Drosophila subobscura. However, there is a lack of knowledge on the genetic variation in genes within inversions, possibly targets of climatic selection, across a geographic latitudinal gradient. In the present study we analysed four candidate loci for thermal adaptation, located close to the breakpoints, in two chromosomal arrangements of the sex (A) chromosome of Drosophila subobscura with different thermal preferences. Individual chromosomes with A2 (the inverted arrangement considered warm adapted) or AST  were sequenced across four European localities at varying latitudes, up to ~ 2500 Kms apart.The role of chromosomal arrangements in adaptation is supported by the repeatable clinal variation in inversion frequencies across continents in colonizing species such as ST orientation but not in the distal ones, independently of their location inside or outside the inversion. This can be possibly due to variation in the levels of gene flux and/or selection acting in these regions.Importantly, we found very low differentiation for each specific arrangement across populations as well as no clinal patterns of genomic variation. This suggests wide gene exchange along the cline. Differentiation between the sex chromosome arrangements was significant in the two more proximal regions relative to the AGene flow appears to have homogenized the genetic content within-arrangement at a wide geographical scale, despite the expected diverse selective pressures in the specific natural environments of the different populations sampled. It is thus likely that the inversion frequency clines in this species are being maintained by local adaptation in face of gene flow. The differences between arrangements at non-coding regions might be associated with the previously observed differential gene expression in different thermal regimes. Higher resolution genomic scans for individual chromosomal arrangements performed over a large environmental gradient are needed to find the targets of selection and further elucidate the adaptive mechanisms maintaining chromosomal inversion polymorphisms.The online version of this article (10.1186/s12862-018-1178-1) contains supplementary material, which is available to authorized users. There is increasing evidence of the role of chromosomal inversion polymorphisms in adaptation and speciation , 2. SeveSeveral hypotheses have been advanced to explain the adaptive character of inversions (see ), with tD. subobscura found an extensive genetic differentiation between arrangements occurring regardless of the distance to the nearest breakpoint, with selection likely implicated [In(3R)Payne of Drosophila melanogaster, both in North American and Australian localities, implicating a role of this inversion in the response to climatic selection [Empirical studies on the molecular variation associated with inversions have frequently detected high levels of genetic differentiation between chromosomal arrangements ; with an enrichment for candidate genes located inside arrangements [D. pseudoobscura with multiple genes presenting expression differences among arrangements [D. subobscura inversions as well as address their variation across the species geographic gradient to search for targets of climatic selection.One of the most emblematic examples of the adaptive role of chromosomal polymorphisms comes from  America . Thermalde scale . Also, sat waves  as well at waves –38 and bat waves , 39. Impngements . This sungements . ConsequD. subobscura and found no differentiation for the same arrangement between Barcelona and Greece indicating high levels of gene flow and the absence of geographic varying selection at least in those two southern regions. In a study in D. subobscura involving several localities across western Europe, Simões et al. [2), suggesting adaptation at neighbouring loci.Pegueroles et al.  analyseds et al.  using mis et al. , 43). NeDrosophila subobscura across a geographic gradient. Specifically, we intend to (1) characterize genetic variation and differentiation between arrangements, addressing the impact of inverted regions at the genetic level; (2) analyse patterns of within-arrangement differentiation, searching for evidence of different epistatic combinations in different populations; (3) identify latitudinal patterns of genetic variation, which might indicate targets of climatic selection at a geographical scale. In this study we targeted AST and A2, the two most frequent inversions of the A chromosome in our sampled localities. These two arrangements present clinal variation of frequencies in Europe, as well as in the two recently colonized American hemispheres, with A2 increasing frequency towards warmer locations and AST showing the corresponding opposite pattern [PhKgamma, Ubc-E2H, Hfw and Nipsnap). These genes are good candidates to search for evidences of climatic latitudinal selection associated with the Drosophila subobscura inversion clines as they (1) have been previously implicated in thermal adaptation , and the only two arrangements present in all analysed locations  with an estimated age of 160.000 years [2   chromosome . This p2 or AST) had been previously scored as described above. By amplifying and directly sequencing males we avoided the problems associated with sequencing of diploid data. The karyotype of these males was inferred from their descendants (see above) making it possible to obtain both genetic and karyotypic data for the same individual. Genomic DNA extraction was performed following the protocol described in [Nipsnap that was localized nearby but outside the inversion  and Nipsnap  are located outside the A2 inversion, and Ubc-E2H  and Hfw  within that inversion  is involved in lateral inhibition and protein polyubiquitination, PhKgamma (Phosphorylase kynase enzyme) is involved in embryonic morphogenesis and glycogen metabolism and Hfw  is involved in intracellular signal transduction and pupariation and no involvement in biological processes is yet ascribed to the Nipsnap gene although a putative role in vesicular transport has been proposed (the function of each gene is according to Flybase). Primers used for amplification and sequencing reactions are listed in Table PhKGamma and around 0.574 Mb for the other 3 genes  whose chromosomal arrangements , number of polymorphic sites (S), the nucleotide diversity (π), nucleotide diversity in synonymous sites and non-coding positions (πsil), heterozygosity in silent sites (ϴsil) and divergence per silent site (Ksil) using D. pseudoobscura as outgroup [ST [xy [PhKgamma and Ubc-E2H gene regions, pairwise FST values were used to compute a Principal Coordinate Analysis (pcoa in R package ape version 3.5 (http://ape-package.ird.fr/). A locus-by-locus analysis of Molecular variance (AMOVA) was performed on Arlequin v3.5.2 [CT) between arrangements at each specific polymorphic site. A hierarchical structure was considered with two groups corresponding to the two arrangements (A2 vs AST), with the different localities and individuals sampled within these groups.Several standard parameters for each locality and arrangement were estimated using DnaSP v5 : the numoutgroup , with throup [ST  and Dxy  [ST [xy  as well  [ST [xy  were obt [ST [xy  was appln v3.5.2  to test ZnS was estimated for informative sites. LD was also estimated between polymorphic informative sites through r2 estimates. Deviations from neutrality were tested using Tajima’s D [2 [D. subobscura genes whether located inside or outside chromosomal arrangements (see [Gene conversion tracts (GCTs) were detected using the algorithm defined by . Linkagejima’s D  for eacha’s D [2 . Statistnts (see ). All nents (see  was usedST and A2), we analysed the four genomic regions in the two most geographically distant localities (Málaga and Groningen), and two gene regions for the two central localities (Rasquera and Dijon) . No differences in the divergence per silent site values (Ksil) relative to D. pseudoobscura were found between the two arrangements . Higher nucleotide diversity was found for Hfw and Nipsnap  and Ubc-E2H .As low levels of within-population variation – as is the case in stimates , 64 we hST individuals from different locations in non-coding regions, but not for A2 individuals. Considering each gene region, we found high differentiation between arrangements in upstream regions for Ubc-E2H and both upstream and intronic regions for PhKgamma while no relevant differentiation was observed for the other two genes in any region analysed  between arrangements at each specific polymorphic site using a hierarchical structure (see Methods). Again, differentiation between arrangements was observed exclusively in non-coding regions  and non-coding (non-exon) regions for the four genes concatenated data set for Málaga and Groningen , showed that the significant differentiation between groups of individuals carrying different arrangements was due solely to variation in non-coding regions , or AST . Similar results are also obtained for the two genes separately . For Ubc-E2H, one fixed difference was found between the two arrangements (position 60 in the 5’UTR). No fixed differences were found in the other two genes.Gene conversion tracts (GCT) were detected only in 2) showed only 35 significant comparisons out of a total of 2415 . Importantly, the majority of the significant LD comparisons were observed between sites from the same gene region, indicating short-range LD . In the analysis including only the individuals carrying the A2 inversion just 3 out of a total of 496 comparisons indicated significant LD after Bonferroni correction . However, in these three cases, the significant LD comparisons were always between sites within the same gene region  were found between arrangements and sites in PhKgamma  and also in Ubc-E2H . Interestingly, all positions in PhKgamma are also in complete LD among themselves . All these sites are located in non-coding/intronic regions. The fixed position in the Ubc-E2H gene is located 547 bp upstream from the first exon. The fixed positions at 358–360 bp in the PhKgamma gene are located in an intronic region just 26 bp downstream of the first exon. Comparison against D. pseudoobscura orthologous gene indicates more similarity to AST sequences. The fixed position between arrangements at 205 bp is within the 5’UTR region 36 bp upstream of the start coding site of PhKgamma, and possibly within the core promoter region, despite the absence of characteristic sequence motifs such as TATA box and the TFIIB recognition element [In general, levels of linkage disequilibrium (LD) were low in all gene regions and arrangements analysed  presented significant differentiation between arrangements particularly in non-coding regions, regardless of the arrangement’s geographical origin. Several molecular studies in Drosophila have documented significant genetic differentiation between chromosomes carrying different arrangements, particularly within inverted regions [2 - around 160.000 years [PhKgamma and Ubc-E2H did not show similar patterns [Our study shows that these arrangements shape the patterns of genetic variation likely by reducing recombination between certain regions within chromosomes. In fact, the two proximally located regions in A regions , 33, 66  regions , 21, 23) regions , 18. Fur regions ). In add regions , particu regions , 69. Thi regions . In our  