Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations .Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.Microviridae family, can be among the most abundant viral genomes in a sample.Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems. It is now increasingly clear that microorganisms play a central role in all of Earth’s ecosystems and processes. In every biome—from the human gut to the oceans, soils, and extreme environments that challenge life to succeed—microbes drive the nutrient and energy transformations that fuel these ecosystems . MicrobiRecent technical and theoretical advances are now also revealing that these microbial roles are modulated by co-occurring and co-evolving viruses . VirusesBecause natural community samples typically yield limiting input DNA, multiple displacement amplification (MDA) or whole genome amplification (WGA) are commonly used prior to sequencing library preparation in viromics studies . While tThe recently available Swift Biosciences 1S Plus kit for preparing sequencing libraries incorporates an adaptase step prior to linker ligation and amplification, which makes it efficient for both ssDNA and dsDNA templates . Here wePseudoalteromonas or Cellulophaga baltica , a range of genome length (35–130  kb) and GC% (30–45%). The two ssDNA phages included were phiX174 and alpha 3, representing two distinct clades in the well-characterized Microvirus genus (from the Microviridae family), both grown on Escherichia coli  corresponding to two contrasting situations with either low abundance of ssDNA viruses  or high abundance of ssDNA viruses .Each virus to be included in the mock community was grown on its specific host, and viral capsids were obtained from lysates. The concentration of viral capsids was determined through SYBR Gold counting , using tFor each mix, DNA was extracted with the QIAamp DNA Mini Kit (Qiagen 51304). Triplicate viromes were generated using DNA extracted from these two mock communities with three different sequencing library protocols . This experimental design allowed us to evaluate the potential influence of both the DNA extraction step  and the DNA amplification step (different biases between methods) at three different levels: (i) the relative proportion of ssDNA vs dsDNA viruses, (ii) the relative proportion of individual genomes within ssDNA and dsDNA virus communities, and (iii) the coverage variation within a genome. The MDA library was generated using the GE HealthCare GenomiPhi v2 DNA Amplification Kit followed by NexteraXT DNA Library Preparation Kit, the TAG library using the standard NexteraXT DNA Library Preparation Kit, and the A-LA library with the Swift 1S Plus DNA Library Kit for Illumina. All samples were sequenced on the Illumina MiSeq platform.Raw reads were curated with Trimmomatic to remove adaptors, trim reads as soon as the base-calling quality dropped below 20 on a 4 bp sliding window, and remove reads shorter than 50 bp . TrimmedWithin dsDNA viruses, the influence of mock community (A or B), library preparation method , and genome on relative abundance  was investigated with Kruskal–Wallis tests. Each genome’s relative abundance was compared between A-LA and TAG libraries using a Wilcoxon two-sided test . SimilarFor freshwater lakes, three integrated water column samples were taken in April 2013 in Lake Superior (SU08M), Lake Erie (ER15M), and Lake Michigan (MI41M). Samples from three different depths  were combined, since the lakes were not stratified at the time of the sampling. For each lake, 33 to 45L of water were 0.22 µm-filtered and viruses were concentrated from the filtrate using iron chloride flocculation  followede-value <  10−3) against RefSeqVirus  and one significant hit (score >  50) against PFAM  were considered as cellular and thus excluded . This allowed us to keep in the dataset both sequences similar to known viruses, and sequences entirely new , which in a virome are most likely viral.For freshwater and seawater samples, trimmed and filtered reads  for all libraries  were pooled for each sample and assembled with Spades 3.6.2 with the “sc” and “careful” options . All cone-value <  10−3). Contigs with best BLAST hits to only ssDNA or dsDNA viruses were considered ssDNA or dsDNA viruses respectively, while the genome type of contigs with no hits or mixed affiliations  was predicted based on their coverage in the different libraries: contigs detected in TAG libraries were considered dsDNA, while contigs only detected in MDA and/or A-LA were predicted as ssDNA. In order to take into account the fact that dsDNA genomes will provide twice as many templates than ssDNA genomes per single virus in A-LA and MDA viromes (because the first step of the protocol is dsDNA denaturation), the coverage of all affiliated and predicted dsDNA contigs was divided by 2, so that the relative proportion of contigs are approaching the relative proportion of viral particles in the sample.QC’d reads from individual libraries were then mapped back to the contigs with Bowtie 2  to evalhttp://mirrors.iplantcollaborative.org/browse/iplant/home/shared/iVirus/DNA_Viromes_library_comparison), as well as https://bitbucket.org/MAVERICLab/dna_viromes_library_comparison (for scripts).All scripts and datasets used in this study are available on iVirus  or majority (MCB) of ssDNA viruses were established from 2 ssDNA and 10 dsDNA viruses , Table 1As qualitatively observed previously , and herp-value <  2.2e–16, p-value >  0.01, effect size < 0.8) for six of the 10 genomes between TAG and A-LA  in A-LA than TAG samples , with highly variable coverage in TAG datasets for low GC genomes  of ssDNA contigs (conservatively identified by best BLAST hit to ssDNA virus genomes) was 0.03–4.68% and 0.005–0.03% in freshwater and seawater viral communities, respectively . MeanwhiMicroviridae family) and eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses  than A-LA in four out of six samples . The twoMicroviridae family. Novel and unusual ssDNA viruses continue to be isolated, particularly from eukaryotic and archaeal hosts . For comparing the efficiency of different methods for each group of phage , relative abundance within each group were computed from this table . The raw coverage for each genome in each sample is indicated in the bottom table.Table S2: Accuracy and efficiency of mock community genome coverage and assembly in A-LA viromes. The coverage of each genome across the different contigs is indicated for each library (% covered) alongside the coefficient of variation in read-based coverage along the genomes (Coverage CV) and the number of contigs matching this genome (# contigs). A star (∗) denotes libraries for which the assembly had to be run on a subset of the QC’d reads  to avoid biases linked to the over-coverage of ssDNA genomes.Table S3: Cross-assembly result of freshwater and seawater viromes. For each sample, the number of contigs assembled (≥500 bp) classified into ssDNA contigs, dsDNA contigs, predicted ssDNA contigs, and predicted dsDNA contigs is indicated. Predicted ssDNA or dsDNA contigs are sequences for which a gene-base affiliation was not possible  and which genome type were defined based on the coverage across libraries: the sequence was considered