considering experimental groups, results obtained in flow cytometry assays are represented in Interestingly, higher percentages of blood PMNs were positively correlated with a worse neurological outcome 48 hours after MCAO ; , but no Bacterial growth from lung homogenates plated on agar was detected in both sham and MCAO groups, as shown in As expected, all naive rats (4/4) showed normal lung histology consisting of definite alveoli with thin walls lined by two types of cells: pneumocyte type I, the predominant cell type, and pneumocyte type II, which are large and cuboidal and show short microvilli on their cell surface. The interalveolar septa appeared basically thin, although normal focal areas of thick septum could be observed. In lung tissue from naive rats we also observed that the BALT (bronchus-associated lymphoid tissue) which corresponds to the lung immune system, was generally located in bronchi bifurcation. .On the oResults obtained in this second set of experiments using a transient model with a nylon filament were equivalent to those derived from eMCAO model.The mean infarct volume of ischemic rats submitted to a tMCAO (n = 7) was 40.54±12.56%, and no lesion was detected in sham animals (n = 7). The mean neurological score was 4.14±1.07 for ischemic rats and 0 for sham rats.Regarding blood leukocyte counts, we observed that ischemic animals showed an increase in blood PMNs (63.78±22.28%) as compared to sham (38.88±26.65%) or naive (22±6.5%) groups. In this case, only the comparison between naive and ischemic groups reached significance after Bonferroni correction (p<0.01) . Also inIn relation to bacteriological analysis, CFU growth was observed only after plating sham and ischemic lung homogenate samples. We could detect significant differences when comparing naive to sham animals  and also naive to ischemic animals  . No CFUsPlasma levels of CXCL1 were evaluated 48 hours after ischemia or sham surgery and also in naive rats (n = 7/each group). Sham animals showed upregulated levels  as compared with naive rats , although a more pronounced release was seen in ischemic animals . After correction, only significance was reached when comparing naive and ischemic animals (p<0.001) . CorrelaExperimental stroke induces systemic inflammatory responses followed by global immunosuppression Blood analysis showed differences between sham and ischemic animals in circulating PMNs. According to previous studies, it has been well characterized that brain injury produces a secondary inflammatory response, accompanied by an increase on systemic neutrophils, the most representative PMN subtype Remarkably, our results showed that leukocyte changes were in the same direction in both sham and ischemic groups. To explain this, we support that both isoflurane and sham surgery might disturb leukocyte responses, a hypothesis recently demonstrated in a published study Pneumonia and bacteraemia are the most common infections in stroke experimental studies Lung evaluation showed normal histology in naive rats whereas both sham and ischemic animals presented non-specific lesions such as distension of alveolar units, thickened alveolar septa, cellular consolidated areas, presence of inflammatory infiltrates, oedema and haemorrhage. The severity of these lesions was extraordinarily variable among animals of the same group in both sham and ischemic animals. Again, surgery stress, anaesthesia and inflammatory response due to the cranial trepanation could also explain these non-specific lung lesions found in both sham and ischemic animals.Compiling our results, we have found many unexpected changes in sham animals in the present study. The animal (species and strain) or the model we have performed to induce ischemia could have had a big influence in our final results. To our knowledge, this is the first study inducing ischemia in rats by embolic MCAO and assessing bacterial analysis and lung histology thus we can speculate that the strong severity of the model  could contribute to an important systemic inflammatory response. Despite our efforts in reducing the pain and discomfort of the rats subjected to an embolic stroke, the mortality associated to this experimental model is around 40%, as similarly shown in other studies On the other hand, the fact of obtaining similar results after submitting the rats to a tMCAO model introducing a nylon filament and submitting them to an embolic MCAO model makes us consider the cranial trepanation and the cervicotomy as responsibles for the immunodepression state detected also in sham animals. In this direction, some authors concluded that even being sterile, brain surgeries may contribute to a general immunodepression et al. et al. per se triggers an additional chemokine effect. Thirdly, that in this animal model, as found previously in others In agreement with Campbell Consequently, an important result to highlight from our study is that including a sham animal group in stroke-induce infection studies is extremely important to identify surgery-related responses and avoid misinterpretation of the data from ischemic animals. Additionally, it might be of great importance to check the sanitary status of the animals (presence of opportunistic and not only pathogenic microorganisms) previously to the surgery if the aim of the study is to evaluate spontaneous infections.In conclusion, our study could not discriminate ischemic from sham animals in terms of infection evaluation after embolic MCAO. Therefore, we cannot affirm that changes observed in ischemic animals are the consequence of the ischemic insult. The initial hypothesis of our study was that stroke-induced immunodepression in the rat would make ischemic animals more prone to respiratory infections. Considering very recent publications which report some neuroprotectants to ameliorate stroke-induced peripheral immunodepression


Multiple Sclerosis (MS) is an inflammatory disease of the Central Nervous System (CNS) that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons and axons. Historically, MS has been thought of as a T cell-mediated autoimmune disease of CNS white matter. However, recent studies have identified gray matter lesions in MS patients, suggesting that CNS antigens other than myelin proteins may be involved during the MS disease process. We have recently found that T cells targeting astrocyte-specific antigens can drive unique aspects of inflammatory CNS autoimmunity, including the targeting of gray matter and white matter of the brain and inducing heterogeneous clinical disease courses. In addition to being a target of T cells, astrocytes play a critical role in propagating the inflammatory response within the CNS induced NF-κB signaling. Here, we will discuss the pathophysiology of CNS inflammation mediated by T cell—glial cell interactions and its contributions to CNS autoimmunity. Multiple Sclerosis (MS), an inflammatory T cell-mediated autoimmune disease, is the most common neurological disease of young adults. MS causes the demyelination of nerve cells and destroys oligodendrocytes, neurons and axons  autoimmunity  genes and genes associated with T cell activation and homeostasis; however, the strongest genetic linkage occurs with certain alleles of MHC class II, which suggests a direct relationship between autoreactive CD4in vivo study of the migratory behavior of pathogenic T cells  further supports the hypothesis that myelin-reactive CD4 T cells have a central role in MS disease pathogenesis  and S100β, have been studied as targets for autoreactive T cells. GFAP, an intermediate filament protein, is an archetypal astrocyte-specific antigen that is expressed throughout the gray matter and white matter of the brain and spinal cord , to follow the fate of naïve GFAP-specific T cells. To determine if BG1 mice maintain quiescence to GFAP over their lifetime, a cohort of WT, Rag1−/− and Gfap−/− BG1 mice were analyzed for clinical signs of CNS disease as they aged. We observed that BG1 mice do not maintain ignorance of GFAP: ~50% of WT BG1 mice and 100% of Rag−/− BG1 mice succumb to spontaneous clinical signs of CNS autoimmunity by 6 months of age. The majority of diseased BG1 mice develop balancing defects, lethargy, uneven gait and ataxia—such symptoms are referred to as atypical disease  cells that populate peripheral tissues following viral challenges  and μMT−/− (B cell-deficient) BG1 mice were generated. Spontaneous CNS disease in IAbβ−/− BG1 mice was similar in frequency and severity to WT BG1 mice. In contrast, μMT−/− BG1 mice were found to be highly susceptible to spontaneous CNS disease, with ~80% of μMT−/− BG1 mice developing chronic clinical disease, a fundamentally distinct disease course as compared to the relapsing-remitting disease most often observed in WT BG1 and Rag−/− BG1 mice (Sasaki et al., In both WT and Rag1In addition to immune cells, we have demonstrated that non-immune cells, including vascular endothelial cells and glial cells, play critical roles in the induction of chronic inflammatory diseases such as EAE. Glial cells of the CNS can secrete large quantities of chemokines, growth factors and IL-6 in response to inflammatory stimuli, all of which can activate the NF-κB and STAT signaling pathways (Ogura et al., in vivo (Arima et al., The inflammation amplifier is regulated by the production of several neurotransmitters, including norepinephrine and ATP. These findings led us to hypothesize that the inflammation amplifier may link the onset and severity of CNS diseases to mental and physical stress. Indeed, regional neural activity created by gravity of the Earth on the soleus muscles enhances chemokine expressions within the CNS, resulting in inflammation occurring around the dorsal vessels of the fifth lumbar cord, during early stages of EAE. CNS inflammation and the upregulation of chemokine expression can similarly be induced artificially using electric stimulation of peripheral muscles, formally demonstrating that neuronal activity can regulate the inflammation amplifier The inflammation amplifier can be turned on or off in response to acute inflammation, as well as to mental and physical stress. Thus, the temporal regulation of NF-κB and STAT signaling pathways in glial cells may regulate episodic cycles of relapsing/remitting clinical disease in MS patients. Mechanistically, one way this may occur is by recruiting or limiting immune cell migration through the “gateway” present within the spinal cord and potentially through other sites within the brain. Clarifying these mechanisms, and identifying how different immune cell lineages and subsets respond to and regulate the inflammation amplifier, will provide insights into the pathogenesis of relapsing/remitting CNS diseases, and identify drug-targetable molecular pathways that can be exploited to minimize MS disease