regions  the age patterns  - namelyST/A2 complex, Nóbrega et al. [2. In our study, we show that the reduction of recombination is not homogeneous in regions around the vicinity of the breakpoints of the AST/A2 complex, with significant genetic differentiation and linkage disequilibrium between inversions occurring only in the proximal region of AST. Interestingly, linkage disequilibrium occurred in one region (PhKgamma) located outside the inversion, although near the breakpoint. Other studies have suggested that suppressed recombination can extent to 2.5 Mb or more outside inversion breakpoints [PhKgamma and Ubc-E2H is in fact adaptive.In the specific context of the Aa et al.  also fouakpoints , 71. FunPhKgamma and Nipsnap have shown to be positively associated to latitude in semi natural populations of D. subobscura from a similar geographic gradient, but not when the same populations were maintained in a common garden [We did not find evidence for geographical (i.e. climatic) selection within arrangements, as very low within-arrangement genetic differentiation was detected across localities. It is possible that the differences in gene expression described previously for these genomic regions between thermal regimes  were conn garden . Thus, aD. subobscura found very limited evidence for geographical molecular variation within inversions. Pegueroles et al. [D. subobscura and found no significant differentiation within each arrangement for individuals sampled in Barcelona (Spain) and Mount Parnes (Greece). Evidence for similar patterns of generally low within-arrangement differentiation was also described in other D. subobscura studies [D. melanogaster Payne [Other studies in s et al.  also ana studies , 74 as wogaster Payne , 34, perAtpα gene, another candidate gene for thermal adaptation, Pegueroles et al. [Within-arrangement differentiation across localities would be expected if different epistatic interactions evolved  in different localities due to adaptation to the specific local conditions as proposed by the Dobzhansky’s coadaptation model . The lacs et al.  found stD. subobscura localities of the European cline found very low within-arrangement differentiation across localities as well as no clinal patterns of genetic variation. This indicates that gene flow across the European continent has homogenized the genetic content of these potentially selected regions at a wide geographical scale. Given the strong effect of gene flow in this species, it is possible that considerable within-arrangement differentiation at a geographical scale is only maintained at relatively few loci subjected to stronger selective pressures in the different environments. In this scenario, one can argue that restricted gene flow cannot explain the observed inversion frequency clines and that local adaptation is maintaining the clinal variation in this species, counteracting the homogenizing effects of the high gene flow. We also found an important effect of the AST/A2 complex in shaping genetic variation and preventing relevant gene exchange in different genomic regions, although its impact was not uniform across the different regions analysed. More generally, our study adds to a body of evidence showing that inversions act as barriers to recombination even outside inversions and can potentially have a role in the spread of favourable genic combinations and consequent adaptation to local habitats.Our genetic analysis of candidate loci for thermal adaptation  across gAdditional file 1:Nipsnap gene. (PDF 108 kb)New hybridization location for Additional file 2:PhKgamma and Ubc-E2H. A) Genetic differentiation (FST) between all localities and arrangements in PhKgamma  and Ubc-E2H . B) Genetic differentiation (Dxy) between all localities and arrangements in PhKgamma  and Ubc-E2H . (XLSX 12 kb)Measures of genetic differentiation within and between arrangements for Additional file 3:Nucleotide diversity (π) for all genes, arrangements and localities. (PDF 178 kb)Additional file 4:ST) between Malaga and Groningen in Hfw, Nipsnap and the concatenated dataset (four loci). (XLSX 10 kb)Genetic differentiation  for coding and non-coding regions in the concatenated dataset for all 4 genes (A) and for coding, intronic and 5’ UTR regions for PhKgamma (B). (XLSX 13 kb)Genetic differentiation  for all genes. 5’UTR, intronic and exonic regions are discriminated  and Ubc-E2H (B). (PDF 344 kb)Genetic differentiation between arrangements for each locality in Additional file 8:Linkage Disequilibrium parameters for all regions and the concatenated dataset. (XLSX 10 kb)Additional file 9:2) against the nucleotide distance between compared sites. Significant comparisons after Bonferroni correction are presented in red . A) LD between arrangements; B) LD within AST; C) LD within A2. (PDF 248 kb)Linkage disequilibrium (LD) between pairs of polymorphic sites


Streptococcus thermophilus in dairy plants, DNA sequence information relating to these phages remains limited. Genome sequencing is necessary to better understand the diversity and proliferative strategies of virulent phages. In this report, whole genome sequences of 40 distinct bacteriophages infecting S. thermophilus were analyzed for general characteristics, genomic structure and novel features. The bacteriophage genomes display a high degree of conservation within defined groupings, particularly across the structural modules. Supporting this observation, four novel members of a recently discovered third group of S. thermophilus phages (termed the 5093 group) were found to be conserved relative to both phage 5093 and to each other. Replication modules of S. thermophilus phages generally fall within two main groups, while such phage genomes typically encode one putative transcriptional regulator. Such features are indicative of widespread functional synteny across genetically distinct phage groups. Phage genomes also display nucleotide divergence between groups, and between individual phages of the same group (within replication modules and at the 3′ end of the lysis module)—through various insertions and/or deletions. A previously described multiplex PCR phage detection system was updated to reflect current knowledge on S. thermophilus phages. Furthermore, the structural protein complement as well as the antireceptor  of a representative of the 5093 group was defined. Our data more than triples the currently available genomic information on S. thermophilus phages, being of significant value to the dairy industry, where genetic knowledge of lytic phages is crucial for phage detection and monitoring purposes. In particular, the updated PCR detection methodology for S. thermophilus phages is highly useful in monitoring particular phage group(s) present in a given whey sample. Studies of this nature therefore not only provide information on the prevalence and associated threat of known S. thermophilus phages, but may also uncover newly emerging and genomically distinct phages infecting this dairy starter bacterium.Despite the persistent and costly problem caused by (bacterio)phage predation of  Streptococcus thermophilus in the dairy industry has been well described -Cas phage-resistance system relies on the acquisition of short genomic regions from the infecting phage  could be assigned to either the so-called cos-containing or pac-containing groups  of a representative 5093 group phage was defined, significantly updating the genomic annotation of this phage group.It is clear that there is a need to expand the amount of genetic information available in this field, particularly in light of the emergence of genetically divergent phage groups described above. Here, we present whole genome sequences of 40 phages infecting Streptococcus thermophilus strains were routinely grown from single colonies or from 10% Reconstituted Skimmed Milk (RSM) stocks overnight at 42°C in M17 Broth  supplemented with 0.5% lactose . L. lactis NZ9000 was maintained at 30°C in M17 broth containing 0.5% glucose , while NZ9000 derivatives containing pNZ8048-based constructs were maintained in GM17 with the addition of 5 μg/ml chloramphenicol . Phage enumeration was performed using a standard method  and either 10 g/L (solid agar base) or 4 g/L (semi-solid overlay) Technical Agar . Whey samples from dairy plants producing fermented milk products were obtained and analyzed for the presence of phages against S. thermophilus using spot and plaque assay methods as described above. Single plaque isolates were then propagated as follows: 10 ml LM17 broth was inoculated with 100 μl of the appropriate host strain and grown at 42°C for 1.5–2.0 h. Using a sterile pipette tip, a single, well defined plaque was added to the growing culture, thoroughly mixed and incubated for a further 2–4 h or overnight. The lysed culture was centrifuged and the supernatant filtered (0.45 μm). The filtered supernatant was stored at 4°C and used as stock for subsequent assays. This process was repeated at least twice prior to DNA sample preparation and subsequent genome sequencing.g for 15 min (to concentrate) and resuspension in 5 ml TBT Buffer , 10 mM MgCl2, 20 mM CaCl2; Sigma-Aldrich). The suspension was extracted at least twice using an equal volume of chloroform  and phages were purified by a discontinuous (3 M/5 M) cesium chloride (Sigma-Aldrich) gradient centrifugation at 76,000 × g for 2.5 h. Translucent blue bands visible at the interface of the gradient after centrifugation were carefully removed using a syringe and dialyzed against 50 ml TBT overnight at 4°C. Phage preparations were stored at 4°C until required for electron microscopy and DNA extraction.Individual phages Table  were proPhage DNA was prepared using existing methods  was incubated at 37°C for 17 min prior to electrophoretic examination as described above.Unique phages were distinguished (prior to DNA sequencing) by restriction profile analysis using EcoRV or HaeIII FastDigest (Thermo Scientific) enzymes, whereby a reaction mixture containing 8 μl phage DNA, 1 μl enzyme, 2 μl 10X FastDigest Buffer and 9 μl sterile distilled water  random shotgun sequencing using pyrosequencing technology was performed  using a 454 FLX instrument yielding at least 65-fold coverage for each genome, with the exception of phage 7632, which yielded 17-fold coverage. Individual sequence files were assembled using GSassembler , generating a consensus sequence for each phage; (ii) The remaining genomes were determined by GenProbio srl  using a MiSeq sequencer . Genomic libraries were constructed employing the TruSeq Nano DNA LT Kit (Illumina) and using 200 ng of genomic DNA, which was fragmented with a Bioruptor NGS ultrasonicator  followed by size evaluation using Tape Station 2200 (Agilent Technologies). Library samples were loaded into a Flow Cell V3 600 cycles (Illumina) according to the technical support guide. The generated paired-end reads (2 × 251 bp read lengths) were depleted of adapter sequences, quality filtered and assembled through the MIRA software program , and its attached 6xHis N-terminal-specifying purification tag and appropriate restriction enzyme recognition sequence was PCR amplified ], cell lysis, and sonication as previously described  and resuspended in 225 μl 5 pfu/ml were added to the cells or control and the mixture was incubated at 42°C for a further 12 min before centrifugation at 20,000 × g for 3 min