dsDNA if covered in TAG library, ssDNA otherwise . Sample codes are the same as in Table S4: Relative abundance of the four different types of contigs  in each virome. Relative abundances are based on normalized coverage of the contigs . Sample codes are the same as in Table S5: Affiliation of the most abundant affiliated ssDNA virus contigs in sample SU08M (A-LA library). Contigs were ranked based on their read coverage in the library. Affiliations are based on best BLAST hit against NCBI RefSeqVirus, and presented for the 17 contigs affiliated to ssDNA viruses within the 100 most abundant contigs. CRESS-DNA: circular Rep-encoding single-stranded DNA.Table S6: Click here for additional data file.10.7717/peerj.2777/supp-2Figure S1Examples of spots counted as individual viruses are indicated with an arrow (5 for each panel).Click here for additional data file.10.7717/peerj.2777/supp-3Figure S2The relative abundance of each dsDNA viral genome within the dsDNA viral community  was estimated for each virus in each sample. These relative abundance values are plotted for grouped by sample (left panel), or grouped by genome (right panel). Sample methods  are indicated via coloring of the boxplot (left panel) or dot (right panel). Genomes with relative abundance significantly different between TAG and A-LA libraries (Wilcoxon two-sided test) are indicated with a star above the boxplot (right panel).Click here for additional data file.10.7717/peerj.2777/supp-4Figure S3x-axis, genomes are ordered according to their overall GC content. All pairs of distributions are significantly different (Wilcoxon two-sided test) except for the ones indicated as “ns”.The coverage was estimated for each genome on sliding windows of 1,000bp separated by a 100bp step. The variability of coverage was then estimated for each sample and each genome by calculating the coverage coefficient of variation (standard deviation divided by average). On the Click here for additional data file.


Inside an fMRI scanner, participants performed the saturation adaptation task, where they rated how much vivid/faded visual objects looked like real/unreal ones using a visual analog scale. We found that participants experienced greater unreality when they perceived fadedness than vividness despite physically identical saturation. The combined fMRI and PET analyses revealed that the faded perception-related activities of the dorsolateral prefrontal and parietal cortex were positively correlated with striatal D2 receptor availability. This finding may help to understand the neuromolecular mechanisms of faded perception associated with feeling unreal in derealization symptoms.Perceptual changes in shape, size, or color are observed in patients with derealization symptoms; however, the underlying neural and molecular mechanisms are not well understood. The current study explored the relationship between neural activity associated with altered colorfulness perception assessed by fMRI and striatal dopamine D Derealization is observed not only in a wide range of psychiatric disorders, such as depersonalization disorder, schizophrenia, depression and anxiety disorder4, but also in healthy people. Population surveys revealed that derealization as well as depersonalization that is the experience of unreality in sense of the self5 were experienced in healthy subjects with the prevalence of about half in a sample population of college students7.Derealization is an abnormal subjective experience in which the external world appears strange or unreal. DSM-V defines the perceptual alteration in derealization as “experiences of unreality or detachment with respect to surroundings 8. In particular, the perceptual alteration of colorfulness is produced by saturation adaptation, in which, after being exposed with highly saturated stimuli, the following object looks faded, while the same object looks vivid after perceiving low saturation stimuli9. Thus, application of saturation adaptation in fMRI may reveal the neural activities associated with subjective faded/vivid perception toward physically identical saturation objects.Perceptual alterations of shape, size, or color in derealization have been less studied, and thus little is known about the neural mechanisms of altered visual perception associated with feeling unreal. In the field of vision studies, however, there is ample experimental evidence of perceptual changes of visual objects induced by adaptation techniques in healthy individuals13, improves the cortical neuronal signal-to-noise ratio (SNR)16, and enhances the aftereffects of visual adaptation17. A recent study using positron emission tomography (PET) further revealed that the dopamine D2 receptor availability in striatum was positively correlated with subjective visual experience18. Thus, it is speculated that striatal dopaminergic transmission may affect how visual information, including the aftereffects of saturation adaptation, is processed in cerebral cortex.Dopamine is a key modulator for learning and cognitive control, but also for visual experience. It has been shown that dopaminergic activation facilitates visual perceptual performance2 receptor availability, measured by [11C]raclopride PET, in healthy subjects.Here, we set out to assess the subjective colorfulness and unreality experience of visual objects, and to examine the relationships between the neural representation of subjective colorfulness and unreality, measured by fMRI, and striatal Dn = 14, paired t-test, t(13) = −9.1, p < 0.001, Fig. HSMS, subjective faded condition) and middle saturation stimuli preceded by low saturation stimuli  were 55.0 ± 11.0 and 65.2 ± 8.7, respectively. The ratings of the subjective faded condition (HSMS) were significantly lower than those of the subjective vivid condition (LSMS)  = −4.8, p < 0.001, Fig. HSMS) than the subjective vivid condition (LSMS) despite their saturation levels being physically identical.The reality ratings of low saturation stimuli  (35.4 ± 15.9) were significantly lower than those of high saturation stimuli  (70.6 ± 15.9)  in the contrast of color pictures  versus scramble images. This revealed that the average activity of right and left V4 was significantly higher for the objective vivid condition (HS) than any other conditions 20, the contrast of the subjective faded condition (HSMS) versus the subjective vivid condition (LSMS) showed increased activations in the right thalamus, left amygdala and left inferior frontal cortex  versus the subjective faded condition (HSMS) did not show any activated regions at this liberal threshold.The contrast of the objective faded condition (LS) versus the objective vivid condition (HS) recruited the activity of parietal cortex ) and the left parietal cortex (including left angular gyrus (BA 39), left inferior parietal lobe (BA 40), and left superior parietal lobule (BA 7); voxel level of p < 0.001; cluster level of p < 0.05 (FDR corrected))  versus the subjective faded condition (HSMS). Analyses of the contrasts of physically different saturation levels (LS versus HS) did not yield any significant regions associated with D2BPND values of any subdivisions of striatum, but the D2BPND value of left executive striatum in the objective