relapses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Astyanax mexicanus, is a unique model system consisting of cave-adapted and surface-dwelling morphotypes that diverged >1 million years (My) ago. This remarkable natural experiment has enabled powerful genetic analyses of cave adaptation. Here, we describe the application of next-generation sequencing technology to the creation of a high-density linkage map. Our map comprises more than 2200 markers populating 25 linkage groups constructed from genotypic data generated from a single genotyping-by-sequencing project. We leveraged emergent genomic and transcriptomic resources to anchor hundreds of anonymous Astyanax markers to the genome of the zebrafish (Danio rerio), the most closely related model organism to our study species. This facilitated the identification of 784 distinct connections between our linkage map and the Danio rerio genome, highlighting several regions of conserved genomic architecture between the two species despite ∼150 My of divergence. Using a Mendelian cave-associated trait as a proof-of-principle, we successfully recovered the genomic position of the albinism locus near the gene Oca2. Further, our map successfully informed the positions of unplaced Astyanax genomic scaffolds within particular linkage groups. This ability to identify the relative location, orientation, and linear order of unaligned genomic scaffolds will facilitate ongoing efforts to improve on the current early draft and assemble future versions of the Astyanax physical genome. Moreover, this improved linkage map will enable higher-resolution genetic analyses and catalyze the discovery of the genetic basis for cave-associated phenotypes.The Mexican tetra,  Anoptichthys (“bony fish without eyes”)  . Breedint eyes”) , b. Bothresearch  focusingresearch , developresearch , physiolOca2) (brown phenotype (Mc1r)  phenotypes evolved through heritable genetic changes. These studies centered on both Mendelian and complex phenotypes, including eye regression , and int1 × Pachón cave backcross pedigree using markers generated from random amplified polymorphic DNA (RAPD) fingerprinting  technology. This technology enables accurate and high-throughput collection of massive amounts of sequence data  bred from a male surface fish and female cavefish from the Pachón cave. In addition, surface (n = 4), Pachón cave (n = 4), and surface × Pachón F1 hybrid (n = 4) specimens were used to evaluate and code GBS markers for use with JoinMap software , but were not included in linkage mapping calculations. Parental specimens belonged to laboratory populations originally sourced from the El Abra region of northeastern Mexico and all fish used were generously provided to our laboratory by Dr. Richard Borowsky (New York University). All live fish used in this study were maintained as previously described (see 2 population (n = 129) was individually reared in a 1-liter tank. All phenotypic data from the “Asty12” F2 population (n = 41) were obtained from paraformaldehyde-preserved specimens.Linkage mapping and QTL studies were performed using genotypic and phenotypic data obtained from two separate F1 and F2 hybrid Astyanax mexicanus specimens using the DNeasy Blood and Tissue Kit (Qiagen) as previously described . Samples were processed by the Institute for Genomic Diversity (Cornell University), where genomic libraries were constructed and GBS was performed as described elsewhere  surface or cave individuals had the identical nucleotide at a particular locus, then the genotype was assigned to the consensus parental population. Likewise, a true “hybrid” genotype was assigned if three or more F1 individuals harbored the same heterozygous condition  at a given locus. Those genotypes with an ambiguous morphotypic origin were denoted “NA.”Genotypes for each of 7956 GBS markers (each consisting of a single SNP in a 64-bp-long sequence fragment) were screened in cave and surface  forms to assign the morphotypic origin of each allele. Fi.e., both the surface and cave genotypes were scored “NA”) or if the assigned surface and cave genotypes were identical; 6006 genomic markers were deemed suitable and prepared for linkage map calculation using the “cross-pollination” (CP) segregation coding used in JoinMap. At this stage, 107 markers were found to be uninformative  and discarded from further analysis. We screened the remaining set (n = 5899) to identify markers failing to conform to predicted genotypic ratios ; 2896 markers demonstrated a χ2 value more than 50, implying significant departure from the predicted genotype ratio and were discarded from further analysis. Our final GBS marker set included 3003 markers evaluated in 170 F2 individuals.Markers were then screened for suitability in linkage calculations. Markers were deemed unsuitable and discarded from further analysis if neither parental genotype could be assigned . Our workflow used program default settings, with the following exceptions: the maximum grouping independence LOD value was set to 50.0; linkage groups were calculated using regression mapping; and linkage mapping was performed using the Kosambi method . LinkageAstyanax mexicanus has karyotypic number of 25; The first round of mapping produced 28 linkage groups comprising a total map length of 2956 cM. At this stage, one linkage group  failed to assemble into a consolidated group and was therefore eliminated from further analysis. The remaining individual linkage groups ranged in length from 27.25 to 187.46 cM, containing between 10 and 225 markers with an average intermarker distance between 0.51 and 6.40 cM. After this initial round of mapping, we further screened existing linkages to target the most optimal 25 groups  was eliminated. A densely populated group with a high independence LOD  was split and 12 linkage groups (103.982 ≤ n ≤ 142.783 cM) were trimmed.Astyanax mexicanus and the zebrafish genome and used to map albinism as a proof of concept. Albinism was scored as a binary phenotype wherein presence of melanin (0) or absence of melanin (1) was assigned to each of the members of our experimental F2 pedigrees. All QTL analyses of albinism were conducted using R/qtl .At present, physical genome resources for 0 My ago . Despitee.g., three different Danio rerio chromosomes). In cases where a single marker sequence aligned multiple times with the same target, raw results were filtered by e-value, retaining the lowest e-value alignment for each marker-target pairing. There are two 64-bp sequences for each GBS marker, differing only in that each contains one of the two alleles for the imbedded SNP. Because both of these sequences were included when BLAST searches using the 64-bp marker sequences were conducted, this filtering step also served to collapse these results into a single set of results, retaining the better of the two alignments for each marker-target pairing.These and all subsequent searches were performed using a BLASTN script run on the Ohio Supercomputing Cluster (OSC). All quality control defaults, including an expect value  cutoff of 10, were maintained. The script permitted the return of alignments between a given 64-bp marker sequence and regions of up to three distinct targets  for a given marker sequence agreed with the target reported for one or more other markers on the same linkage group that returned only a single robust hit, then the top hit for the marker in question was considered “supported” and retained. If the top hit was not supported in this fashion but a different BLAST result was, then the latter “not top hit, supported” result was retained instead. If none of the results returned for a marker sequence were supported, then the top hit was retained, despite the lack of support. In rare cases, there was no way to resolve which result should be retained. Results for these “unresolved” markers were discarded.In some instances, a single queried sequence returned alignments with multiple targets. These instances were resolved by sorting results to determine the “top hit,” which was defined as having the lowest e-value and highest percent identity  to a particular target sequence. If the target of the top hit . The collective sequence data for the Astyanax genome (GenBank Assembly ID GCA_000372685) were downloaded from Ensembl, along with the transcript sequences for 23,042 predicted genes. BLAST searches were used to determine putative locations for the 64-bp sequences of the 2235 GBS markers comprising our final linkage map in the Astyanax genomic and transcriptomic data sets. After initial searches were performed as described, ∼2000-bp stretches of genomic sequence harboring our 64-bp GBS marker sequences were aligned with the Danio genome. Similarly, full sequences for predicted Astyanax transcripts to which our GBS markers aligned were queried against a Danio cDNA database downloaded from Ensembl. Both data sets were then filtered (as described), yielding a single “best” Danio alignment for each informative query. This process enabled us to leverage draft genomic and transcriptomic data to augment the amount of sequence information associated with our 64-bp GBS markers and to identify homologous genomic positions in a well-characterized model system.When using BLAST searches to align our 64-bp markers directly to the Astyanax and Danio rerio genomes.After BLAST searches using the direct, genomic, and transcriptomic alignment methods were completed, the filtered results for all three were combined. When multiple methods returned results for the same marker, a single result was chosen and retained using the same filtering process applied to single data sets (above). The Circos program  for each data set were aligned with Astyanax genome scaffolds using the same BLAST and filtering protocols used for our own data (above). Both previous studies included markers located in candidate genes. The locations of Astyanax orthologs of these candidate genes were identified using Ensembl.Previous maps published by http://dx.doi.org/10.5061/dryad.6s718).GBS marker sequences and genotyping data are available from the Dryad Digital Repository  comprising 2235 markers spanning 2110.7 cM, with an average intermarker distance of 1.052 cM (Table S1). The strategy we used enables application of powerful, cost-effective, next-generation sequencing technology to facilitate genetic studies in emerging or nonmodel systems.Here, we present a dense linkage map for Astyanax genomic and transcriptomic resources, followed by searches of the homologous sequences in Danio (Astyanax genomic (26.5%) or transcriptomic (13.3%) sequences as an intermediary  comprising ancient syntenic blocks shared with each of 25 zebrafish chromosomes . However, other Danio chromosomes appear scattered across several linkage groups, without a consensus representation for any particular group .Our analysis of synteny