to pellet bound phages with the cells. Remaining phages were quantified by plaque assay (as described above). Adsorption to wild-type and antireceptor-incubated cells was calculated as follows: (control phage titer − sample phage titer)/(control phage titer) × 100. Adsorption inhibition, expressed as a percentage of phage adsorption to wild type cells, was calculated as follows: (% adsorption on WT − % adsorption on incubated cells)/(% adsorption on WT) × 100.Adsorption inhibition assays were performed using a method adapted from two previously reported methods , which is consistent with previous observations for phages infecting this species .A total of 40 phage isolates Table  were genS. thermophilus strains), and (iii) diversity (as determined by host range and restriction profiling of phage genomic DNA). For the purpose of this study, the results (and discussion thereof) below pertain to the subset of 40 phages whose genomes were sequenced, unless otherwise indicated.Three main criteria were used to select phages for sequencing: (i) phages which did not return a PCR product using the above mentioned phage grouping PCR and which were therefore considered novel, (ii) persistence and/or prevalence , a dominance also observed—though to varying degrees—in previously analyzed phage collections  and 5093  phage groups produce products of different sizes , these newly designed primer sets are fully compatible with each other and with the cos and pac phage-detecting pairs , tail-associated lysin (TAL), antireceptor and baseplate components) was carefully performed in this study, in particular for members of the 5093 group, and also exhibit functional synteny between phage groups despite substantial nucleotide divergence , confirming the genetic conservation of members of the 5093 lineage. The amino acid divergence between proteins encoded by the structural regions of this and other groups, previously observed by Mills et al.  two distinct groups, namely the Sfi21- and 7201-like, or Group I and II  is often characterized by insertions, deletions, and point mutations leading to a large degree of heterogeneity  DNA recombination are encoded by 12 phages in the present collection-either singly or in combination Table . These pcos-sites) was found to correlate with mode of DNA packaging in each phage. Those phages utilizing the cos mode of DNA packaging possessed a terminal HNH endonuclease, whereas those predicted to utilize the pac mode did not , this conclusion cannot be drawn, despite the exponent value of <0.5  sequenced in this study, the 987 group phages as well as 13 previously sequenced  5 Figure . The incS. thermophilus  MTase-positive phage  systems , as well as those sequenced as part of this study were observed to encode an Ltr-like protein, which either belongs to the ArpU family or the Ltrb family  in phages infecting Gram-positive bacteria have been implicated in DNA packaging and lysis  is purported to be the main host range determinant , and found .The structural protein complement of phage 0095, as a representative of the 5093 phage group, was determined by mass spectrometry Figure  and CHPCS. thermophilus phages (discussed above) also applies to phage 5093, ORF295093 is predicted to encode the TMP, known to be responsible for phage tail length , based on a HHPred-mediated match for this product to the Dit protein of Bacillus phage SPP1 (P = 6.2 × 10−31). The product of the next gene (ORF315093) shows similarity to phage endopeptidases using BLAST and Pfam searches, and its position on the genome is consistent with a common lactococcal phage tail structure, namely the tail-associated lysin or TAL  in this group of phages s Figure . These s0095 was heterologously expressed and purified  was included as a negative control to indicate the specificity of RBP0095 in inhibiting the adsorption of phage 0095  between the gene products  undertaking of https://www.ncbi.nlm.nih.gov/genbank/, under accession numbers KY705251-KY705290, inclusive.The datasets generated and/or analyzed during the current study are available in the GenBank repository, BM was involved in the experimental design and work and prepared the manuscript. JM was involved in the experimental design and work and data analysis interpretation. LH performed the initial processing of whey samples and isolation of phages. HN performed the electron microscopy. JN performed the mass spectrometry. GL and MV performed the Illumina sequencing of phage genomes. TK was involved in the experimental design and manuscript editing. DS was involved in the experimental design, data interpretation and manuscript editing and preparation. All authors read and approved the manuscript.A patent application describing elements of this work has been submitted. BM is funded by DSM Food Specialties, and LH and TK are employees of DSM Food Specialties. The other 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.


Dear Editor,1 Despite appropriate therapy, acute OM can progress to chronic OM (COM).2 One form of COM is chronic suppurative OM (CSOM), which is generally caused by Pseudomonas aeruginosa (PA).3 The most common sequelae of CSOM is sensorineural hearing loss (SNHL) characterized by auditory hair cell damage.4 However, the molecular mechanisms underlying auditory hair cell damage that causes hearing loss during CSOM are not known. We argued that inflammatory mediators generated during PA infection of the middle ear epithelial cells (MEECs) can pass from middle to inner ear and affect the viability of auditory hair cells. To examine this hypothesis, filtered supernatants from 2- and 16-h PA-infected human MEECs (HMEECs)5 were used to treat the primary organ of Corti explant cultures from 3-day-old rats6 for 24 h, and then the explants were subjected to confocal and scanning electron microscopy (SEM). As a control, a supernatant from uninfected HMEECs was used. The samples were stained with fluorescein isothiocyanate phalloidin to visualize hair cells (HCs). The explant cultures treated with supernatant from uninfected HMEECs showed three rows of completely preserved outer (OHCs) and one row of inner (IHCs) hair cells  comprises a group of complex inflammatory and infectious disorders affecting the middle ear.ir cells . In contand 16-h . Quantifand 16-h . Abnormaand 16-h . This wa7 MyD88 is used by almost all TLRs (except TLR3) to start the signaling cascade.8 Therefore, to determine whether MyD88 pathway is involved in auditory HC damage, the expression of MyD88 was silenced in HMEECs by transfecting it with MyD88 siRNA and then infecting with bacteria. HMEECs transfected with scrambled siRNA and infected with bacteria served as a positive control group, whereas supernatants from uninfected and untransfected cells served as a negative control. Intriguingly, the explant cultures treated with supernatants from PA-infected HMEECs transfected with MyD88 siRNA or uninfected cells showed little or no damage to OHCs and IHCs , stimulates the release of inflammatory mediators.and IHCs . Howeverin vitro cell culture model can be used to understand the pathogenetic mechanisms employed by otopathogenic PA to cause inner ear damage. This cell culture model will also be of immense significance to understand middle–inner ear interaction during the pathogenesis of OM. Further studies are warranted to characterize the role of MyD88 in CSOM that will open up avenues to design novel treatment modalities against the disease.In summary, this study for the first time demonstrated that interaction of PA with MEECs triggers the generation of inflammatory mediators that causes auditory HC damage, which is dependent on the MyD88 pathway. We believe that this


To maintain thermal homeostasis, specific thermogenic tissues are under the control of central thermoregulatory networks that regulate the body's response to thermal challenges. One of these mechanisms involves non-shivering thermogenesis in brown adipose tissue (BAT), which is activated in cold environments in order to defend the body against physical damage as a result of hypothermia. The objective of our study was to assess the interaction between CNS thermoregulatory pathways and sympathetic innervation in BAT during a cold exposure paradigm. Our results show that an innocuous whole-body cooling paradigm induces significant differences in fMRI BOLD signal at the location of the right anterior insula and the red nucleus/substantia nigra region, between lean subjects with high levels of sympathetic innervation in supraclavicular BAT (BAT+ group), and subjects with low levels of sympathetic innervation (BAT− group). Specifically, results indicate significantly larger fMRI BOLD signal changes between periods of cooling and warming of the skin in the BAT+ (as compared to BAT−) group at the location of the right anterior insula. In contrast, the BAT+ group showed significantly smaller fMRI BOLD signal changes in the midbrain between periods of skin cooling and warming. Our findings are consistent with a hierarchical thermoregulatory control system that involves the initiation of inhibitory signals from the right anterior insula toward midbrain areas that normally exert tonic inhibition on the medullary raphe, from where BAT is directly innervated. Our data suggests that exposure to cold elicits differential neuronal activity in interoceptive regulatory centers of subjects with high and low level of sympathetic innervation. As a result, the variability of cold-activated BAT mass observed in humans might be, in part, yoked to different sensitivities of interoceptive cortical brain areas to skin temperature changes. We have previously studied sympathetic innervation in human supraclavicular BAT (both at rest and during cold exposure) using PET/CT imaging with 11C-meta-hydroxyephedrin, a structural analog of the neurotransmitter norepinephrine  is to protect the body against damage caused by hypothermia. One of several thermoregulatory mechanisms contributing to the generation of heat in order to maintain thermal homeostasis is the activation of peripheral brown adipose tissue (BAT) by the sympathetic nervous system (SNS). The presence of BAT in supraclavicular regions of humans that can be activated by cold exposure has been well documented  induced experimenter-controlled changes in thermoregulatory defense will induce differential changes in BAT and (b) sub-groups with differential effects in cold-activated BAT will also be characterized by systematic differences in the range of their fMRI responses to cooling and rewarming. These motivations drove the current multi-modal imaging study in which we acquired within-participant data using two modalities (PET and fMRI).We first employed 18F-fluorodeoxyglucose (FDG) PET imaging to stratify subjects into groups according to their cold-activated BAT mass, an indicator of peripheral sympathetic innervation levels  subjects were studied (Table 2 (kg/m2). Percent body fat (%) was calculated from the sum of skinfold measurements at the biceps, triceps, subscapular and suprailiac sites using Lange calipers. The lean body mass  was subsequently calculated as body weight less fat mass. This study was carried out in accordance with the recommendations and approval of the Wayne State University Human Investigation Committee. All subjects gave written informed consent in accordance with the Declarations of Helsinki. The