faded condition (LS) versus the objective vivid condition (HS) contrast correlated with the left parietal cortex ) at a marginally significant level , which overlapped with the left parietal cortex found in subjective faded condition , in contrast to the identical object adapted to a low level of saturation (subjective vivid condition). This behavioral result suggests that subjective fadedness accompanies the subjective experience of unreality.Previous vision study observed that the subjective experience of colorfulness changed following the adaptation to the different levels of saturation2 receptor availability in executive striatum were positively associated with activities of the right middle frontal gyrus (BA46), a part of the dorsolateral prefrontal cortex (DLPFC), and the left parietal cortex for the subjective faded condition. Dopamine has been proposed as playing a key role in prediction and the specification of own expectation22, which helps to build adaptive internal models of the body and the world. This scheme is proposed particularly in motor control23 but may also be employed for the formation of visual world, based on the current findings as well as several previous studies that demonstrated the influences of dopamine on the enhancement of visual perceptual performance and acuity13. In addition, impaired color perception has frequently been reported in patients with Parkinson’s disease that is the case of dopaminergic dysfunction24, and their abnormal color vision can be reversed by treatment with levodopa that is an amino acid precursor of dopamine25. Our previous study using [11C]raclopride and L-[β-11C]DOPA revealed that postsynaptic D2 receptor binding potentials were inversely related with presynaptic dopamine synthesis26. Therefore, the current finding indicates that individuals with lower endogeneous dopamine synthesis show higher activity in the frontoparietal network for subjective faded perception, and the lower dopamine may invade to build internal models of the predicted world, observed as the unreality feeling in the current study. Enhancement of dopamine is supposed to treat depersonalization theoretically27, and the current study may provide the possible role of dopamine on unreality experience associated with faded perception.We discovered that the individual differences of dopamine D28 and both are functionally connected with striatum29, compose the top-down attentional control system31. Previous neuroimaging studies of depersonalization disorder patients observed increased activities in the frontal and parietal cortex35, and proposed that the emotional numbing or detachment in these patients was associated with the hyperactive top-down control system that inhibits emotional responses. DLPFC is also associated with the experience of presence in a virtual reality environment36, where the higher activity in DLPFC was associated with the lower experience of presence and reality37. Moreover, derealazation is observed as ictal phenomena in patients with temporal and parietal lobe epilepsy38. Taken together with our findings, these results indicate that the increased neural activity in frontal and parietal cortex plays a role for unreality feeling associated with subjective fadedness.Frontal and parietal cortex, where the former is anatomically connected with executive striatum39. A number of studies have shown that frontoparietal activity is associated with changes in the contents of visual consciousness40. Taking together both the observations of the striatal dopaminergic transmission and the proposed frontoparietal system, it could be speculated that the unreality experience based on subjective faded perception is influenced by the interaction between striatal dopaminergic transmission that plays a role in broadcasting the expectation-based sensory information to the cortex and the frontoparietal network, which in turn are necessary for conscious perception of fadedness and for the top-down allocations based on the subjective faded perception induced by the experimental settings of the current study.Furthermore, over the last two decades, a strong association of frontal and parietal activity and visual awareness has been established2 receptor availability in striatum.Lastly, it should also be noted that while the objective vivid condition recruited more activation in visual areas including the color processing region, these regions were not modified by subjective faded and vivid conditions or by individual differences of dopamine D41, we still cannot deny the possibility of retinal adaptation. In particular, it is known that dopamine receptors in retina have impacts on visual adaptation42 and that Parkinson’s disease has also shown some evidence of retinal dysfunction43. Thus, future study will hopefully clarify the association and dissociation between retinal and neural visual information processing, which are relevant to the emergence of unreality experience, together with the role of dopaminergic transmission. Second, the current data strongly suggest the involvement of striatal dopamine transmission in the neural activity of subjective faded and unreality experience, but do not determine the precise functional role of dopamine nor clarify the nature of the underlying neural processes. One line of future experimentation will be to apply the pharmacological approaches that have been used to study the impact of dopamine transmission on visual perceptual performance. At last, [11C]raclopride has equal affinity for D2 and D3 receptors. However, the previous study showed putamen and caudate are almost filled with all D2 receptors44. Executive striatum is composed of mostly putamen and caudate; thus, the current finding is regarded to reflect D2 receptor availability rather than D3 receptors.One of the major limitations of the current study is that the findings of aftereffects may be explained not at neural levels but rather at retinal levels. Although studies indicate that saturation adaptation is a product of the brain2 receptor availability were associated with the function of the frontoparietal network in a healthy population. Although these results are only correlational and a causal relation will still need to be established, we demonstrated that striatal D2 receptor availability is positively related to the neural networks of faded perception and unreality experience. The findings may suggest the underlying mechanisms of the relatively neglected phenomena of derealization, and help in the consideration of possible treatment targets for this population.In conclusion, the current findings highlight the neural underpinnings of the unreality experience induced by subjective faded perception, and the individual variabilities of striatal dopamine DSeventeen right-handed healthy male subjects (mean age = 23.9 ± 5.4 (mean and standard deviation) years) participated in the study. All participants had no history of neurologic or psychiatric disorder, and they were not taking any medications that could interfere with task performance or PET/fMRI data. Three participants were excluded from the analysis owing to insufficient quality of data recording: two were excluded due to excessive head movements in fMRI scan (more