between e groups . CertainDanio chromosomes within our GBS marker set. We examined this possibility by assessing the number of syntenic links between our GBS-based linkage map and each Danio chromosome. We would anticipate that longer chromosomes would naturally harbor more syntenic links. Values were therefore expressed as a ratio of syntenic links per megabase (mean = 0.59 GBS markers/Mb). Although the mean value for chromosomes that were not strongly represented on any particular linkage group in our map  was lower than that for chromosomes demonstrating strong synteny with a particular linkage group , there was not a significant difference between the two groups . This leads us to conclude that, although representation of particular chromosomes in our data set may be a contributing factor, it is unlikely that this is the primary cause of the differences in chromosomal representation patterns observed.We believe these findings most likely reflect genomic rearrangements that have occurred since the divergence of these two species. However, this finding could also be attributed to low representation of particular Astyanax GBS markers  may include paralogous genes or otherwise ambiguous results that could lead to erroneous links between a linkage group and a Danio chromosome. Although we cannot rule out this possibility, we feel our strategy prioritized the “optimal” BLAST result among multiple hits for a single marker leading to alignments that agree with nearby unambiguous results  of final calls were unsupported by the results for other markers belonging to the same linkage group (Table S1). Given that chromosomal arrangements have occurred over the ∼150 My since divergence, we feel our systematic approach best identifies paralogous genes and other potential sources of ambiguity.Alternatively, BLAST results for  results . As a reMaterials and Methods); however, we relied on a relatively small number of cave, surface, and F1 hybrid individuals (n = 4 each) to identify parental allelic origin. Similarly, the relatively small number of meiotic events represented by the 170 F2 individuals may have resulted in linkage map inaccuracies  of the 2235 GBS markers present in our map. These markers were localized to positions spread across 598 different Astyanax genome scaffolds. Our 25 Astyanax linkage groups contain markers representing between 12 (linkage groups 8 and 22) and 55 (linkage group 3) genome scaffolds each, with a map-wide average of 27.64 scaffolds/linkage group. Individual genome scaffolds contained between 1 and 31 GBS markers appearing in our final map, with an average of 3.50 markers per scaffold. GBS markers located on the same genomic scaffold colocalized to a single linkage group 87.3% of the time. This suggests that our recombination mapping successfully recapitulated the true genomic positions of the markers used to construct our map.Positional locations in the current draft of the Astyanax and Danio. Metrics such as the number of linkage groups, total map length, number of markers, and marker density are commonly used to compare linkage maps within species. Both our GBS-based map and the RAD-seq and microsatellite-based map published by Astyanax mexicanus karyotype number of 25. The microsatellite-based map presented by Danio  per Danio rerio chromosome  and Danio chromosome = 6.92, minimum = 1, maximum = 20). Additionally, although instances of synteny strongly represented in previous maps were also identified in this analysis, our map demonstrated increased representation of certain Danio chromosomes poorly represented in previous maps. For example, Danio rerio chromosome 11; however, we identified 36 links between our map and chromosome 11. Similarly, Danio chromosomes 17 and 19 are each represented once in the map of Our map contains a total of 784 links between our linkage groups and the romosome . This reAstyanax genomic scaffolds and each linkage map. We examined the five strongest syntenic links between single linkage groups in our GBS-based map and single Danio chromosomes and then identified analogous connections between those chromosomes and specific linkage groups in the maps presented by Our linkage map uses an entirely different marker set than those used in previous maps. Therefore, it was not possible to make direct comparisons with the linkage groups across prior studies. However, we could indirectly compare maps by examining connections between Astyanax genomic scaffolds harboring markers associating each linkage group with a particular Danio chromosome were then compared  GBS marker sequences. As expected, based on the significant divergence between these species, we recovered varying levels of synteny between portions of our Astyanax linkage groups and regions of the Danio genome. As a proof of concept, we successfully mapped a strong QTL associated with albinism and demonstrated significant conserved genomic architecture in the regions surrounding the gene Oca2, between Astyanax and Danio. We successfully anchored emerging Astyanax genomic information to our GBS-based linkage map, identifying the putative location of thousands of anonymous GBS marker sequences within unplaced Astyanax genome scaffolds. This strategy revealed significant colinearity between genomic scaffolds and our linkage map, and it demonstrated the utility of high-density, GBS-based linkage maps to inform and improve nascent genomic resources. Multiple comparisons with previously published maps suggest that our GBS-based map offers a higher level of resolution and a greater number of connections between Astyanax and Danio genomes. We hope that this resource and technology will accelerate the search and identification of genes mediating cave-associated traits in Astyanax, facilitate the genomic assembly for this system, and prove useful to other natural model systems of evolutionary and biomedical relevance.We constructed a high-density linkage map for


Experimental data from animal models of sepsis support a role for a transcription factor, nuclear erythroid-related factor 2 p45-related factor 2 (Nrf2), as a master regulator of antioxidant and detoxifying genes and intermediary metabolism during stress. Prior analysis of a pediatric septic shock transcriptomic database showed that the Nrf2 response is a top 5 upregulated signaling pathway in early pediatric septic shock.We conducted a focused analysis of 267 Nrf2-linked genes using a multicenter, genome-wide expression database of 180 children with septic shock 10 years of age or younger and 53 healthy controls. The analysis involved RNA isolated from whole blood within 24 h of pediatric intensive care unit admission for septic shock and a false discovery rate of 5 %. We compared differentially expressed genes from (1) patients with septic shock and healthy controls and (2) across validated gene expression–based subclasses of pediatric septic shock (endotypes A and B) using several bioinformatic methods.We found upregulation of 123 Nrf2-linked genes in children with septic shock. The top gene network represented by these genes contained primarily enzymes with oxidoreductase activity involved in cellular lipid metabolism that were highly connected to the peroxisome proliferator activated receptor and the retinoic acid receptor families. Endotype A, which had higher organ failure burden and mortality, exhibited a greater downregulation of Nrf2-linked genes than endotype B, with 92 genes differentially regulated between endotypes.Our findings indicate that Nrf2-linked genes may contribute to alterations in oxidative signaling and intermediary metabolism in pediatric septic shock.The online version of this article (doi:10.1186/s13054-015-1052-0) contains supplementary material, which is available to authorized users. The prevalence of pediatric septic shock is rising –3, and mEvidence for mitochondrial dysfunction in pediatric septic shock is supported by a recent report that nuclear-encoded mitochondrial genes are differentially expressed early in pediatric septic shock compared with healthy controls and across septic shock endotypes . MoreoveA mechanistic link between sensing of oxidative stress, immune dysregulation, and mitochondrial bioenergetic failure can be made with a nuclear transcription factor, nuclear erythroid-related factor 2 p45-related factor 2 (Nrf2) , 20. UndWe sought to further investigate the differential expression of Nrf2-linked genes by performing a focused analysis of a U.S. multisite genome-wide expression database of children with septic shock . In our The study protocol was approved by the institutional review boards of each participating institution. The name of each institutional review board corresponds to the name of each respective institution: Cincinnati Children’s Hospital Medical Center, The Children’s Hospital of Philadelphia, University of California Benioff Children’s Hospital Oakland, Penn State Hershey Children’s Hospital, Children’s Mercy Hospital, Children’s Hospital of Orange County, Akron Children’s Hospital, Children’s National Medical Center, Miami Children’s Hospital, Texas Children’s Hospital, and C.S. Mott Children’s Hospital at the University of Michigan. Children 10 years of age or younger admitted to the PICU who met pediatrics-specific criteria for septic shock were eligible for enrollment . Age-matWritten informed consent to participate in this study was obtained from the parents or legal guardians of all children with septic shock and all control subjects. Blood samples were obtained within the first 24 h of meeting criteria for septic shock. Total RNA was isolated from whole blood using the PAXGene Blood RNA System . Microarray hybridization was performed as previously described using the GeneChip Human Genome U133 Plus 2.0 Array  .Nrf2-linked genes chosen for our focused analysis were derived from data compiled in independent studies that used microarray analyses and chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq) experiments . We identified 267 genes linked to Nrf2, as defined by Nrf2 target genes containing putative antioxidant response elements or xenobiotic response elements in their promoter regions and genes that directly or indirectly regulate expression of Nrf2. These 267 genes correspond to 566 gene probes on the GeneChip Human Genome U133 Plus 2.0 Array, as shown in Additional file We analyzed existing normalized microarray data. The original analyses were performed using one patient sample per chip. Image files were captured using a GeneChip Scanner 3000 (Affymetrix). Raw data files (.CEL) were subsequently preprocessed using robust multiarray average (RMA) normalization and GeneSpring GX 7.3 software . All signal intensity–based data were used after RMA normalization, which specifically suppresses all but significant variation among lower-intensity probe sets . All chit test