protocol was approved by the Wayne State University Institutional Review Board.A total of 20 lean healthy subjects  PET imaging during mild cold condition in order to assess the presence of cold-activated supraclavicular BAT. Whole body changes in daily energy expenditure (DEE) between the cold and thermoneutral condition were assessed using indirect calorimetry and related to changes in BAT DEE. In addition, to assess functional brain responses to cold stress, all subjects underwent a functional MRI (fMRI) scan paradigm. During fMRI, subjects underwent a carefully manipulated oscillating whole body temperature paradigm. The paradigm induced periods of mild hypothermia which was interspersed by periods designed to return the body to basal core temperature. The fMRI study was performed on a separate day and within a week of the PET scan. Finally, all studies were performed during the spring/summer months, thus differential activation of BAT due to outside day temperature is unlikely.A histogram of cold-activated BAT mass showed two distinct peaks with maxima at 10–20 g and at 90–100 g. To account for the bimodal distribution, subjects were divided into two groups (High-BAT or BAT+ and Low-BAT or BAT−), with the threshold separating the two groups set to 20 g of activated BAT  was injected intravenously, and following a 50 min uptake period (during which cooling was maintained), a four bed-position PET/CT scan was acquired, covering the torso from the shoulder to the lower abdomen. All PET images were corrected for attenuation as well as tracer decay and reconstructed using the OSEM iterative reconstruction, yielding a final spatial resolution of ~7 mm full-width-at-half-maximum (FWHM).3. The final BAT ROI was chosen at the location of the largest contiguous group of voxels that survived the masking operation. In case that no voxels survived the masking operation (no BAT activation), a volume of ~10 cm3 (1.5 × 1.5 × 4.0 cm3 ROI) was selected at a typical location of supraclavicular BAT. The volume of BAT ROIs (cm3) was converted into weight (g) by assuming a density of 0.90 g/cm3.Activated BAT was defined as tissue areas that were more than 5 mm in diameter and had a minimal standard uptake value  of FDG of at least 2.0. This cutoff represented more than 2 SD above the maximal SUV seen in typical depots of white adipose tissue . Subsequently, a gradient echo EPI fMRI data acquisition was conducted . These parameters allow acquisition of fMRI data with high in vivo spatial resolution. The total study time was ~1 h.Structural and functional MRI data acquisition was performed on a 3T Siemens Verio system using a 12-channel volume head coil. Initially, a 3D T1-weighted anatomical MRI sequence was acquired  resulting in a sequence of 26 min duration blocked into five 5-min epochs  that reflects high rates of skin temperature change in response to the stimulus and (b) periods when skin temperature remains relatively stable , most likely reflecting physiological adaptation. These distinct epochs constituted separable physiological predictors of the BOLD response, and were employed to construct epochs of interest for fMRI analyses. Each epoch was modeled with a temporal radius of 1.5 min centered at either (a) points of the highest rates of skin temperature change (in the negative or positive direction) or (b) at points of relatively constant skin temperature . Thus, in each participant these first level models estimated neuronal responses during (relatively) rapid skin temperature transitions when thermoregulatory demands were maximal, separately from periods of relatively stable skin temperature that reflected adaptation following temporary relaxation of the stimulus.The resulting skin temperature curve Figure  is chara2; l/min), carbon dioxide production  and respiratory quotient  were all measured for 10 min. Whole body energy expenditure was calculated from VO2 and RQ values using the BreezeSuite software . Energy expenditure was averaged over 8 min  and then multiplied with a factor 1,440 min/day, yielding a daily energy expenditure .Measurement of energy expenditure  under resting (prior to cold exposure) and cold (90 min into cold exposure) conditions was performed using the MedGraphics VO2000 Portable Metabolic Testing System . For both conditions, subjects were measured while lying in a relaxed position, in a fasting state (≥6 h). A neoprene face masks was fitted for each subject and heart rate , oxygen consumption . Normally distributed continuous variables were compared between the BAT+ and BAT− groups using an independent sample 3). To remove low-frequency scanner drift, a high-pass filter (cutoff 1/128 s) was applied. Subsequently, an established SPM analysis pipeline was applied, as reported previously   young (23.3 ± 3.8 years of age) healthy controls (10M/10F) (Table p < 0.01). Overall, we determined a significant decrease in heart rate (HR: −5.8 ± 4 bpm) and significant increase in mean arterial pressure (MAP: 11 ± 8 mmHg) during cold exposure. However, there was no significant difference with respect to HR and MAP between the BAT+ and BAT− groups.The study population consisted of lean (BMI = 23.0 ± 2.6 kg/mcontrols 0M/10F (Tp < 0.01), whereas it remained on average virtually constant (−2.8%) in the BAT− group   to mild cold exposure. In the BAT+ group the baseline DEE was lower than in the BAT− group, however during mild cold exposure the DEE increased in the BAT+ group by about 17%  was marginally lower in the BAT+ as compared to the BAT− group , suggesting greater levels of resting fat oxidation in this group . In the second level analyses we compared activations between the two groups to identify clusters in core thermoregulatory and interoceptive regions. The identified clusters reflect differential responses between the two groups to body temperature changes during cooling and warming periods. To supplement the spatial profiles of the observed differences in activation, in conjunction we supply the temporal profiles of the smoothed  showed a significantly greater range of the fMRI response between periods of cooling and warming of the skin in the right insula, as compared to the time series for the BAT− group  than in the BAT− group  relative to participants with lower sympathetic innervation (BAT−): (a) BAT+ showed greater CNS sensitivity to skin temperature changes in the right anterior insula but (b) decreased CNS sensitivity of suprapontine midbrain and cerebellar structures. These effects are discussed in greater detail in the Discussion section.R2 = 0.273, σ = 0.574, s for residual = 0.573), relative to the BAT− group  or cooling (cold). These analyses discovered a single cluster in the substantia nigra Figure  characteOur work demonstrates differences in core interoceptive and regulatory brain areas that effect sympathetic innervation in cold-activated supraclavicular BAT. Differences in neuronal activity between subjects was assessed using co-acquisition of fMRI BOLD signals and skin-temperature changes during an oscillatory thermoregulatory challenge. The applied challenge induced oscillatory and dynamic skin temperature changes during cooling and warming periods, interspersed with periods when skin temperature remained relatively stable (following adaptation as the applied paradigm shifted between cooling and warming).We restate our CNS results that were obtained comparing subjects with high and low peripheral sympathetic innervation in supraclavicular BAT: (1) The group with increased peripheral sympathetic innervation (BAT+ group) showed increased neuronal activity (and activity range) in the anterior insula during cooling relative to warming of the skin Figure ; (2) TheIt is well established that BAT thermogenesis is mediated by relatively old cytoarchitectonic brain structures that insure autonomic control of BAT; these include most importantly the preoptic anterior hypothalamus (POAH) and various nuclei of the brainstem  that is under the control of higher brain areas such as the insula, orbitofrontal cortex and superior parietal cortex  displayed a higher response range of fMRI BOLD signal to cold in the BAT+ group. Consequently, the observed decrease of midbrain BOLD signal in the BAT+ group can be interpreted as the removal of a tonic inhibitory mechanism on infrapontine brain stem areas , resulting in BAT activation through disinhibition-induced stimulation of the central sympathetic nervous system.Previous evidence for the involvement of midbrain nigro-striatal structures to thermoregulatory control provides further support for our findings. For example, lesions in rodents at the location of the substantia nigra result in decreased metabolism and hypothermia when exposed to cold, although no deficiencies in thermoregulation were detected at both moderate and hot environmental temperature  to cold in the BAT+ group appears to be responsible for the dominant pattern of DEE increases during cold exposure. In contrast, the lower sensitivity of the anterior insula to cold accompanied by continuous tonic inhibition of infrapontine brainstem areas responsible for BAT activation in the BAT− group might be the underlying basis for the observed mixed DEE response during cold. Our data both duplicates as well as provides a rationale for previous findings that showed significant increase in DEE during cold exposure in a BAT+ group but not in a BAT− group . Thus, the possibility for these effects to influence activation cannot be excluded, despite the fact that post-experimental debriefing indicated that subjects perceived the maximum stimulus as “very cold,” but not painful.Because we did not directly measure temperature from Additional limitations of our study are related to some temporal and spatial constraints that are common to nearly all fMRI studies. Our spatial resolution was high relative to many studies (2 × 2 mm in-plane), yet precise anatomical location of sub-cortical nuclei remains difficult due to the small size of the underlying anatomy. Cellular differences in midbrain nuclei are not easily distinguishable using conventional imaging methods, explaining the absence of well resolved anatomical masks. Thus, the designation of the exact anatomical location with respect to the observed significant activations in the midbrain region is challenging and one can only speculate with respect to the exact underlying mechanisms.A region heavily implicated in thermoregulation by rodent literature, but only infrequently identified in human fMRI studies are the hypothalamic nuclei (Freedman et al., Results of our study suggest differential response to cold exposure in interoceptive and regulatory CNS regions between subjects with high and low amount of sympathetic innervation in supraclavicular BAT. The cold-dependent differential response in higher-order interoceptive centers generates varying levels of inhibition over lower-order autonomic midbrain structures, which subsequently regulate sympathetic activation of BAT in an activation-dependent manner. Thus, our data indicates that the high variability of cold-activated BAT mass observed in humans might be, in part, related to different sensitivities of higher-order interoceptive brain regions to skin temperature changes.OM and VD jointly designed, performed, analyzed and interpreted the research data.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.