than 1 voxel), and one was excluded due to inadequate understanding of the task procedure. Therefore, the final sample comprised fourteen participants (mean age = 23.4 ± 4.2). Each subject underwent fMRI and PET scans. All participants provided written informed consent before participating in the study, which was approved by the Ethics and Radiation Safety Committee of the National Institute of Radiological Sciences in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.11C]raclopride (228.4 ± 8.1 MBq with specific activity of 158.4 ± 53.5 GBq/µmol), three-dimensional dynamic images were acquired on a PET camera  for 60 min. All PET images were reconstructed by filtered back-projection method  corrected for attenuation, randoms, scatter and head motion.After intravenous injection of [45, which provides parametric images of the binding potentials (BPND). All parametric images were spatially normalized to the MNI152 standard space based on the transformation parameters from the MR images estimated by DARTEL toolkit of SPM8. A connectivity-based probabilistic atlas of the striatum28 was applied to the spatially normalized parametric images to extract weighted-means of BPND values among corresponding voxels for the three sub-regions of left and right striatum . Contribution of the bilateral sub-regions to the total striatal volume was 20, 49, and 24%, respectively28.The PET image analysis was performed using PMOD software 3.5 . Quantitative analysis was performed using the multilinear reference tissue model (MRTM2)We used 20 pictures of flowers from the image library . The level of saturation was adjusted to yield high saturation, middle saturation and low saturation levels, using Adobe Photoshop software . The level of saturation for high saturation stimuli (HS) was increased by 79.0% (±7.7%) from middle saturation stimuli (MS), and that for low saturation stimuli (LS) was decreased by 73.3% (±11.5%) from MS based on our pilot experiment.HSMS, subjective faded condition) was supposed to induce low saturation perception, and “MS presented after LS”  was supposed to induce high saturation perception. Our pilot examination showed that saturation perception was significantly lower for the subjective faded condition (HSMS) than the subjective vivid condition (LSMS)  = −3.40, p = 0.012, Supplementary Fig. p-values ≤ 0.001, Supplementary Fig. HSMS) was associated with higher (or lower) reality in the subjective vivid condition (LSMS) for the current fMRI experiment.The target MS images were presented after both HS and LS to induce different saturation perception and a sense of reality for the targets. “MS presented after HS” , a picture was presented for 8 seconds, and participants rated how much they felt reality using a visual analog scale (VAS), ranging from 0  to 100 .HSMS), objective faded condition (LS), and subjective vivid condition (LSMS) , subjective faded condition (MS) Fig. . The objHSMS) and the subjective vivid condition (LSMS) to test the effects of physically different saturation and of saturation adaptation on reality experience, respectively. Two-tailed paired t-tests were performed using SPSS . p < 0.05 was considered statically significant.We examined the differences of reality ratings between the objective vivid condition (HS) and the objective faded condition (LS) and between the subjective faded condition . A high-resolution T1-weighted magnetization-prepared gradient echo sequence  was also collected for spatial normalization and localization.www.fil.ion.ucl.ac.uk/spm). EPI images were corrected for slice timing and rigid head motion. Head motion parameters were examined, and we confirmed that all trials had less than one voxel of translation and 2.0° of rotation in each participant. T1 images were segmented and spatially normalized to the same stereotaxic space by using the diffeomorphic anatomical registration through exponentiated Lie algebra (DARTEL) algorithm46. The voxel values of segmented and normalized gray matter images were modulated by the Jacobian determinants obtained from nonlinear normalization steps. After normalization, images were spatially smoothed using a Gaussian kernel with a full-width at half-maximum of 6 mm. High-pass temporal filtering (filter width of 128 seconds) was also applied to the data.Functional image analysis was performed using Statistical Parametric Mapping  using a general linear model (GLM). For each condition, the whole period from the onset of the stimuli (8 seconds) was modeled and was convolved with SPM8’s standard canonical hemodynamic response function. Six realignment parameters and two derivatives were used as covariates. All artifacts in fMRI time-series data were detected and corrected with Robust WLS Toolbox in SPM847.The first-level analysis contained five regressors of conditions , a cluster level of p < 0.05 ), as significant results48. Brain figures were created using MRIcron (http://people.cas.sc.edu/rorden/mricron/index.html)49.For the group analyses, the individual contrast images of LS versus HS and of et al.19, and extracted the contrast coefficient  of each condition. These regions corresponded to V4 areas defined by the SPM Anatomy Toolbox (v2.2). One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test was used to determine statistical significance (p < 0.05) using SPSS.To compare the activity of bilateral V4, we constructed the bilateral V4 mask image (MNI = −30/−84/−8 and 34/−80/−8 at 4-mm sphere, based on Rottschy Supplementary Information


SOX2, OCT3/4, and NANOG to evaluate their diagnostic and performance values in high-grade gliomas. Forty-four specimens were obtained from glioblastoma patients . Quantitative real-time polymerase chain reaction was applied for relative gene expression quantification. In silico network analysis was executed. NANOG and OCT3/4 mRNA expression levels were significantly downregulated while that of SOX2 was upregulated in cancer compared to noncancer tissues. Receiver operating characteristic curve analysis showed high diagnostic performance of NANOG and OCT3/4 than SOX2. However, the aberrant expressions of the genes studied were not associated with the prognostic variables in the current population. In conclusion, the current study highlighted the aberrant expression of certain longevity-associated transcription factors in glioblastoma multiforme which may direct the attention towards new strategies in the treatment of such lethal disease.Glioblastoma multiforme (GBM) (grade IV astrocytoma) has been assumed to be the most fatal type of glioma with low survival and high recurrence rates, even after prompt surgical removal and aggressive courses of treatment. Transcriptional reprogramming to stem cell-like state could explain some of the deregulated molecular signatures in GBM disease. The present study aimed to quantify the expression profiling of longevity-related transcriptional factors  Tumors of the brain were considered one of the ten most common causes of cancer-related mortality . AccordiSeveral emerging evidences support the reactivation of pluripotent transcription factors in many types of cancer –12. As aAmong these pluripotent transcription factors overexpressed in high-grade gliomas are “sex-determining region