and corrections for multiple comparisons using a Benjamini-Hochberg false discovery rate (FDR) of 5 %. We did not include predetermined fold expression filters in the analysis, because the biological implications of a specific threshold change in our target gene set have not previously been established. Thus, to account for the possibility that even a modest change in the expression of genes from a common metabolic pathway could yield dramatic variability in flux through that pathway [Differences in mRNA abundance between study groups were determined using Welch’s  pathway , we repoGene lists of differentially regulated genes were analyzed using the Ingenuity Pathway Analysis (IPA) application  to explore potential associations with specific domains of Nrf2 function. IPA is a database generated from peer-reviewed scientific publications that provides a tool for discovery of signaling pathways and gene networks within the uploaded gene lists. Adjunct analyses of gene lists were conducted using the ToppGene application .Gene expression mosaics representing the expression patterns of differentially regulated genes were generated using the Gene Expression Dynamics Inspector (GEDI) . GEDI pr2 test .Ordinal and continuous clinical variables not normally distributed were evaluated by analysis of variance on ranks. Dichotomous clinical variables were analyzed using a χt test starting with all 54,675 gene probes on the array and corrected for multiple comparisons using a Benjamini-Hochberg FDR of 5 %. There were 23,912 gene probes differentially regulated between children with septic shock and healthy control pediatric subjects.We first determined how many gene probes were differentially regulated between children with septic shock (n =180) and healthy control pediatric subjects (n =53). The demographic characteristics of the two study groups were reported previously by Weiss et al. . We condWe then conducted a Venn diagram analysis to determine how many of the 566 gene probes corresponding to the Nrf2-linked genes were found in this list of 23,912 differentially regulated gene probes. This analysis yielded 281 gene probes corresponding to 178 unique genes, of which 123 were upregulated and 55 were downregulated in children with septic shock relative to controls. The complete list of differentially expressed Nrf2-linked genes is provided in Additional file Uploading the 281 differentially regulated Nrf2-linked gene probes to the IPA platform resulted in the top 5–scoring canonical pathways listed in Table We next determined whether the Nrf2-linked genes were differentially regulated across previously validated gene expression-based subclasses of pediatric septic shock: endotype A and endotype B . The clit test starting with all 54,675 gene probes on the array and corrected for multiple comparisons using a Benjamini-Hochberg FDR of 5 %. There were 11,630 gene probes differentially regulated between endotype A and endotype B patients.We first determined how many gene probes were differentially regulated between endotype A and endotype B patients. We conducted a Welch’s We then conducted a Venn diagram analysis to determine how many of the 566 gene probes corresponding to the Nrf2-linked genes were found in this list of 11,630 differentially regulated gene probes. This analysis yielded 138 gene probes corresponding to 92 unique genes, of which 40 were upregulated and 52 were downregulated in endotype A patients, relative to endotype B patients. The complete list of differentially expressed Nrf2-linked genes is provided in Additional file We used the GEDI program to build gene expression mosaics to provide a global representation of the 92 differentially expressed genes in endotype A and endotype B patients. The GEDI mosaics in Fig. Among the 92 differentially expressed genes, the top 5 canonical pathways from IPA are shown in Table The Nrf2 pathway has been shown in previous microarray analyses of pediatric sepsis to be in the top 5 signaling and metabolic pathways upregulated in whole blood–derived RNA, with 24 genes represented . The preNrf2 plays a role in degradation of triglycerides and/or phospholipids and enzymes involved in fatty acid oxidation. In addition, Nrf2 negatively regulates many genes encoding enzymes involved in lipid biosynthesis, fatty acid desaturation, and fatty acid transport through unknown mechanisms . LangleyConcurrent measures of oxidative stress and metabolite data are not available to determine how the observed Nrf2 pathway gene expression changes might alter the total antioxidant status, fatty acid metabolism, and ATP production in early pediatric sepsis. It is likely that changes in intermediary metabolism and oxidative state affect Nrf2-linked gene expression, and researchers in future studies should measure the effects of oxidative stress, metabolite concentration, and flux through various metabolic pathways influenced by Nrf2, such as glycolysis, the citric acid cycle, the PPP, fatty acid oxidation, and fatty acid synthesis.The present study is constrained by the fact that we limited the analysis to 267 genes linked to Nrf2, as determined by previous human and murine studies. Linkage was defined as either genes regulated by Nrf2 or genes that regulate Nrf2 function. It is certainly possible that our selection method excluded some relevant genes. Limiting our working gene list in this manner is a potential source of bias. We attempted to mitigate this bias by conducting the initial statistical analyses, with corrections for multiple comparisons, using all gene probes available on the array. We subsequently searched the resulting gene lists for our selected Nrf2 genes and found that many of these genes were differentially regulated when we considered all available gene probes.Nrf2-linked genes involved in cellular lipid metabolism were predominantly upregulated early in pediatric septic shock compared with healthy controls. Genes corresponding to the oxidative stress response mediated by GSH-binding enzymes and NADPH-oxidase generation of superoxide were predominantly downregulated in a distinct, previously defined endotype of pediatric septic shock having higher organ failure burden and mortality. Our findings indicate that Nrf2-linked genes may contribute to alterations in oxidative signaling and intermediary metabolism in pediatric septic shock. Studying the temporal changes in expression of Nrf2 pathway genes and correlation of gene expression with metabolomic analysis of intermediary metabolites should provide insight into the energy and oxidative states of children over the time course of sepsis. We propose that future work should be focused on the hypothesis that differential regulation of Nrf2-linked genes may be an important mechanism contributing to the host ability to combat oxidative stress and reprogram metabolic pathways to provide necessary energy to survive childhood sepsis and MODS. The oxidative stress transcription factor Nrf2-linked genes were differentially expressed in children with septic shock compared with healthy controls. Nrf2-linked genes can be used to differentiate two previously validated subclasses of children with septic shock, with a greater degree of repression in children with a higher mortality and more organ failure. Nrf2 may be an important transcription factor contributing to alterations in oxidative signaling and intermediary metabolism in pediatric sepsis.


MMSET (WHSC1/NSD2) expression and is a poor prognostic factor in multiple myeloma (MM). MMSET encodes two major protein isoforms. We have characterized the role of the shorter isoform (REIIBP) in myeloma cells and identified a clear and novel interaction of REIIBP with members of the SMN  complex that directly affects the assembly of the spliceosomal ribonucleic particles. Using RNA-seq we show that REIIBP influences the RNA splicing pattern of the cell. This new discovery provides novel insights into the understanding of MM pathology, and potential new leads for therapeutic targeting.The chromosomal translocation t deregulates  FGFR3 and MMSET. FGFR3 over-expression has weak transforming properties and is eventually lost in 30% of patients MMSET in promoting t myeloma which could be therapeutically targeted.The t chromosomal translocation, present in 10–15% of MM cases, is associated with a significantly worse prognosis compared to other genetic subgroups MMSET is a member of the NSD family of SET domain containing oncogenes known to be important in acute leukaemia and other malignancies ACA11, encoded within intron 18–19 of the MMSET gene  The SMN complex is a multi-subunit protein complex, which catalyses the assembly of snRNPs  that generate the RNA splicing machinery MMSET isoforms together with the difficulty of knocking down REIIBP alone. In this work we have evaluated the role of REIIBP using a multi-tagged REIIBP construct artificially transduced into a MM cell line background. This work demonstrates that REIIBP binds to the SMN complex and that REIIBP methyl transferase activity alters snRNPs cellular abundance, affecting RNA splicing. This novel cytoplasmic mechanism for MMSET opens new perspectives in understanding the pathogenesis of t myeloma.Attempts to understand the functional role of REIIBP have been hindered by the lack of antibodies able to distinguish different The cDNA for the MMSET isoform REIIBP was cloned in frame with the C-terminal double tags 6×HIS-3×FLAG and, through an auto-catalytic E2A peptide, linked to green fluorescence protein (GFP). The ΔSET constructs were generated by point mutating Asparagine 359, present in the SET domain, to Alanine (Agilent Technologies). The construct was cloned into a lentivirus system (pLVTHM or pRRLSIN) substituting the original GFP gene.H929 and HeLa cell lines were obtained from ATCC, and were grown in RPMI1640 containing GlutaMax (Life Technology), and DMEM (Life Technology) respectively, both media were supplemented with 10% heat-inactivated fetal calf serum (PAA Laboratories). Cells were cultured at 37°C in a humidified gas chamber with 95% air and 5% carbon dioxide. All cell lines used were mycoplasma-free as confirmed by PCR. Cell identities were confirmed by short tandem repeats analysis. Proliferation assays, western blotting, apoptosis assays and cell cycle analysis were conducted as described previously 8 H929 cells were transduced with tagged REIIBP (H929::REIIBP) or H929 untouched used as control, and then harvested in 50 mM NaH2PO4 pH 7.4; 50 mM NaCl; 0.05% Tween; protease inhibitor (ROCHE). Cells were lysed by 3 cycles of freeze/thawing on dry ice and passed through a 10 gauge needle. Lysates were cleared by passing through 0.45 µm filter, spinning at 13000 rpm for 10 minutes and finally by incubating with 1 mL Agarose resin (Thermo Scientific). Equal amounts of protein from sample and control cleared lysates were loaded into chromatography columns filled with M2 anti-FLAG resin (SIGMA) and agitated at 4°C for 1 hour. Columns were washed with 50 mM NaH2PO4 pH 7.4; 300 mM NaCl; 0.05% Tween; protease inhibitor. Immuno complexes were eluted with 100 µg/ml 3×FLAG peptide (SIGMA). Eluates from the anti-Flag column were incubated for 1 hour in NiNTA agarose resin (Thermo Scientific) and washed with 50 mM NaH2PO4 pH 7.4; 300 mM NaCl; 20 mM Imidazole; 0.05% Tween; protease inhibitor. NiNTA columns were eluted with 250 mM Imidazole.Around 5×10homo sapiens subset database , customised to include the REIIBP construct sequence, using Mascot v2.2 (www.matrixscience.com). A precursor ion tolerance of 5 ppm and fragment ion tolerance of 0.25 Da was applied. Relative protein enrichment was inferred from the spectral counts observed for each protein between the control and H929::REIIBP samples Reagents were purchased from Sigma-Aldrich  unless otherwise stated. 