Since the elucidation of the genetic code almost 50 years ago, many nonrandom aspects of its codon organization remain only partly resolved. Here, we investigate the recent hypothesis of ‘dual-use’ codons which proposes that in addition to allowing adjustment of codon optimization to tRNA abundance, the degeneracy in the triplet-based genetic code also multiplexes information regarding DNA’s helical shape and protein-binding dynamics while avoiding interference with other protein-level characteristics determined by amino acid properties. How such structural optimization of the code within eukaryotic chromatin could have arisen from an RNA world is a mystery, but would imply some preadaptation in an RNA context. We analyzed synonymous (protein-silent) and nonsynonymous  mutational impacts on molecular dynamics in 13823 identically degenerate alternative codon reorganizations, defined by codon transitions in 7680 GPU-accelerated molecular dynamic simulations of implicitly and explicitly solvated double-stranded aRNA and bDNA structures. When compared to all possible alternative codon assignments, the standard genetic code minimized the impact of synonymous mutations on the random atomic fluctuations and correlations of carbon backbone vector trajectories while facilitating the specific movements that contribute to DNA polymer flexibility. This trend was notably stronger in the context of RNA supporting the idea that dual-use codon optimization and informational multiplexing in DNA resulted from the preadaptation of the RNA duplex to resist changes to thermostability. The nonrandom and divergent molecular dynamics of synonymous mutations also imply that the triplet-based code may have resulted from adaptive functional expansion enabling a primordial doublet code to multiplex gene regulatory information via the shape and charge of the minor groove.The online version of this article (10.1007/s00239-018-9828-x) contains supplementary material, which is available to authorized users. All known life on Earth uses nearly the same genetic code to control the templated reproduction of polypeptides from nucleic acid polymers. Its triplet-based codon organization appears to serve two rather distinct functions. The most primary of these is the specification of amino acids by the correct combination of tRNA activation and codon–anticodon binding interaction guided by the first and second nucleobase. A less obvious secondary function is the accommodation of nucleic acid sequence level variation for reasons that are still largely unknown. This degeneracy in the code is achieved through the prevalence of twofold and fourfold synonymous sites which occur mostly at the third codon position and are frequently accommodated by nonspecific base pairing and tRNA wobble at the ribosome Crick . Thus, tIt has recently been demonstrated that codon organization supports the optimization of binding interactions in eukaryotic exonic DNA  the code is highly optimized towards an ability to multiplex additional ‘parallel’ information related to RNA duplexing and protein-binding interaction  nonrandom physicochemical properties of its constituent alphabets  of the relative synonymous and nonsynonymous impacts calculated under the real genetic code (in step 3).Our computational pipeline consists of five steps visually outlined in Fig. All MD simulations were carried out sequentially on 2 Ubuntu 16.04 linux servers (Dell T430 Intel Xeon) mounting a 2880 core water-cooled 980ti and a 3072 core Titan-X GPU running pmemd.cuda released with Amber16  for implicit solvation , root-mean-square deviation (RMSD), root-mean-square fluctuations (RMSF or ‘FLUX’), and atomic correlation (AC or ‘CORR’) averaged over time. Base pair parameters, RMSD, RMSF, and AC values per position were averaged over each data set. The calculations used all backbone atoms within a 5-mer mask centered on each simulation of 25 bp sequence. Mutational impact scores in our MD simulations were based on atomic fluctuations  and atomic correlations among single atom vector trajectories calculated along a single strand of the backbone using the 5-mer mask centered on each codon type . Mutational impacts  physical mask centered on the codon and filtering only trajectories of the backbone carbon atoms. For all single-based substitutions within the total 64 × 64 codon transition matrix, a list of differences in average correlation and fluctuation (dCORR and dFLUX) calculated over the physical mask for each codon type was derived for both the RNA and DNA contexts. Using the degenerate organization of the real genetic code, overall synonymous and nonsynonymous mutational impacts were taken as the sum of the absolute dFLUX and dCORR over all transition types split into each category . Alternate coding schemes were derived by repeatedly reorganizing an associative array combining all 20 amino acids with all possible letter-base matrix combinations within a 4 × 4 × 4 cube until all (4! x 4! x 4! − 1) = 13,823 alternate coding combinations were attained. The degree of degeneracy for each amino acid was maintained for each of these alternative codes. Synonymous and nonsynonymous mutational impacts were also derived for each alternative code. To assess how the codon organization of the canonical genetic code nonrandomly affected the MD of RNA and DNA polymers, the sum total values of synonymous and nonsynonymous dCORR and dFLUX for the real genetic code were compared to histograms representing all possible alternative codes and subsequently, empirical L is the length of a given sequence, and MN are the respective TRX values for the MN dinucleotides defining the phosphate linkages along this length [Table For all single-based substitutions within the 64 × 64 codon transitions described above, a list of differences in average DNA flexibility  were also calculated for each codon type. For calculating DNA flexibility, we used the dinucleotide-based TRX score proposed by  for each alternative code and similarly used to create distributions of dTRX under random genetic codes to which the dTRX under the standard code could be compared.r = 0.58, p < 0.0001) and between dCORR values in DNA and dFLUX values in RNA . In Supplemental Files A–D, we plot all the RMSD matrices for the equilibration steps of each of the 64 codons in DNA and RNA context. All RMSD plots show evidence of stability prior to each production phase of each MD run.We report that in the context of double-stranded aRNA and to a lesser extent bDNA, the genetic code minimizes the total MD impacts of single-base synonymous mutations Fig. a, b. We We report moderate to very strong patterns of potential functional optimization of nucleic acid MD within the standard codon organization of the genetic code. These impacts are defined via general atomic fluctuations and correlations in atomic vector trajectories on the carbon backbone , as well as movements that are specific to defining DNA flexibility [i.e., combined twist, roll, and x-displacement of adjacent base pairs predicted by the TRX score of . A much smaller category of synonymous mutations is symmetric . We find that these asymmetric synonymous changes are highly optimized under the standard code while symmetric synonymous changes clearly are not. We do not have a definitive explanation for this, but might suggest that the optimization of the code regarding MD of duplexed RNA arose within a simpler doublet code whereupon fewer base substitutions were synonymous prior to later expansion to the triplet code we have today.The nonrandom pattern of optimization in synonymous mutation we observed was quantitatively and qualitatively consistent regardless of the method of solvation within the MD simulation we chose. This result is also highly consistent regardless of whether we defined the impact of mutation through its effect on the carbon backbone via atomic fluctuation  or atomic correlations . The optimization of we observe using an empirically based sequence-dependent metric of DNA flexibility (TRX score) corroborates our earlier findings that changes to DNA flexibility are mitigated by synonymous mutation affecting GC3 in coding regions  heatmaps for all 64 equilibration MD runs in (A) implicitly solvated DNA, (B) implicitly solvated aRNA, (C) explicitly solvated DNA, (D) explicitly solvated aRNA. (PDF 10193 KB)Supplementary material 2 (PDF 10308 KB)Supplementary material 3 (PDF 8801 KB)Supplementary material 4 (PDF 8100 KB)