Y-Box (SOX2), octamer-binding transcription factor 4 (OCT 4), and Nanog homeobox (NANOG)” , 15.SOX2 gene encodes a transcriptional factor (TF) of 317 amino acids which contains a high-mobility group DNA-binding domain  assessed retrospectively from archived formalin-fixed paraffin-embedded section (FFPE) specimens of the Pathology Department, Mansoura University Hospitals, Egypt, from 2010 to 2013. They had glioblastoma multiforme grade 4, subjected to surgical removal and postoperative irradiation, and followed up for more than 36 months. Specimens were collected before receiving chemotherapy or radiotherapy prior to surgery. They were compared to 10 FFPE noncancerous brain specimens obtained from patients undergoing brain tissue resection for other reasons collected from the same hospital. Guidelines in the Declaration of Helsinki were followed, and an approval by the Medical Research Ethics Committee of Faculty of Medicine, Suez Canal University, was obtained before taking part. Written informed consent was obtained from all participants before providing the archived tissue samples as part of their routine register in our University Teaching Hospitals.Extraction of total RNA from FFPE specimens was done using RNeasy FFPE Kit  according to the protocol of the manufacturer. RNA concentration and purity were assessed with NanoDrop ND-1000 spectrophotometer , followed by agarose gel electrophoresis (1%) check for RNA integrity.Complementary DNA (cDNA) was obtained by total RNA conversion using the High-Capacity cDNA Reverse Transcription Kit  with RT random primers on T-Professional Basic, Biometra PCR System , as previously described . ApproprTBP) (Hs00427620_m1) which has been proved in our previous work [μl total volume using “StepOne™ Real-Time PCR System (Applied Biosystems)” as previously described in details [The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed for the real-time PCR reactions. Pluripotent gene relative expressions were assessed using “Universal PCR master mix II, No UNG (2×)” , TaqMan assay  and compared to the endogenous control TATA box binding protein  value with the following equation: relative quantity = 2Cq−ΔΔ [Data analysis was done using PC-ORD ver. 5 software package and Statistical Package for the Social Sciences (SPSS) for windows software (version 22.0). Two-tailed statistical tests were used for continuous and categorical variables. Correlation analysis between the variables was performed via Pearson's correlation coefficient. = 2−ΔΔCq . The dia= 2−ΔΔCq .TBP. Our results revealed that the expression levels of NANOG and OCT3/4 were significantly downregulated  while that of SOX2 was significantly upregulated (p = 0.0027) in tumor specimens compared to noncancer tissues . No statistically significant association was found with any other parameters . In addition, elder age of patients was associated with poor overall survival (OS)  and disease-free survival (DFS)  (Higher rameters . Correla= 0.023) .Linear regression analysis was performed to evaluate potential factors affecting overall survival of patients. None of the genes or clinicopathological variables was determined as a good prognostic marker for patients' survival in the study population . HoweverExploratory multivariate analysis by principle component and hierarchical cluster analyses classified patients into 3 groups based on the relative expression of the combined genes . HoweverSOX2, NANOG, and OCT3/4) by examining their mRNA expression in GBM tissues relative to noncancer tissues. Our analyses revealed that the expression level of SOX2 was significantly upregulated. This finding was consistent with several independent cohorts [β), Sonic Hedgehog (SHH), epidermal growth factor receptor (EGFR), and fibroblast growth factor receptor (FGFR) pathways. In addition, SOX2 gene amplification and DNA promoter hypomethylation have been reported in a group of GBM patients to expand the mechanism responsible for SOX2 upregulation [The presence of a significant heterogeneity in certain types of solid tumors including GBM is becoming obvious. Hence, it will be rational to search for and evaluate specific molecular markers that could assist in diagnosis and/or prognosis of these tumors and could act as targeted molecular markers for personalized therapy . Here, w cohorts –30 and i cohorts  who dete cohorts . When ov cohorts , 28, 31, cohorts . At the  cohorts  proposedgulation .NANOG and OCT3/4 were significantly downregulated in GBM tissues. Our finding might seem contradictory to the stemness role these pluripotent transcription factors play; however, it is worth to emphasize that the mechanistic functions of SOX2, OCT4, and NANOG in cancer cells are a little different in each stage of tumor progress. Kallas et al. reported high levels of SOX2, OCT4, and NANOG transcription factor expressions at the beginning of their tested human embryonic stem cell differentiation. However, on progress of the differentiation process, a decline in OCT4 and NANOG expression levels was observed, while expression of SOX2 was kept at a high level [Despite that our in silico analysis revealed that the expression of the studied stem-related factors has similar colocalization and physical interactions with each other , they segh level . They suThe other possible reasons for inconsistency of gene expression for the three stem cell marker studies could be sampling bias and/or relatively low expression levels of these factors within the individual GBM tissue examined in the current study . This coJi et al. reported that unlike normal stem cells, OCT4 could be dispensable for self-renewal, survival, and differentiation of transformed cells. They provided direct evidence for the functional divergence of OCT4 from the pluripotent state following the cancer tissue transformation . This coNANOG and NANOGP8) in human, it has been found that NANOGP8 is the most abundantly expressed of the two NANOG-encoding genes in GBMs, accounting for more than ninety percent of all NANOG-encoding mRNAs in a number of previously tested cases [In contrast to the finding of Zbinden et al.  that NANed cases . HoweverCorrelating the available clincopathological features including the survival data of GBM cases with the gene expression results revealed that poor overall survival and disease-free survival were found significantly among patients as reported by previous studies , 45. DesThe current study findings highlighted the dysregulated longevity-related gene expression in GBM Egyptian cases that could have a potential role in carcinogenesis and procuration of stemness-like properties in this type of tumors. The current study could be limited by the relatively small sample size and the fact that all patients have grade IV gliomas, although this last issue increases the specificity of the study results that confined to one stage of GBM. Additional large-scale studies including different glioma grades are recommended to evaluate the relation of the studied longevity-related gene expression with different WHO grades as well as to confirm their putative role as diagnostic and/or prognostic biomarkers. These could be an interesting era for future individualized molecular-targeted therapy for GBM patients.