1D SDS-PAGE gel lanes corresponding to double affinity purification from H929::REIIBP and H929 were excised equivalently and in their entirety as 9 sections each. The gel sections were digested in-gel with trypsin and analysed by liquid chromatography-mass spectrometry as previously described 2; 0.1% NP40; 10 mM Hepes pH 7.4; Protease inhibitor), and lysed by passing through a 19 gauge needle. Lysate was cleared by centrifugation and through filtering with a 0.45 µm strainer. Equal amount of protein lysate was immunoprecipitated with 2 µg of anti GEMIN5, SMN and normal mouse IgG (Millipore) for 2 hours at 4C. Immune complexes were harvested using Protein G conjugated to magnetic beads (Thermo Scientific) using manual recommendations.Myeloma cell lines were harvested in RSB buffer . Equal amounts of input lysate were harvested and RNA extracted. Immune complexes were purified and RNA extracted and reverse transcribed as described above. cDNA was analysed by quantitative RTPCR (qRT-PCR) using syber green and a relative standard curve method. Data was normalized against mRNA actin values present in the inputs.http://tophat.cbcb.umd.edu/) with default parameters, and Cufflinks 2.1.1 (http://cufflinks.cbcb.umd.edu/) was used to estimate transcript abundance and calculate differential expression between REIIBP and control samples. The results were visualized using cummerbund 2.0.0 (http://compbio.mit.edu/cummeRbund/). See also Experimental procedures in Total RNA was purified using the Trizol (Life Technology) method. Total RNA from 3 replicates of HeLa cell line transduced with cDNA coding for REIIBP and 3 replicates of the control cell line (HeLa) was extracted. These 6 libraries were pulled down and split between 2 lanes of an Illumina HiSeq by Oxford gene technology. Raw fastq files were aligned using Tophat 2.0.8  for 20 minutes at room temperature. Suspension cells were fixed in 4% formaldehyde and then cytospun at 300 rpm for 5 min onto microscope slides. Fixed cells were permeabilised in PBST buffer  for 30 minutes in a humidified chamber, then blocked with 5% goat serum (SIGMA) in PBST. Permeabilised cells were stained for 1 hour at room temperature with one or two of the following primary antibodies: FLAG ; coilin (Santa Cruz); SMN, GEMIN5 (Millipore); Fibrillarin ; REIIBP 0.1×10In order to study REIIBP physiology we virally transduced HeLa cells with an REIIBP construct (HeLa::REIIBP) or an REIIBP construct with an inactivating mutation in the SET domain (HeLa::ΔSET). Both constructs carried HIS-FLAG tags . HeLa cein vivo lacked this effect It has previously been reported that REIIBP can dimethylate H3K36 in isolated nucleosomes In order to characterize the role of REIIBP, we carried out a series of biological assays on transduced cells. We could not detect any difference in the number of cells in apoptosis or proliferation rate as determined by a WST-1 assay in the transduced cells. We found a small but significant increase in the number of HeLa::REIIBP cells in S phase of the cell cycle .The lack of a distinct cellular phenotype for HeLa::REIIBP cells suggests that the biological relevance of REIIBP may be limited to specific cellular backgrounds, such as plasma cells. We sought to investigate this further by identifying REIIBP binding partners present in myeloma cells.We characterized REIIBP interacting partners by tandem affinity purification followed by mass spectrometry. In these experiments an REIIBP construct carrying HIS/FLAG tags was transduced into the t positive H929 myeloma line (H929::REIIBP). In order to avoid artefacts introduced by differences in REIIBP expression, we selected clones expressing tagged REIIBP at lower levels than the endogenous REIIBP by FACS.Mass spectrometry analysis on these cells  identifiIn order to validate the REIIBP/SMN interaction, we carried out western blotting on H929 and H929::REIIBP following anti-FLAG pull-down  using anIn order to demonstrate that this interaction was not mediated via binding through the FLAG or the HIS tags, and at the same time to rule out the possibility that the interaction was due to REIIBP overexpression, we investigated the interaction in an untransduced H929 line. GEMIN5 co-immuno-precipitation (Co-IP) pulled down GEMIN3, a member of the SMN complex . ImportaTo support the Co-IP data we used confocal microscopy to analyse H929 cells immuno-stained with SMN and REIIBP antibodies. Analysis of several optic fields showed that REIIBP co-localized with SMN in the cytoplasm .Collectively, these results confirmed the novel interaction of REIIBP with the SMN complex.Previous reports have demonstrated that knock down of the SMN protein causes a reduction in mature snRNPs and changes the RNA splicing patterns To confirm here the co-localization between the SMN complex and REIIBP we double stained HeLa::REIIBP and HeLa cells with antibodies against FLAG, SMN and the GEMIN5 epitopes . ConsistIF and confocal analysis of HeLa::ΔSET also showed co-localization with GEMIN5  suggestiWe hypothesised that the REIIBP/SMN interaction could influence the formation of new splicing snRNPs. In order to address this question we immunoprecipitated cellular splicing snRNPs in HeLa::REIIBP, HeLa::ΔSET and HeLa lines, using an antibody against the Sm protein SmB/B′, we extracted total RNA from the immunoprecipitates and compared the level of snRNAs  by qRT-PCR . ResultssnRNPs make up the bulk of the spliceosome machinery structure. In order to test if the decreased number of snRNPs in HeLa::REIIBP deregulates downstream RNA splicing patterns, we used RNAseq of the REIIBP transduced cells compared to control HeLa cells. We focused on changes in splicing patterns looking for gene specific alternative isoforms as well as for the presence of new transcripts generated by exon skipping or intron retention. We found a wider variety of transfrags showing intron retention in HeLa::REIIBP, with a statistically significant difference between the proportion of unique transfrags corresponding to annotated exons and at least 10 bp of intron identified between the HeLa samples and HeLa::REIIBP samples . A list of the genes most affected by intron retention is shown in In addition to the altered splicing, REIIBP overexpression affects the expression of a number of genes. Gene ontology analysis of these genes using the DAVID bioinformatic tool We have demonstrated that the interaction of REIIBP with the SMN complex occurs in myeloma and possibly in HeLa cell lines as well. The technical difficulty of knocking down REIIBP alone in myeloma cells, without affecting MMSET II expression, prevented us from analysing the effect of the interaction between REIIBP and the SMN complex in t MM cells. We, therefore, addressed this issue using a dominant negative approach. We overexpressed a mutant SET domain REIIBP construct in H929 cells (H929::ΔSET), this out competed endogenous REIIBP for gaining access to its natural methylation targets and therefore counteracting its function. Analysis of splicing snRNPs levels in H929::ΔSET showed a significant increase compared to H929 . This obMMSET gene which is consistently deregulated by the t translocation in myeloma. It has previously been shown that MMSET II enhances t cell proliferation U2AF1, SF3B1, U2AF2 cause miss-annealing of snRNP at the correct 3′ splice site, leading to intron retention or exon skipping. Interestingly, in the context of the current report, knockdown of these factors leads to specific alterations in splicing patterns for only a subset of genes rather than causing global effects In this work we have characterized a novel role of REIIBP, the only protein product from the Regulation of SMN complex activity has already been shown to be relevant during development and cellular differentiation In this report focusing on the role of REIIBP, we show that as a result of its overexpression, there are significant changes in the pattern of RNA splice variants at a limited number of genes, mostly affected by intron retention rather than occurring as a result of alternative splicing. This is in agreement with observations in other systems where a reduction in the level of snRNPs does not necessarily correlate with shifts in the patterns of alternative splicing but rather can lead to an increase in aberrant splicing products. These changes are seen in a limited subset of genes Intron retention is an important mechanism both in normal tissue regulation and in disease states. It is associated with granulocyte differentiation In terms of the sub-cellular location, we show that REIIBP co-localizes with the SMN complex in the cytoplasm . Within ACA11, encoded within intron 18–19 of the MMSET gene, has been suggested to modulate the control of oxidative stress and to downregulate ribosomal protein genes and is also found in the nucleolus ACA11 could work in the same pathway, as it would be expected for a snoRNA and its host gene ACA11 and REIIBP interact with the nucleolar MYBP1A protein Further insight into the potential pathologic relevance of REIIBP overexpression comes from its localization within the nucleolus. MMSET isoforms, we could not detect a change in histone methylation pattern when REIIBP was over-expressed. The MMSET II isoform is known to alter histone methylation patterns and the difference with REIIBP possibly reflects the fact that they have different nuclear localization and that REIIBP lacks the upstream PWWP and PHD domains which mediate DNA binding ACA11 were unaffected ACA11 seem to be important in RNA processing.In terms of defining the specific biological roles of different The observation that REIIBP does not mediate histone methylation suggests that the different patterns of gene expression we demonstrate as a result of REIIBP overexpression are the result of a different mechanism. Insight into this mechanism comes from analysis of the patterns of deregulated genes. We show that