Human studies have demonstrated that a higher number of iNKT cells in the graft or in the peripheral blood of the recipient post-transplantation are associated with a reduction in GVHD risk, importantly without a loss of graft-versus-tumor effect. In two separate analyses of many immune cell subsets in allogeneic grafts, iNKT cell dose was the only parameter associated with a significant improvement in GVHD or in GVHD-free progression-free survival. Failure to reconstitute iNKT cells following allogeneic transplantation has also been associated with an increased risk of relapse. These data demonstrate that iNKT cells hold promise for future clinical application in the prevention of GVHD in allogeneic stem cell transplantation and warrant further study of the immunoregulatory functions of iNKT cells in this setting.Invariant natural killer T (iNKT) cells serve as a bridge between innate and adaptive immunity and have been shown to play an important role in immune regulation, defense against pathogens, and cancer immunity. Recent data also suggest that this compartment of the immune system plays a significant role in reducing graft-versus-host disease (GVHD) in the setting of allogeneic hematopoietic stem cell transplantation. Murine studies have shown that boosting iNKT numbers through certain conditioning regimens or adoptive transfer leads to suppression of acute or chronic GVHD. Preclinical work reveals that iNKT cells exert their suppressive function by expanding regulatory T cells  Hematopoietic cell transplantation (HCT) remains the only curative treatment option for patients with many hematologic malignancies and hematologic disorders. Despite many improvements to support patients following transplantation, graft-versus-host disease (GVHD) continues to be one of the leading causes of morbidity and mortality . GVHD isInvariant natural killer T cells are a rare subset of T lymphocytes which are characterized by the coexpression of natural killer and T cell markers. They express a T cell receptor (TCR) which is semi-invariant  and which recognizes glycolipid antigens presented by the non-polymorphic MHC Class I-like molecule CD1d with high affinity .Despite their rarity, iNKT cells exert potent immunomodulatory functions bridging the innate and adaptive immune systems by rapidly producing vast amounts of cytokines and chemokines. This results either in enhanced immune responses  via the production of Th1 cytokines such as interferon (IFN)-γ or in suppression of autoimmune and alloimmune reactions by the production of interleukin (IL)-4 and IL-10   with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) results in a relative expansion of host iNKT cells which protect from GVHD, while preserving the graft-versus tumor effect , 7. The −/−) or IL-4−/− recipient mice were used, indicating that host iNKT cells and their production of IL-4 play a critical role in tolerance induction  on the day of transplantation significantly reduced mortality and morbidity from GVHD –13. One nduction . Furthernduction . Interesnduction . As desc+ iNKT expanded in secondary lymphoid organs and migrated to GvHD target organs, similar to conventional T cells (4) and even cells from a third-party source are capable of these effects  effect , 15–17.  effects .ex vivo expansion before adoptive transfer has been explored. Several studies demonstrated that it is feasible to expand iNKT cells in vitro with a combination of α-GalCer and IL-2. In these studies, the expanded iNKT cells provide protection from GVHD which is dependent on the production of IL-4 by iNKT cells , play an important role in the interplay between iNKT cells and Tregs.Most of the studies described above had two intriguing findings in common. First, iNKT cells give rise to a bias in donor T cell polarization toward a Th2 cytokine pattern with significantly reduced production of IFN-γ and tumor necrosis factor (TNF)-alpha , 15, 17,Myeloid-derived suppressor cells are a heterogenous cell subset known to play a major role in the regulation of immune responses in cancer and other pathological conditions , and sev+ DCs. It was shown previously that these cells are the major DC subset to present a variety of glycolipids through the CD1d molecule to iNKT cells leading to their activation (+205+DCs induce Tregs in a transforming growth factor-beta (TGF-β) and retinoic acid-dependent manner  were significantly reduced in extensive cGVHD. Importantly, steroids had no impact on iNKT counts. Although limited by quite high numbers of missing values, this study represents the first human report to explore the link between iNKT recovery and development of GVHD.One of the earliest studies delineating iNKT reconstitution following allogeneic HCT demonstrated a correlation between increased peripheral blood iNKT count and reduced acute and chronic GHVD . In this+ T cells from patients who underwent TLI/ATG conditioning as compared to conventional RIC or normal subjects. In the five patients for whom peripheral blood iNKT count was measured following conditioning, iNKT percentage was increased by a factor of 10. Building on this success, a second report of 111 additional patients, including a few mismatched unrelated donors (MMUD) demonstrated that the overall probability of aGVHD was substantially lower than typically observed with RIC at 5.4%, and slightly lower for extensive chronic GVHD at 28% (An early human study from our group translated the encouraging murine TLI/ATG results in 37 patients receiving human leukocyte antigen-matched (MRD or MUD) granulocyte colony-stimulating factor-mobilized PBSCT for hematologic malignancies . Only twD at 28% . One limD at 28%  and whendecreased number of iNKT cells (defined as CD3+CD56+Vα24Jα18+Vβ11+) at day +40, though by day +100 no difference was noted. The ATG group also had a dramatically lower incidence of aGVHD with no change in cGVHD. Another study failed to find an association of iNKT with GVHD in a MAC plus ATG setting +] predicted aGVHD and overall survival (OS) , and OS was also significantly improved (90 versus 52% at 2 years). Significant reduction in NRM (6.1 versus 36.7%) was noted to be due to fewer deaths from aGVHD and infection. Impressively, the d15 ratio (with a cutoff of 0.58 × 10−3) could discriminate the risk of aGVHD with an area under the curve of 0.812 (odds ratio 0.06 for aGVHD in high iNKT/T group). Interestingly, CD4+ iNKT chimerism of five patients revealed all to be of donor origin.However, in a study of 71 heterogenous patients , early post-transplantation iNKT recovery  were noted to be higher in PBSC than BM grafts owing to the one log higher total T cell content. Although lower total and CD4− iNKT frequency was significantly associated with the occurrence of aGVHD in BMT and PBSCT, lower CD4− iNKT expansion factor was only associated with aGVHD in the PBSCT group. Importantly, this remained true when the expansion of iNKT cells was analyzed in donor peripheral blood prior to mobilization and at defined timepoints in patients post-transplant. The expansion factor was not predictive of relapse rate, cGVHD, NRM, or OS. Although, as the authors recognize, the ability to implement the expansion technique as a GVHD predictive tool across all centers is unlikely, this work highlights the critical need for high frequencies of functional iNKT cells for suppression of aGVHD. It also raises the question of whether iNKT cell dose or frequency relative to total T cells  is the most important value in predicting aGVHD, which remains to be fully answered.A third study investigated 15 immune populations from a mix of fresh and cryopreserved graft products for 117 transplants from a variety of donors (MRD/MUD/MMUD), sources (PBSCT/BMT), and conditioning regimens  . Doses oincreased risk of cGVHD in a univariate analysis (+CD56+CD16−CD117−) and this finding did not pan out in multivariate analysis. The same study also found that increased iNKT cell content was associated with lower risk of relapse in AML/MDS patients.Interestingly, another study of immune cell content in 238 allografts for a very heterogenous patient population showed a significant association of increased iNKT content with analysis . However+CD25hiCD127low). These responders were enriched for patients who had received sirolimus with their GVHD prophylaxis , suggesting a synergistic effect . Importantly, rates of grades II–IV aGVHD were 12.5% in responders and 52.4% in non-responders, though as noted by the authors based on the study design conclusions cannot be drawn as to whether RGI-2001 reduces GVHD incidence. Future work optimizing the dosing strategy, comparing patients receiving RGI-2001 to those who do not, and investigating why some respond and others do not will be required. Despite these limitations, this study represents a critical first step in the intentional boosting of iNKT cells to suppress GVHD.Because of this strong evidence for GVHD suppression by iNKT cells, attempts to intentionally expand the iNKT cell compartment in allogeneic HCT patients have begun. A recently published phase 2A study explored pharmacologic expansion using a single dose of RGI-2001, the liposomal formulation of α-GalCer, on the day of transplantation with two dosing cohorts (identified from phase 1 testing) for a total of 29 patients (most undergoing RIC with PBSC grafts) . A totalAs demonstrated in the aforementioned murine and human studies, iNKT cells may be the holy grail sought in stem cell transplantation that is capable both of suppressing allogeneic immune reactions (GVHD) and enhancing anti-tumor immune reactions (GVT). The murine studies all demonstrated a strong benefit of iNKT cells on GVHD reduction. Although a minority of human studies (primarily in the MAC plus ATG setting) did not find a significant association between iNKT and GVHD, or even found higher iNKT associated with worse GVHD –40, most+ iNKT suppressed aGVHD in a major mismatch model  (+ or CD4− iNKT population is best suited to reduce GVHD (and possible differences between acute and chronic GVHD) highlights the need for further study. Recently, several subsets of iNKT cells have been described skewing toward phenotypes resembling Th1 cells, Th2 cells, and Th17 cells , and ev in Ref. . CD4−CD8unctions . The conce versa  calls ince versa –12. In ace versa , 14–16. pression , 19. MosThe first approach being investigated in humans utilizes delivery of α-GalCer to recipients at the time of transplantation . Though Despite these lingering questions, harnessing the power of iNKT cells remains an attractive approach to improving HCT outcomes. Although extremely rare in number, iNKT cells may represent the most versatile and critical cell population for suppressing GVHD, fighting infection, and reducing relapse in patients undergoing allogeneic hematopoietic cell transplantation.MM and KB contributed equally to this manuscript. MM and KB did the literature study, wrote the manuscript, and critically revised the manuscript. RN provided senior supervision, helped in writing, and critically revised the manuscript. All coauthors approved the final manuscript.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.