CCL8/CXCL11), we screened 92 asthmatic and 69 healthy children without illness for respiratory virus infections.Respiratory illness caused by viral infection is associated with the development and exacerbation of childhood asthma. Little is known about the effects of respiratory viral infections in the absence of illness. Using quantitative PCR (qPCR) for common respiratory viruses and for two genes known to be highly upregulated in viral infections  contains supplementary material, which is available to authorized users. The detection of viruses from human biological samples and the determination of their influence on the host cell transcriptome are paramount for understanding complex human diseases that are initiated or exacerbated by virus infections and their sequelae. Even virus detection alone has proven challenging for current methods. First, PCR-based viral amplicon detection strategies are susceptible to cross-reactivity, lack strain specificity, and are inefficient for broad screening due to the lack of a common gene shared across virus species –3. SolutAnother strategy is to perform shotgun sequencing of nucleic acids isolated from the entire, unseparated clinical sample . This method has been previously applied to total nucleic acids (RNA and DNA) extracted from clinical samples for viral detection –13. AlthUpper respiratory tract infections are among the most prevalent of all viral infections in humans and render a dramatic cost on the health care system in terms of lost economic productivity and general well-being. Beyond their acute effects, respiratory virus infections are strongly associated with the development, symptoms, and exacerbation of asthma , 18. ForIn this study, we detail methods for the detection of common respiratory viruses in asthmatic and healthy children using polyA-selected whole transcriptome sequencing. Using these efficient methods, we found that a large proportion of children without respiratory illness were carrying pathogenic and transcriptionally active respiratory viruses. Virus detection using a dual RNA-seq workflow resulted in 86% sensitivity when compared to quantitative PCR (qPCR)-based screening. Additionally, we detected respiratory viruses not targeted by viral qPCR primers in two subjects with high expression of viral response genes, demonstrating the versatility of this method. Despite absence of respiratory illness, viral carriers exhibited a dramatically altered nasal epithelial transcriptome, reflective of active viral infection. Virus levels determined by RNA-seq correlated strongly with host airway viral response gene expression levels. Even virus levels at the limits of detection by qPCR and RNA sequencing had a discernible effect on the host airway epithelial transcriptome. We detail modules of activated gene expression in viral carriers reflective of epithelial cell subtypes, as well as immune cell types. We also report modules of asthmogenic genes that are strongly associated with viral infection. We conclude that viral infection, even in the absence of observable illness, induces epithelial responses in children with and without asthma.CCL8 and CXCL11, were previously reported as among the most upregulated during the peak stage of acute respiratory illness [CCL8 and CXCL11 gene expression and virus levels . Two subjects that were ranked in the top fifth percentile of expression for both biomarker genes were negative for virus by qPCR, and were therefore suspected to be carriers of viral species not covered by the qPCR panel  cohort subjects  were infected with a respiratory virus, despite the absence of respiratory illness  to 164,870 reads  generated per sample. Viral genome read depth plots were generated for all virus-infected samples with >20 viral reads . Therefore, RNA-seq viral detection at this low read depth is highly quantitative, is nearly as sensitive as qPCR viral assay, and has likely unlimited breadth of detection. Examining all subjects either qPCR or RNA-seq positive for virus, we observed a skew in the frequency of virus-infected subjects by season (P = 0.042). This skew was driven by a significant decrease in viral infection frequency in summer  and a trend toward increased infection in the fall .Examining the two qPCR-negative, but suspected virus carriers based on nasal airway epithelial viral biomarker expression, we detected human parainfluenza virus 4a (HPIV-4a) in one sample and influenzavirus B in the other sample. Neither viral species was tested in our qPCR assays. Examining all samples with virus detected by both methods, there was a strong positive correlation between qPCR viral signal and the proportion of viral reads in RNA-seq data  and (2) Virus-Low: remaining subjects harboring a respiratory virus (n = 10)  and many interferon-induced genes . Moreover, Gene Ontology (GO) analysis of the top differentially expressed genes  resulted in hits for defense response to virus (P = 4.5 × 10−23) and the virus responsive, interferon-gamma-mediated signaling pathway (P = 2.1 × 10−21)  Virus-High subjects: with viral genome coverage of > =1x  [We next examined whether genes previously shown to be upregulated in acute viral infection of the nasal airway are discriminatory for infected subjects without illness. We found 55 of the 56 genes previously reported as upregulated during the peak stage of acute viral respiratory illness were upregulated in our Virus-High subjects  .P < 2.2 × 10−16). These results indicate that respiratory virus infection of the airway epithelium without clinical illness can mediate epithelial expression changes characteristic of active acute infection.Secondly, we determined the in vitro primary airway epithelial transcriptional response to acute respiratory virus infection to draw comparisons to the transcriptional signature exhibited in vivo among the Virus-High subjects without illness. To accomplish this, we performed whole transcriptome paired differential expression analysis of HRV-A16-infected and mock-infected mucociliary differentiated airway epithelial cell cultures from three donors ). Closer investigation of those genes revealed downregulation of the ribosomal subunit genes, which predicted activation of EIF2AK2. The EIF2AK2 gene encodes for a kinase which is activated by virus infection to inhibit expression of translational machinery, as a host viral defense mechanism [EIF2AK2 expression was among the 58 significantly upregulated genes in the Virus-Low subjects. The upregulated genes showed a significant enrichment in non-activated (P = 3.89 × 10−09) and activated neutrophil (P = 1.82 × 10−07) signatures, as well as an activated macrophage signature (P = 3.20 × 10−02). These 100 genes separated the Virus-Low subjects from No-Virus subjects in an MDS analysis  between the Virus-Low and No-Virus subjects   to the 8CCL8 among the 199 genes, suggesting that this gene is only expressed by the immune cells infiltrating the nasal airway. We found strong over-representation of these 199 in vivo infection-specific genes in the blue and pink modules by hypergeometric testing , with no over-representation of these genes found among the remaining expression modules. We then explored which activated leukocyte populations