REIIBP overexpression increases the expression level of a number of developmental related genes and homeotic genes. In terms of this potential mechanism, it is reported that SF3B1 and U2 snRNP, both of which are components of the splicesomal machinery, interact with polycomb proteins in a splicing independent mechanism that mediates the repression of homeotic genes MMSET in modulating the function of the RNA splicing machinery via its interaction with the SMN complex. This could have an impact on downstream cellular functions such as homeotic gene expression and an important role in the pathogenesis of myeloma. We demonstrate that the SET domain is critical for the role of MMSET in RNA processing as well as its previously recognised role in modulating histone methylation patterns. MMSET overexpression is an important early event in myeloma that has multiple downstream effects and is associated with poor outcome. This could potentially be reversed by inhibiting its methyltransferase activity via the SET domain. Blocking the interaction between the SMN complex and REIIBP could also be investigated as a possible therapeutic target.Overall we describe a novel role for the REIIBP isoform of Figure S1Characterization of HeLa cells transduced with REIIBP constructs. A) Schematic Protein structure of the transduced tagged REIIBP. B) Transduced HeLa lines correctly express REIIBP. The label “REIIBP” represents the HeLa cell line transduced with REIIBP double tagged with 6×HIS-3×FLAG. “ΔSET” is the HeLa cell line transduced with the same construct but with an inactivating mutation in the catalytic SET domain. Cytoplasm and nuclear fractions were isolated and analysed by western blot, using an antibody which recognizes both MMSET II and REIIBP isoforms or an antibody which recognizes Histone 3 (H3). KMS11 is a myeloma cell line used as positive control. The position for each of the respective protein species is indicated. C) IF and confocal analysis. REIIBP-Flag (green) co-localizes with the nucleolus marker Fibrillarin (red) in HeLa cells overexpressing REIIBP-Flag. The merged image includes DAPI (blue) staining. White arrows point to the same cellular region in all three frames. Single cell zoomed in as inset in each frame. Colocalization profile of Fibrillarin and Flag-REIIBP is show under the “merged” frame. The fluorescence profile is generated from the area covered by the red arrow in the merged picture. The blue plot represents the intensity of the DAPI stain; the green plot, the intensity of FLAG and the red the intensity of Fibrillarin. D) IF and confocal analysis on H929 t MM cells. REIIBP (red) localizes in the cytoplasm and in the nucleolus. Single nucleolus zoomed in as inset in each frame.(TIF)Click here for additional data file.Figure S2REIIBP co-localizes with the SMN complex in myeloma t cells. Immuno fluorescence and confocal analysis on H929 cells. A) REIIBP (green) co-localizes with SMN (red) in the cytoplasm. B) SMN (red) colocalizes with Coilin (green) at level of cajal bodies in the nucleus. The merged images include DAPI (blue) staining and white arrows show the nuclear position for the cajal bodies. Of note the antibody against REIIBP also recognizes the MMSET II isoform.(TIF)Click here for additional data file.Figure S3REIIBP-Flag co-localizes with SMN and GEMIN5 in HeLa cells overexpressing REIIBP-Flag. Immuno fluorescence and confocal analysis of HeLa::REIIBP-FLAG cells. A) Colocalization of FLAG (red) with SMN (green). Open arrows show the colocalization signal from the cytoplasm. Closed arrows indicate the nuclear position of Cajal bodies. B) Colocalization of FLAG (green) with GEMIN5 (red). Co-localization profile of GEMIN5 and REIIBP is shown at the bottom. The fluorescence profile is generated from the area covered by the red arrow in the merged picture. The blue plot represents the intensity of the DAPI stain; the green plot, the intensity of FLAG, the red the intensity of GEMIN5. C) SMN (red) co-localizes with Coilin (green) at level of cajal bodies in the nucleus. Merged images in A), B) and C) include DAPI (blue) staining.(TIF)Click here for additional data file.Figure S4REIIBP-Flag with an inactivating mutation in the SET domain co-localizes with GEMIN5 in HeLa::ΔSET. Immuno fluorescence and confocal analysis of HeLa::ΔSET cells. Colocalization of REIIBPΔSET-FLAG (green) with GEMIN5 (red). Merged image includes DAPI (blue) staining. Arrows show the colocalization signal from the cytoplasm.(TIF)Click here for additional data file.Table S1Transcript isoforms alternatively spliced between HeLa and HeLa::REIIBP. RNASeq result showing a list of differently expressed transcripts within genes where the overall expression was unchanged.(DOCX)Click here for additional data file.Table S2Gene ontology analysis of genes overexpressed in HeLa::REIIBP. List copiled using DAVID bionformatic resources (http://david.abcc.ncifcrf.gov/). Fold enrichment measures the magnitude of enrichment compared to the human genome. Fold enrichment >1.5 was considered as interesting. The percentage is the total number of genes involved in given term divided by the total number of input gene. P-value examine the significance of gene-term enrichment. P value <0.05 was considered significant.(DOCX)Click here for additional data file.Table S3Gene pathway analysis of genes overexpressed in HeLa::REIIBP cells. KEGG_PATHWAY analysis of genes overexpressed in HeLa::REIIBP cells. The list was copiled using DAVID bionformatic resources (http://david.abcc.ncifcrf.gov/). Fold enrichment measures the magnitude of enrichment compare to human genome. Fold enrichment >1.5 was considered as interesting. The percentage is the total number of genes involved in a given term divided by the total number of input genes. P-values examine the significance of gene-term enrichment. P value <0.05 was considered significant. PANTHER and REACTOME pathway analysis gave similar results.(DOCX)Click here for additional data file.Table S4Polycomb regulated genes deregulated in HeLa::REIIBP cells. RNASeq result showing differently expressed transcripts which are known to be silenced by the Polycomb complex.(DOCX)Click here for additional data file.Text S1Supplemental Experimental Procedures.(DOC)Click here for additional data file.


Vulpes (true foxes) comprises numerous species that inhabit a wide range of habitats and climatic conditions, including one species, the Arctic fox (Vulpes lagopus) which is adapted to the arctic region. A close relative to the Arctic fox, the red fox (Vulpes vulpes), occurs in subarctic to subtropical habitats. To study the genetic basis of their adaptations to different environments, transcriptome sequences from two Arctic foxes and one red fox individual were generated and analyzed for signatures of positive selection. In addition, the data allowed for a phylogenetic analysis and divergence time estimate between the two fox species.The genus de novo assembly of reads resulted in more than 160,000 contigs/transcripts per individual. Approximately 17,000 homologous genes were identified using human and the non-redundant databases. Positive selection analyses revealed several genes involved in various metabolic and molecular processes such as energy metabolism, cardiac gene regulation, apoptosis and blood coagulation to be under positive selection in foxes. Branch site tests identified four genes to be under positive selection in the Arctic fox transcriptome, two of which are fat metabolism genes. In the red fox transcriptome eight genes are under positive selection, including molecular process genes, notably genes involved in ATP metabolism. Analysis of the three transcriptomes and five Sanger re-sequenced genes in additional individuals identified a lower genetic variability within Arctic foxes compared to red foxes, which is consistent with distribution range differences and demographic responses to past climatic fluctuations. A phylogenomic analysis estimated that the Arctic and red fox lineages diverged about three million years ago.The Transcriptome data are an economic way to generate genomic resources for evolutionary studies. Despite not representing an entire genome, this transcriptome analysis identified numerous genes that are relevant to arctic adaptation in foxes. Similar to polar bears, fat metabolism seems to play a central role in adaptation of Arctic foxes to the cold climate, as has been identified in the polar bear, another arctic specialist.The online version of this article (doi:10.1186/s12864-015-1724-9) contains supplementary material, which is available to authorized users. Vulpes (foxes) comprises of twelve extant species that inhabit diverse habitats, including subtropics, deserts, temperate climatic zones as well as the Arctic [Vulpes lagopus) is the only fox species occurring in the Arctic and is adapted to its extreme climatic conditions. Arctic foxes have to cope with extremely cold and dark winters without being able to hibernate, followed by short summer seasons to reproduce. Several evolutionary adaptations such as insulating and camouflage colored fur, a compact body, and reduced metabolism during starvation or extremely cold weather enable Arctic foxes to survive under these harsh conditions [The genus e Arctic . The Arcnditions .Vulpes vulpes). The two species diverged from their most recent common ancestor approximately 2.9 million years ago (Ma) [A closely related species of the Arctic fox is the red fox (ago (Ma) . In contago (Ma) . The cloThe adaptations of the two fox species are expected to have left signatures in the protein-coding genes. A widely used method to identify candidate genes that are under positive selection is calculating the dN-dS ratio (ω). This is the ratio of the number of non-synonymous substitutions per non-synonymous site (dN) and the number of synonymous substitution per synonymous site (dS). An ω > 1 indicates positive selection, an ω < 1 indicates negative (purifying) selection, while an ω ≈ 1 implies neutral evolution . Differede novo sequence assembly of transcriptome data is less complex than for whole genomes. Therefore, the aim of this study is to produce transcriptomes of Arctic and red foxes for identifying candidate genes and metabolic pathways that might be involved in arctic adaptation and to estimate phylogenomic based divergence times. Genes under positive selection will be chosen from transcripts with high abundance in terms of Fragments Per Kilobase per transcript per Million mapped reads (FPKM) and transcriptomes from two fox individuals will be used to verify the sequence data. Compared with positive selection analyses of data from a single individual, this stringent approach reduces sequencing and assembly errors that can introduce a bias in positive selection analysis [Canis lupus), and we provide a resource of potential variable