Guillain-Barré syndrome is an acquired polyradiculo-neuropathy, often preceded by an antecedent event. It is a monophasic disease but a recurrence rate of 1–6 % is documented in a subset group of patients. Patients with Guillain-Barré syndrome show cerebrospinal fluid albuminocytologic dissociation. Normal cerebrospinal fluid protein levels during both initial and recurrent episodes of Guillain-Barré syndrome is a rare occurrence and has not been described earlier in the literature.Twenty-five-year-old Sri Lankan female with past history of complete recovery following an acute inflammatory demyelinating polyneuropathy (AIDP) variant of Guillain-Barré syndrome 12 years back presented with acute, ascending symmetrical flaccid quadriparasis extending to bulbar muscles, bilateral VII cranial nerves and respiratory compromise needing mechanical ventilation. Nerve conduction study revealed AIDP variant of Guillain-Barré syndrome. Cerebrospinal fluid analysis done after 2 weeks were normal during both episodes without albuminocytologic dissociation. She was treated with intravenous immunoglobulin resulting in a remarkable recovery. Both episodes had a complete clinical recovery in three and four months’ time respectively, rather a faster recovery than usually expected.Recurrence of Guillain-Barré syndrome can occur in a subset of patients with Guillain-Barré syndrome even after many years of asymptomatic period. Normal cerebrospinal fluid profile does not exclude Guillain-Barré syndrome and may occur in subsequent recurrences of Guillain-Barré syndrome arising the need for further studies to identify the pathophysiology and the possibility of a different subtype of Guillain-Barré syndrome. Guillain-Barré syndrome (GBS) is an acquired heterogeneous group of disorders due to an immune-mediated inflammation and demyelination of the peripheral nervous system, following an antecedent illness in two thirds of the patients, commonly an infection –3. It isGBS is a monophasic illness. Although rare, recurrence has been described following an asymptomatic period of few months to years (4 months – 10 years) in 1–6 % of patients –6. RecurWe present a rare case of RGBS presenting after 12 years, adding to the limited number of cases with a long asymptomatic interval. Such reported cases from South-Asia are rare. Apart from the young age at initial episode, she did not have other risk factors for recurrence and had a rare findings of normal CSF protein concentrations on both presentations. Extensive literature survey including published case reports and case series did not reveal a similar case report of RGBS or occurrence with normal CSF protein concentrations during both initial and recurrent presentations of GBS.A 25-year-old Sri Lankan female presented with weakness of all four limbs in January 2014. She had a similar illness 12 years back.st hour. Nerve conduction studies performed on day 4 of the illness showed focal segmental demyelinating type sensory and motor neuropathy with conduction blocks  with areflexia in all 4 limbs. There were no cranial nerve palsies or features suggestive of autonomic involvement. Sensory system examination was normal. Full blood count, blood picture, serum electrolytes, blood urea and liver enzymes profile were normal. Her erythrocyte sedimentation rate was 13mm during 1Mycoplasma, Campylobacter jejuni, cytomegalovirus, Epstein-Barr virus, hepatitis B & C and retroviral studies were negative. Autoimmune panel including anti-nuclear factor was normal. Due to the rapid progressive nature of weakness and respiratory distress, patient was mechanically ventilated for 12 days. Diagnosis of GBS was made and intravenous immunoglobulin 0.4 g/kg/day was administered for 5 days. In addition, she received physiotherapy to support her recover in motor function of limbs and speech therapy following extubation. She made a good clinical recovery assessed subjectively as well as objectively and was discharged home on day 28 with a muscle power of 4/5 and deep tendon reflexes of +1.In 2014, patient readmitted with numbness and progressive ascending weakness of all four limbs for 3 days duration and had developed poor cough response, dysphagia and difficulty in breathing during the following 2 days. This episode was preceded by an upper respiratory tract infection two weeks back. There was no similar illness noted in any of her family members. On examination, flaccid quadriparesis with generalized areflexia was noted with a muscle power of 1/5 in lower limbs, 2/5 in upper limbs and 2/5 in neck muscles. Weakness progressed to involve bilateral seventh cranial nerves and bulbar muscle without ophthalmoplegia. Rest of the neurological examination including sensory system and other organ system examination were normal except for a resting tachycardia of 130 beats/min without any significant blood pressure fluctuations. Respiratory rate was 30 cycles/min with oxygen saturation of 92 % on air. Nerve conduction studies done on day 10 of the illness revealed focal segmental demyelinating type sensory and motor neuropathy with prolonged distal motor latency, delayed F-wave and conduction blocks, concluding as AIDP variant of GBS  GBS with treatment related fluctuations (GBS-TRF), (2) Chronic inflammatory demyelinating polyneuropathy (CIDP). GBS-TRF which occurs in 6–16 % of patients with GBS is defined as significant deterioration within 2 months after disease onset following post treatment improvement or stabilization . RepeatiDiagnosis of GBS is mainly clinical and supported by evidence from electrophysiological studies and CSF analysis. Characteristically CSF has high protein levels with normal cell counts and sugar levels. Nerve roots that exit from the spinal cord traverse through CSF and when nerve roots are inflamed in GBS, proteins leak in to the CSF. Since the inflammation is confined to the nerve roots, significant numbers of inflammatory cells are not seen. Although normal CSF findings are seen during the first week of disease, albuminocytologic dissociation is seen in 82–90 % of the patients with GBS by the end of second week of the illness . Grand’mStudy done by Gonzalez-Quevedo et al. revealed raised CSF total proteins correlated with the degree of inflammation at the nerve roots and higher level of CSF proteins were related to clinical severity . CorstonIndex patient had a recurrence of GBS after a long asymptomatic period of 12 years as supported by acute onset, rapid progression, disability peaked within 2 weeks followed by complete subsequent recovery following treatment, presence of antecedent infection and supportive electrophysiological evidence. She also had more severe disease during the recurrence. CSF findings were not characteristic and were normal during both episodes occurring 12 years apart, which is a rare finding and not described earlier in patients with RGBS. Mechanism behind normal level of CSF proteins during recurrent episodes remains unclear.Recurrence of GBS is rare but can occur after many years of asymptomatic period and is associated with more severe clinical manifestations. In such a presentation, it is important to distinguish GBS from CIDP as the treatment modalities are different. Normal CSF total protein levels tested after 1 week after the onset of disease can occur in initial and recurrent episodes of GBS, where the mechanism is not fully understood. Research on recurrent GBS is needed to evaluate the higher probability of certain subgroup of patients with GBS for recurrences, to identify the occurrence and pathophysiology of normal CSF profile and the possibility of a different subtype in recurrent GBS patients.