might be represented by these modules by examining enrichment of the module genes in the publicly available expression datasets of specific activated immune cell populations [Five of the detected modules  had highly correlated expression patterns, which were characterized by high module eigengene expression values in Virus-High subjects and low values in the No-Virus samples, regardless of asthma status Table . Enrichmulations  (AdditioP = 1.52 × 10−33) and genes involved in the eIF2 signaling pathway, and had a statistically significant overlap with the genes differentially expressed in the Virus-Low group  [https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/) predicted IL-13, the primary driver of Th2 inflammation [RFX3 transcription factor binding sites. The RFX family of transcription factors is known to drive the expression of genes involved in motile cilia formation of metazoans [The remaining three modules were associated to varying degrees with asthma among non-viral carrying samples Table . In fact ALOX15) , 33 wereammation , to be tammation . Lastly,etazoans .Dual RNA sequencing of host and microbe has been proposed as a groundbreaking method to simultaneously detect/quantify microbes and reveal their influence on the host transcriptome . To date7/sample) was sufficient to detect virus infection at species and even strain level among subjects without illness.We suspect that several features of our study design aided in successful virus detection in these samples. First, our immediate lysis of the complete sample and application of the total lysate onto the RNA column without pre-processing steps allowed us to avoid cumbersome, potentially biased, separation methods that could have resulted in loss of viral RNA. Secondly, we took advantage of the fact that most respiratory viruses have polyadenylated RNA genomes and that their expressed transcripts are also polyadenylated –41. We pDetection of viruses in asymptomatic children has been previously reported by multiple PCR studies, which detected viruses in 33–52% of asymptomatic subjects using nasopharyngeal swabs and washings , 42–45. EIF2AK2, leading to inhibition of viral replication, and infiltration of airway epithelium by activated neutrophils.The dual RNA-seq method applied to host-dominated samples allowed us to determine that a clinically “silent” carriage of respiratory viruses results in dramatic alterations of the airway molecular phenotype with 8126 genes significantly affected. Comparison of acute in vitro experimental viral infection of airway epithelium and GO analyses of viral infection categories revealed high similarity between signatures of viral carriage without illness and active viral response. We did not observe significant differences in airway expression between asthmatic and healthy control subjects carrying a respiratory virus. We did note, however, that three of the nine modules associated with viral carriage were also associated with asthma status in uninfected subjects. Interestingly, we found that genes associated with type 2 airway inflammation, a common airway endotype in asthma, were also expressed much lower in virus-infected asthmatic and healthy children than in subjects without any viral reads detected. The association of viral infection with dampened type 2 inflammation may be due to anti-viral immune cells infiltrating the airways. Supporting this, we found viral gene response modules strongly characteristic of activated macrophage and neutrophil populations. This finding is significant, as it strongly suggests that not only are epithelial cells responding to the presence of a virus, but that the local immune system is being directed by the level of virus carriage we detect. Additionally, we identified a subset of the differentially expressed genes that were significantly altered even in subjects with very few viral reads present. These genes represented host suppression of ribosomal subunit genes by activated We acknowledge multiple factors in our study that limit interpretation of our results and raise important questions beyond the scope of this work. Namely, mild respiratory illness may not have been reported or recognized in some subjects due to community recruitment. Additionally, symptoms and severity of rhinitis were not explicitly scored on study subjects, preventing tests of association here. Additionally, the cross-sectional nature of our study does not allow us to determine if the viral carriage we detect pre-dates, post-dates, or is unrelated to a clinically significant viral infection. It is unlikely that our detection post-dates infection, since a recent respiratory illness (6 weeks prior) was among the study exclusion criteria for the asthmatics. We acknowledge that the lack of recruitment of symptomatic patients to the study prevented us from conducting a comprehensive comparison of the effects of the viral infection without illness to viral infection effects in patients with illness. Moreover, the collection of only a single sample from subjects did not allow us to determine the persistence of this viral carriage. Furthermore, the polyA-enrichment protocol limits this method to detection of viruses with polyadenylated genomes and transcripts and would miss any viruses without those characteristics potentially present in the samples. Finally, while dual RNA-seq was initially proposed for studying host-microbiome interactions in bacterial infections , the appWe applied a protocol and analytical workflow for dual RNA-seq of nasal airway epithelial brushings to concurrently detect respiratory viruses and generate the host airway transcriptome in one sample preparation and sequencing experiment. The viral detection with this method achieved 86% sensitivity when compared with qPCR-based respiratory virus detection. The sensitivity, specificity, and scope of this method resulted in the detection of multiple respiratory viruses and provided sufficient resolution to distinguish between virus species and strains in infected subjects with low total viral read percentages. Co-generation of the host transcriptome data allowed us to determine that molecular function of the airway is greatly altered by a “silent” virus carriage, which is characterized by immune cell infiltration of the airway, downregulation of ciliated cell gene expression, and modulation of type 2 inflammatory and other asthma-associated gene expression patterns.All nasal brushing analysis subjects included in this study were recruited as a part of the Genes-environments and Admixture in Latino Americans II (GALA II) childhood asthma cohort. The asthmatics and controls included in this study were randomly selected. Asthma was defined by physician diagnosis and the presence of at least two asthma symptoms . Study recruitment excluded subjects with respiratory illness as judged by a recruiting nurse evaluating cold symptoms. Additional study exclusion for asthmatics included a reported respiratory illness in the past 6 weeks. Clinical characteristics including medication usage and asthma symptoms in the past 2 weeks are listed in Additional file Cytology brushes were used to collect airway epithelium from the posterior surface of the inferior turbinate from