microsatellites, which will be useful in future population studies.Recent studies analyzing whole genomes of polar and brown bears, an Arctic/non-Arctic species pair with comparable habitat ranges and adaptations to that of Arctic and red foxes, identified a number of candidate genes involved in fatty-acid metabolism, cardiovascular functions, and fur color to be under positive selection . From thanalysis . In addide novo assembly of each of the three fox individuals are shown in Table Three transcriptome libraries were generated from pooled RNA-extracts from different tissues  of two Arctic fox individuals  and one red fox (Red fox). The sequencing produced more than 300 million reads with an average read length of 94 base pairs, bp  term, for Arctic fox1, Arctic fox2 and red fox respectively corresponding to an average of approximately 35 % of each of the final transcripts. The remaining unannotated transcripts might represent non-coding RNAs, 3′ or 5′ untranslated regions, or fox-specific transcripts. GO annotation based on the GOslim database divided the functional annotations into the three main ontology components: “biological process”, “molecular function” and “cellular component”. The distribution of the number of genes in the three different GO subcategories for each species is shown in Fig. After coding sequence (CDS) prediction of the transcripts, only CDS longer than 300 nucleotides (nt) were annotated to avoid spurious hits. All CDS were screened against the human protein database using BLAST and 82 % of the fox genes could be annotated  were identified to be under positive selection by both methods, pairwise and branch-site analyses. The reason for this limited overlap may be a consequence of different methodologies and dataset sizes. The ten candidate genes additionally identified by the branch site test complement the findings by the pairwise analyses. The annotation indicates that the identified positively selected genes of the branch site test are involved in molecular and cellular functions such as lipid metabolism and nuclear chromatin fragmentation ). The GTR + G model was identified as the most likely substitution model for the nt data and MIX + G was the preferred model for the aa data. Both the nt and aa data sets yielded the same tree topology in maximum likelihood analyses Fig. . DivergeIn total, approximately 70 % of the correctly paired reads from each of the three fox individuals could be mapped to the dog genome. Between the dog and each of the three fox individuals, in total 1,007,130 (Arctic fox1), 1,355,055 (Arctic fox2), and 1,162,694 (red fox) substitutions were found, respectively Fig. . Approxide novo assemblies. In total, 17 non-synonymous and four synonymous substitutions were identified among the three individuals. Amplification and sequencing of three additional fox individuals (one Arctic fox and two red foxes) revealed 22 additional substitutions  were selected for Sanger-sequencing to verify the quality of the assembled transcriptome data. Amplification and sequencing of the same fox individuals that were used for the transcriptome sequencing identified no differences between the Sanger and Illumina data confirming the high quality of A total of 952 and 969 di-, tri- and tetra-repeat unit microsatellites were identified in the Arctic and red fox transcripts respectively. While the sequences have not been tested for variability in population samples from the two species yet, they were selected for numerous repeat units. Thus, the identified microsatellite loci will be a valuable resource for future population studies , cardiac gene regulation (SRFBP1), apoptosis (TIA1) and blood coagulation (GGCX).We found that 1 % of the genes appear to be under positive selection (ω > 1), a value that is comparable to transcriptome studies in other mammals  and indiGLTPD1) [AKT2) [AKT2 is also involved in insulin signaling in adipose cells by binding to vesicles containing GLUT4 (glucose transporter) [GLPTD1 and AKT2 may play a role in adaptation to strong seasonal fluctuations as experienced by Arctic foxes. It is noteworthy that fatty-acid metabolism pathways have been identified to be under positive selection in a recent study on polar and brown bear genomes [Specifically, the branch site test for positive selection identified two genes related with fatty-acid metabolism to be under positive selection in the Arctic fox: a) the Glycolipid transfer protein domain containing 1 (GLTPD1)  and b) V) [AKT2) . The AKTsporter) . Insulinsporter) . Arctic sporter) . In addisporter) . Hence,  genomes . Energy  genomes . Thus thThe three fox transcriptomes yielded a large number of protein-coding genes that could be used for evolutionary studies. About 17,000 different protein-coding genes were identified by comparisons to the dog genome from each fox individual by pooling cDNA from three different tissues. This approach yielded 3,000 more genes per transcriptome than a previous study, based only on brain tissue . Thus, rVulpes macrotis) and the swift fox (Vulpes velox), two desert-adapted species occurring in North America [Vulpes corsac), a closely related species that is adapted to arid conditions [The CDS based time estimate for the divergence of the red and Arctic fox from a common ancestor at 3.17 +/− 0.09 Ma is slightly older than a previous estimate based on 22 nuclear loci and 3 mitochondrial (mt) genes and has a much narrower confidence range [ America , 32, whi America  the adapnditions , was estnditions , 34 coinCompared to the red fox, fewer heterozygous sites  were found in the transcriptome of the Arctic fox, a finding that was verified by Sanger sequencing in additional Arctic and red fox individuals. This is in agreement with previous studies that have identified a lower level of nucleotide diversity for mt DNA of Arctic foxes  comparedTranscriptome sequencing is an economic strategy to assemble a large number of protein-coding genes in non-model organisms for which genome sequences are not yet available. This first comparative transcriptome study of the Arctic and the red fox revealed a number of candidate genes that are possibly involved in adaptations to different climatic conditions. Fatty acid metabolism genes are under positive selection in the Arctic fox similar to the polar bear and seem generally to be crucial for mammalian survival under arctic conditions. The SNPs and a large number of microsatellites markers discovered in the fox transcriptome sequences will provide a rich resource to establish new markers for future population genetic studies of nuclear genes that can complement mt DNA based studies , 36, 39.Vulpes lagopus) and one red fox (Vulpes vulpes) within less than 24 h post-mortem and were stored in RNA later (Ambion) –phenol–chloroform extraction method . One ml Poly-adenylated RNA was captured using Oligo-dT magnetic beads . cDNA was generated using first and second-strand synthesis using the SuperScript II reverse transcriptase (Invitrogen) and a nested-poly-T oligonucleotide. A specific oligonucleotide was ligated to the 5′ end to allow for amplification as described by . NormaliDe novo assembly was performed separately for the three samples (two Arctic fox and one red fox samples) as well as for a pool of reads from both Arctic foxes. Large amounts of misassembled transcripts/contigs, erroneous and poorly supported contigs can arise during the assembly. Therefore, all reads were mapped back to their respective assembled transcripts using Bowtie2 [Raw sequence reads were cleaned for RNA adapters using Tagdust . Sickle  Bowtie2 . Abundan Bowtie2  and tran Bowtie2  to furthde novo assembly in the Uniprot database to obtain associated GO terms (ftp://ftp.ebi.ac.uk/pub/databases/uniprot/). Blast2GO assembled the GO terms into three functional GO categories, associated with biological processes, molecular functions and cellular components based on the annotation file from Sma3s [Sma3s program was used to search the transcripts  resultinom Sma3s . To reduom Sma3s  predicteom Sma3s  in the wVulpes vulpes), which are red foxes characterized by a coat-color variant [To identify only those orthologous genes present in all three fox individuals Proteinortho was used with all genes of the species in each orthologous group with algebraic connectivity of one . The dat variant , was dow variant  and cons variant . The silThe identified orthologous Arctic and red fox CDS were pairwise aligned and used for pairwise positive selection analysis to identify genes with an ω > 1. All CDS with dS = 0 were discarded. For the pairwise positive selection analysis, first all CDS sequences were translated and aligned using PRANK, which was shown to perform better compared to other sequence aligners used in positive selection analyses , 57. NexCanis lupus familiaris) and giant panda (Ailuropoda melanoleuca) from Ensembl (http://www.ensembl.org/info/data/ftp/index.html). Similar to the orthology screen in foxes, one-to-one orthologous genes present in all investigated carnivores were determined using Proteinortho [In addition to the pairwise selection analysis, a branch site test was performed to identify positive selection on the branches leading to the Arctic and red fox including two additional carnivorous species, dog (einortho . Ortholoeinortho  and wereeinortho  was usedUrsus maritimus) (http://gigadb.org). Utilizing the two Arctic fox CDS datasets, we generated a common Arctic fox CDS dataset by first identifying CDS present in both individuals using BLAT [We took the advantage of several available sequenced genomes to reconstruct the evolutionary history of Carnivora using a phylogenomic data set. The taxon sampling included the Arctic, red fox and the polar bear , dog , ferret , and cat . Orthologous genes in the seven species were identified using Proteinortho [Canini and Vulpini divergence at 12 Ma [Other carnivorous genomes were downloaded from the Ensemble database including giant panda (einortho . All seqeinortho . The proeinortho  and was einortho : 1) the einortho ; 2) the einortho ; and 3) at 12 Ma , 64. Divat 12 Ma . Mean vaat 12 Ma  bootstraat 12 Ma .The dog genome (canFam3) represents the evolutionarily closest available genome dataset compared to Arctic and red foxes and was therefore retrieved from the UCSC genome database. Single variable sites were obtained by mapping the reads from each of the three fox individuals to the dog genome using the STAR alignment tool including the 2-pass STAR method , 68. GATTo identify heterozygous sites (SNPs) for each fox individual, reads were mapped back to their corresponding assembled transcripts using BWA with default parameters . SamtoolTotal DNA was isolated from muscle samples from red and Arctic foxes  database. Study accession number: PRJEB7790. The Sanger sequences are available at ENA accession numbers LN680791-LN680850.