Glioma is the most common of all primary brain tumors with poor prognosis and high mortality. The 2016 World Health Organization classification of the tumors of central nervous system uses molecular parameters in addition to histology to redefine many tumor entities. The new classification scheme divides diffuse gliomas into low-grade glioma (LGG) and glioblastoma (GBM) as per histology. LGGs are further divided into isocitrate dehydrogenase (IDH) wild type or mutant, which is further classified into either oligodendroglioma that harbors 1p/19q codeletion or diffuse astrocytoma that has an intact 1p/19q loci but enriched for ATRX loss and TP53 mutation. GBMs are divided into IDH wild type that corresponds to primary or de novo GBMs and IDH mutant that corresponds to secondary or progressive GBMs. To make the 2016 WHO subtypes of diffuse gliomas more robust, we carried out Prediction Analysis of Microarrays (PAM) to develop DNA methylation signatures for these subtypes.In this study, we applied PAM on a training set of diffuse gliomas derived from The Cancer Genome Atlas (TCGA) and identified DNA methylation signatures to classify LGG IDH wild type from LGG IDH mutant, LGG IDH mutant with 1p/19q codeletion from LGG IDH mutant with intact 1p/19q loci and GBM IDH wild type from GBM IDH mutant with an accuracy of 99–100%. The signatures were validated using the test set of diffuse glioma samples derived from TCGA with an accuracy of 96 to 99%. In addition, we also carried out additional validation of all three signatures using independent LGG and GBM cohorts. Further, the methylation signatures identified a fraction of samples as discordant, which were found to have molecular and clinical features typical of the subtype as identified by methylation signatures.Thus, we identified methylation signatures that classified different subtypes of diffuse glioma accurately and propose that these signatures could complement 2016 WHO classification scheme of diffuse glioma.The online version of this article (doi:10.1186/s13148-017-0331-9) contains supplementary material, which is available to authorized users. The neoplasia of non-neuronal glial cells in the brain is referred to as glioma and is the most common type of primary central nervous system (CNS) tumors . The difOver the past decades, classification of brain tumors was based on the histopathological and microscopic features in hematoxylin- and eosin-stained sections, like cell type, level of differentiation, identifying necrotic lesions, and presence of lineage-specific markers. According to the WHO 2007-based classification, grade II/diffused astrocytoma (DA) was described as low grade while high-grade glioma comprised of grade III/anaplastic astrocytoma (AA) and grade IV/glioblastoma (GBM) . The vasNearly 12% of GBM patients have been identified to have point mutation in codon 132 (R132H) of the isocitrate dehydrogenase 1 (IDH1) gene located in the chromosome locus 2q33 . IDH1 coIDH mutation has been shown to be associated with alterations in the methylome thus being sufficient to establish glioma hypermethylator phenotype . At presIn the present study, we investigated the altered methylation pattern among the different subtypes of diffuse gliomas as per 2016 WHO CNS tumor classification  and derihttps://cancergenome.nih.gov/) to various statistical tools and validation steps  of 0.4 between groups. The TCGA samples were then divided randomly into two equal groups as training and test sets  set . In GSE58218, the 14 CpG methylation signatures were able to discriminate oligodenroglioma from diffuse astrocytoma samples with an overall diagnostic accuracy of 97.5% Tables . SimilarPAM analysis of differentially methylated CpGs  from the TCGA dataset. For this purpose, we analyzed TP53 mutation, ATRX loss, and 1p/19q codeletion status of all the samples  from the TCGA dataset. For this purpose, we analyzed TP53 mutation, ATRX mutation, and 1p/19q codeletion status in DA, ODG, and discordant samples of LGG  from the TCGA dataset  . With thThe meeting by the International Society of Neuropathology held in Haarlem, Netherland, established guidelines for how to incorporate molecular findings into brain tumor diagnosis thereby setting the platform for a major revision of the 2007 CNS WHO classification . The curInfinium HumanMethylation450K BeadChip array data for astrocytoma , oligodendroglioma, and oligoastrocytoma tumor samples from TCGA dataset was used in this study. By using PAM, we have successfully developed and validated DNA methylation signatures to distinguish LGG IDH mutant from LGG IDH wild-type samples, LGG IDH mutant samples into diffuse astrocytoma and IDH mutant GBM from the IDH WT GBMs. The signatures classified these groups with very high accuracy and also validated successfully in multiple independent datasets. We also used PCA to test the ability of signatures to divide the two groups in comparison into two distinct classes. Further, the 10-fold cross-validation using PAM identified the discordant samples, which upon further analysis revealed that majority of misclassified samples were indeed due to inadequacies of the current methods used for classification.Thus, the present study enabled us to identify DNA methylation fingerprint for each of the groups in comparison . The 2016 WHO classification system fails to classify some samples accurately in occasions like absence of certain molecular markers, errors due to antibody-based scoring, and intra-tumoral heterogeneity. We believe that DNA methylation signatures based on whole tumor developed in this study could complement the 2016 WHO classification of diffuse glioma subtypes.In conclusion, we were able to classify diffuse glioma subtypes with high accuracy. The discordant samples identified by the methylation signature were found to be either due to technical errors or mixed histological types. More importantly, we believe that the high levels of intra-tumoral heterogeneity reported in glioma could also be a reason for their misclassification , 27. Coln = 197), oligoastrocytoma (n = 136), oligodendroglioma (n = 197), and glioblastoma (n = 124) samples, was used. Samples were then segregated according to the WHO 2016 CNS tumor IHC-based grading classification into three distinct groups, namely 1. lower grade glioma IDH wild-type and mutant (LGG IDH WT and mutant), 2. lower grade glioma IDH mutant with intact 1p/19q termed as diffuse astrocytoma and with 1p/19q codeletion termed as oligodendroglioma (DA and ODG), and 3. glioblastoma IDH mutant and wild type (GBM IDH WT and mutant). The clinical information for the same was also procured from TCGA.Glioma TCGA dataset was used for this study. Methylation data for histologically defined WHO classification glioma types, which include astrocytoma (With an aim to identify methylation differences between the diffuse glioma subtypes (based on IDH mutation and 1p/19q codeletion status) of each group, a supervised machine learning approach through PAM   was usedβ value was applied that showed 9,554 significantly differentially methylated  and LGG patients with WT IDH (n = 97) was randomized and 50% of each of the two classes formed the training set, and the remaining 50% was used as the test set. We randomized TCGA dataset ten times to obtain ten different training sets and their corresponding test sets. After performing PAM on each of the ten training sets, the training set that gave least error with minimum number of CpGs was selected for further studies. This process gave a set of 14 discriminatory CpG probes which were further tested through SVM and subset analysis before testing on the test set and external validation sets  versus non-codel (DA), and 3. GBM: IDH mutant versus WT. For the first comparison between LGG IDH mutant and WT, we have performed a Wilcoxon-rank sum test between IDH mutant and WT which yielded 269,442 CpG probes significantly (FDR ≤0.0001) differentially methylated in mutant versus WT. Next, a stringent cutoff of 0.4 absolute Δβ value was applied that showed 2817 significantly differentially methylated  and non-codel (n = 261) was randomized and 50% of each of the two classes formed the training set, and the remaining 50% was used as the test set. We randomized TCGA dataset ten times to obtain ten different training sets and their corresponding test sets. After performing PAM on each of the ten training sets, the training set that gave least error with minimum number of CpGs was selected for further studies. This process gave a set of 14 discriminatory CpG probes which were further tested through SVM and subset analysis before testing on the test set and external validation set  patients Fig. . For thiset Fig. .β value was applied that showed 259 significantly differentially methylated  and WT (n = 117) was randomized and 50% of each of the two classes formed the training set, and the remaining 50% was used as the test set. We randomized TCGA dataset ten times to obtain ten different training sets and their corresponding test sets. After performing PAM on each of the ten training sets, the training set that gave least error with minimum number of CpGs was selected for further studies. This process gave a set of 13 discriminatory CpG probes which were further tested through SVM and subset analysis before testing on the test set and external validation set  using the package pamr available in R software (version 3.1.0) were applied. PAM uses nearest shrunken centroid method for classifying samples. This method “shrinks” each of the class centroids towards the overall centroid by the threshold. In case of selecting a signature, it is ideal to choose a threshold value that would achieve a set of minimum number of genes with maximum accuracy thereby least error. For preparing input files for PAM analysis, the list of significantly methylated probes between each compared groups across all the tumor samples was randomized and 50% of each of the two classes formed the training set, and the remaining 50% was used as the test set. This randomization was performed ten times which resulted into ten different compositions of training set and their corresponding test set. Thereafter, each of these ten training sets was subjected to PAM analysis that uses 10-fold cross-validation to identify a predictive signature. Ten different training sets that were used to construct the PAM classifier resulted in ten non-identical predictive signatures, one for each iteration. The most promising signature which had the maximum training and test set accuracies was chosen. We also performed an internal cross-validation on the training set of the most promising signature as predicted by PAM.For internal cross-validation, we have used Support Vector Machine (SVM) . Many prn = 49), 2. diffuse astrocytoma (n = 131) versus oligodendroglioma (n = 86), and 3. GBM IDH mutant (n = 4) versus WT (n = 59) by random subset sampling. PAM was used to predict the respective accuracies in the random subset sampling.The predictive accuracy of the three signatures was also analyzed in a subset of the following cases: 1. LGG IDH mutant (217) versus WT  uses orthogonal transformation to convert a set of variables into a set of values of linearly uncorrelated variables that are called principal components. The number of principal components can be less than or equal to the number of original variables. The first two principal components account for the largest possible variation in the dataset. PCA was performed using R package (version 3.1.0), on the training and test sets to know how well the identified methylation signature classifies LGG IDH mutant and WT.β was used here to identify significantly differently methylated probes between the two classes).This process was repeated for identifying a methylation signature between IDH mutant DA and ODG and between GBM IDH mutant and WT  set. Table S3A. List of the 14 CpG methylation signatures for LGG IDH mutant versus IDH WT in the test set (TCGA) and validation set (GSE48462). Table S3B. List of the 14 CpG methylation signatures for oligodendroglioma (ODG) versus diffuse astrocytoma (DA) in the test set (TCGA) and validation set (GSE48462). Table S4. List of differentially methylated CpGs between oligodendroglioma and diffuse astrocytoma used as PAM input in the training (TCGA) set. Table S5. List of 259 differentially methylated CpG probes between GBM IDH mutant and WT used as PAM input in the training (TCGA) set. Table S6. Molecular analysis of discordant samples identified by CpG methylation signatures. Table S7A. Molecular status for IDH, TP53, ATRX, and 1p/19q in LGG samples from TCGA used in this study. Table S7B. Molecular status for IDH, TP53, ATRX, and 1p/19q in LGG samples from GSE58218 used in this study. Table S7C. Molecular status for IDH, TP53, ATRX, and 1p/19q in LGG samples from GSE48462 used in this study. Table S8. Patient IDs of the discordant samples derived from all datasets used in this study. Table S9A. Molecular status of IDH, TP53, ATRX, EGFR, and PTEN in GBM samples from TCGA dataset used in this study. Table S9B. Molecular status of IDH, TP53, ATRX, EGFR, and PTEN in GBM samples from GSE38278 dataset used in this study. (ZIP 1601 kb)Sample size and diffuse glioma subsets of various cohorts used in this study. Additional file 2:Has ten additional figures and their corresponding figure legends. (PPTX 830 kb)