Cardiovascular disease is one of the leading causes of death in patients with advanced renal disease. The objective of this study was to investigate impairments in heart rhythm complexity in patients with end-stage renal disease. We prospectively analyzed 65 patients undergoing peritoneal dialysis (PD) without prior cardiovascular disease and 72 individuals with normal renal function as the control group. Heart rhythm analysis including complexity analysis by including detrended fractal analysis (DFA) and multiscale entropy (MSE) were performed. In linear analysis, the PD patients had a significantly lower standard deviation of normal RR intervals (SDRR) and percentage of absolute differences in normal RR intervals greater than 20 ms (pNN20). Of the nonlinear analysis indicators, scale 5, area under the MSE curve for scale 1 to 5 (area 1–5) and 6 to 20 (area 6–20) were significantly lower than those in the control group. In DFA anaylsis, both DFA α1 and DFA α2 were comparable in both groups. In receiver operating characteristic curve analysis, scale 5 had the greatest discriminatory power for two groups. In both net reclassification improvement model and integrated discrimination improvement models, MSE parameters significantly improved the discriminatory power of SDRR, pNN20, and pNN50. In conclusion, PD patients had worse cardiac complexity parameters. MSE parameters are useful to discriminate PD patients from patients with normal renal function. Cardiovascular disease is the leading cause of mortality and morbidity in patients with chronic kidney disease or end-stage renal disease (ESRD) undergoing either hemodialysis or peritoneal dialysis (PD)13Analysis of variations in heart rate oscillation, commonly known as heart rate variability (HRV), is commonly used to assess autonomic function in clinical studies due to its simple and noninvasive approach101213141516Only few studies evaluate heart rhythm complexity in ESRD patients181919Sixty-five patients (39 men) undergoing PD without prior cardiovascular disease and 72 individuals (36 men) with normal renal function (control group) were enrolled in this study. The reason of PD implementation were variable primary glomerulonephritis in 47 patients , diabetic nephropathy in 9 patients, lupus nephritis in 3 patients, polycystic kidney disease in 2 patients and other causes in 4 patients.The clinical data are shown in In linear analysis, the PD patients had a significantly lower standard deviation of normal RR intervals (SDRR), percentage of absolute differences in normal RR intervals greater than 20 ms (pNN20), the percentage of absolute differences in normal RR intervals greater than 50 ms (pNN50), low frequency (LF), high frequency (HF), and low/high frequency ratio than control participants .In DFA anaylsis, both DFA α1 and DFA α2 were comparable in both groups. The quantification of MSE parameters is shown in In analysis of all participants, scale 5, area 1–5 and area 6–20 were significantly correlated with both time and frequency domain parameters . In contIn receiver operating characteristic (ROC) curve analysis, scale 5 had the greatest discriminatory power for the two groups compared to all other linear and non-linear parameters . The areIn both net reclassification improvement (NRI) and integrated discrimination improvement (IDI) models, three MSE parameters  significantly improved the discriminatory power of SDRR, pNN20, and pNN50 . Scale 5The major findings of this study were: 1) the PD patients had worse heart rhythm complexity than those with normal renal function; 2) in all linear and non-linear parameters, scale 5 and SDRR had the greatest single discriminatory power to detect the patients undergoing PD; 3) the combination of linear and non-linear improved the discriminatory power to differentiate PD patients from patients with normal renal function.Traditional linear analysis of HRV is a useful tool to evaluate the autonomic system, and is commonly used to stratify the risk of patients with cardiovascular disease83422et al. demonstrated the superiority of DFA analysis compared to traditional linear parameters to predict 5-year mortality rates in hemodialysis patientsDFA is a scaling analysis method to represent the correlation property of a signal. The physiological background of DFA has been demonstrated to be associated with a delicate interplay between sympathetic and vagal outflowComplexity is a concept that lies between periodicity and randomness. Heart rate dynamics is a complex system. The nonlinear methods viewed the healthy heart beat fluctuation as an output of an integrative control system with multiple interacting physiological processes operating at different time scales which emerge as complex dynamical patterns. When disease status developed, the system breakdown into either periodic-like14142728The usefulness of MSE is not limited to the cardiovascular system, and it has also been shown to be able to predict the outcomes of patients with severe trauma requiring treatment in an intensive care unit and across the diverse spectrum of traumatic injuryet al. found the largest separation between heart failure patients and healthy subjects is obtained for scale 5. Interesting, the strongest separation was also obtained for scale 5 for separation from elder from young subjectsIn the present study, scale 5 had the best discriminatory power in both groups. Similar findings have also been reported in heart failure and older patientsPatients with ESRD carry a higher risk of developing cardiovascular disease including vasculopathy and cardiomyopathy secondary to pressure and volume overload12There are several limitations to this study. First, we only enrolled PD patients, and further studies are needed to validate whether our findings can be applied to hemodialysis patients. Second, this cross-sectional study aimed to investigate impairments in heart rhythm complexity in ESRD patients, and whether MSE parameters can predict the clinical prognosis is unclear.In conclusion, the PD patients had impaired cardiac complexity. Scale 5 in MSE studies had the greatest discriminatory power to differentiate PD patients from patients with normal renal function. In addition, MSE parameters significantly improved the discriminatory power of linear parameters.In this prospective, cross-sectional study, we enrolled 65 Taiwanese patients who received PD with conventional glucose-based lactate-buffered solutions  and 72 participants (control group) who visited for health check-up at National Taiwan University Hospital.The PD patients were enrolled prospectively for a cohort study. Some study results regarding echocardiography or laboratory data were published33The participants in control group were prospectively enrolled for this study. The inclusion criteria were 1) estimated glomerular filtration rate >60 ml/min according to the Chinese Modification of Diet in Renal Disease Study equationA medical history of each participant including demographics and medications was carefully recorded and biochemical parameters were measured at the first evaluation. All patients underwent 24-h ambulatory ECG Holter recording . Standard transthoracic echocardiography  was performed in each patient. This study was approved by the Institutional Review Board of National Taiwan University Hospital, and all subjects provided written informed consent including for storage of their information in the hospital database and usage for research. The methods in the study were carried out in accordance with the approved guidelines.An epoch of four hours of daytime RR intervals (between 9AM and 5PM) was selected for analysisSDRR was calculated and taken to represent the overall variability of autonomic modulation. Two pNNx parameters (pNN50 and pNN20) were calculated as the percentage of absolute differences in normal RR intervals greater than x ms. The threshold of x ms can filter out the RR changes with larger amplitudes, and better evaluate the function of the autonomic systemThe parameters derived from nonlinear methods were used to quantify the important characteristics of the physiological systems beyond variability such as scale-invariant  underlying the signals that vary with time26DFA can be used to evaluate the fractal behavior beneath the RR dynamics originating from well-regulated, interconnected systemsShort-term RR intervals are predominately modulated by respiratory sinus arrhythmia in normal subjects causing drastic changes in the slope between short- and long-term timescales. Crossover can be quantified by the α exponents of RR dynamics over short  and long  timescales to better probe the fractal property of the physiological systemInstead of merely estimating the predictability of a time series with a single scale, MSE provides meaningful information richness embedded in different timescales by the degree of predictable sequential changes over the timescaleTo avoid underestimation of entropy due to external non-stationarity, the RR dynamics were detrended by removing any trend longer than 2 hours before performing MSE. The empirical mode decomposition (EMD) method, which is based on Hilbert-Huang transformation, was used to adaptively extract the trends and subtract them from the original RR interval signals4041Standard transthoracic echocardiography  was performed in each patient. The echocardiographic measurements included two-dimensional, M-mode and Doppler ultrasound recordings. Left ventricular dimension, interventricular septum and posterior wall thicknesses, and left ventricular ejection fraction (M-mode) were measured via a parasternal long axis view.th–75th percentile). Comparisons of continuous data between the PD and control groups were made using the Mann-Whitney U-test. Differences between proportions were assessed using the chi-square test or Fisher’s exact test. Correlation tests were performed using Spearman’s correlation tests.Continuous variables were presented as median , version 2.15.2. Statistical significance was set at p < 0.05.NRI and IDI models were used to assess improvements in prediction using two different logistic regression modelsHow to cite this article: Lin, Y.-H. et al. Heart rhythm complexity impairment in patients undergoing peritoneal dialysis. Sci. Rep.6, 28202; doi: 10.1038/srep28202 (2016).