children with and without asthma (controls) as part of the GALA II study  , respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human parainfluenza virus 1 (HPIV1), human parainfluenza virus 2 (HPIV2), and human parainfluenza virus 3 (HPIV3). Each assay was conducted using RNA extracted from the nasal airway brushings of 161 GALA II study subjects . Specifically, RNA was reverse transcribed using oligo-dT primers and SuperScript III Reverse Transcriptase (Invitrogen). The cDNA synthesized for each sample was used for qPCR assays (5 ng per reaction) using specific primers and probes for each viral species as previously described . The qPCCCL8 and CXCL11 genes was measured by qPCR assays in RNA extracted from the nasal airway brushings of the same cohort of 161 GALA II study subjects  as used for the virus detection qPCR assays. The cDNA was synthesized as described above, and each qPCR reaction was run using 5 ng of cDNA template and custom PrimeTime qPCR assays for the biomarkers CCL8 (Hs.PT.58.39289889.g), CXCL11 (Hs.PT.58.26723814), and the housekeeping gene GusB (Hs.PT.51.2648420) (IDT Technologies). The qPCR amplification cycling was conducted as follows: 3 minutes at 95 °C, followed by 40 cycles of denaturing for 10 seconds at 95 °C, and annealing for 30 seconds at 60 °C. The Ct values for CCL8 and CXCL11 were normalized to GusB expression levels within each sample, and gene expression values were determined using the comparative Ct analysis method [Expression of the CCL8 and CXCL11 expression, and 25 randomly selected samples with no suspected asymptomatic virus carriage. Barcodes and adapters compatible with the Ion Torrent Proton sequencing instrument were used. Sequencing was conducted with P1 chips. The viral pathogen detection pipeline is schematically illustrated in Fig. Kapa Biosystems mRNA-seq library kits  were used to generate sequencing libraries for Ion Torrent whole transcriptome sequencing for the 21 samples with respiratory virus detected by qPCR, 2 samples with suspected viral infection based on high https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/). Enrichment for type 2 inflammation genes was performed with hypergeometric testing using a list of 70 genes from Poole et al., 2014 [Reads uniquely mapping as human with GSNAP  were usel., 2014 . Enrichml., 2014  obtainedl., 2014  and Genel., 2014 , in the l., 2014 . All othl., 2014  for genel., 2014  for genel., 2014  implemen5 cells per insert. At 24 hours post-plating, airway media 1 was replaced with airway medium 2 containing 2% Ultroser G [2; control inserts were incubated with X-Vivo media alone in the apical chamber. Both the apical and basolateral chambers of the inserts were washed three times with warmed PBS, fresh airway media 2 containing USG was added to the basolateral chamber, and the apical chamber was left at the air-liquid interface for an additional 24 hours in a 37 °C incubator. Inserts were washed with PBS, cell monolayers lysed in RLT buffer, and RNA was extracted using an RNA Miniprep Kit (QIAGEN).Human tracheal airway epithelial cells from three donors were cultured using a modified Schlegel method as previously described –67. Basatroser G . CultureP values <0.01, and absolute log2 expression fold changes > =1 were then used to assess whether viral infections without illness in vivo demonstrate similar expression changes as in vitro airway epithelial HRV infection.RNA-seq libraries were prepared using the standard protocol for the Ion AmpliSeq Transcriptome Human Gene Expression Kit. The prepared libraries were multiplexed and sequenced on the Ion Torrent Proton sequencer. Raw sequencing reads were mapped to human transcriptome reference hg19) and gene count tables were generated with ampliseqRNA plugin v.5.0.0.0 and Torrent Suite v5.0.4 (Additional file 9 and genA second nasal brushing collected at the same time as the brushing used for RNA-seq was solubilized, and the cells were cytospun, H&E stained, and imaged by standard methods.


Heph) and Ceruloplasmin (Cp) single or double (Heph/Cp) knockout (KO) mice to study the roles of MCFs in the kidney. Renal iron levels and the expression of iron metabolism genes were examined. The non-heme iron content both in the renal cortex and medulla of Heph/Cp KO mice was significantly increased. Perls’ Prussian blue staining showed iron accumulation on the apical side of renal tubular cells in Heph/Cp KO mice. A significant increase in ferritin protein expression was also observed in the renal medulla and cortex of Heph/Cp KO mice. Both DMT1 and TfR1 protein expression were significantly decreased in the renal medulla of Heph/Cp KO mice, while the expression of DMT1 protein was significantly increased in the renal cortex of these animals. Significant increase in proteinuria and total urinary iron was observed in the double knockout mice, and this was associated with compromised structural integrity. These results suggest that KO of both the HEPH and CP genes leads to kidney iron deposition and toxicity, MCFs could protect kidney against a damage from iron excess.Multicopper ferroxidases (MCFs) play an important role in cellular iron homeostasis. However, the role of MCFs in renal metabolism remains unclear. We used Hephaestin ( Multicopper ferroxidases (MCFs) are known to play a central role in iron nutrition and homeostasis in organisms ranging from yeast to humanset al. suggested that iron was reabsorbed by late proximal tubule segments and early distal tubule segments13818The role of MCFs and a range of other proteins involved in iron metabolism in iron transport in the kidney remains poorly understood910Iron accumulation in the kidney is found in a number of diseases, including Fanconi syndrome, Dent’s disease and chronic renal disease14202021Cp and Heph single, and Cp/Heph double knockout mice to examine the role of these MCFs in the kidney. These studies showed that knockout of both the Heph and Cp genes, but not either alone, leads to kidney iron deposition and toxicity. These findings suggest that MCFs may play an important role of protecting kidney against damage from iron excess and that either oxidase is able to compensate for the loss of the other.In this study we have used Heph/Cp KO mice, hematological data were obtained from mice at six months of age. Heph/Cp KO and Heph KO mice, but not Cp KO, were anemic based on reductions in hemoglobin (Hb), mean cell volume (MCV), red cell number and hematocrit  ratios were significantly increased in r groups . The urir groups . The maie groups , but More groups .Cp single and Heph/Cp double KO mice, and only increased in the kidney of Heph/Cp double KO mice. In Heph KO mice, however, there was a reduction in iron levels in plasma, liver and kidney  in the renal medulla than in the renal cortex may explain this difference in iron load. Previous reports have suggested that most iron is reabsorbed by the proximal tubules, but the distal tubules also play a role in this processHeph/Cp KO mice may help to increase the iron transport and re-absorb when iron accumulated in the renal medulla of Heph/Cp KO mice, which may explain why the DMT1 protein was increased in the Cp/Heph KO mice cortex. Consistent with this, the cortex of the Heph/Cp KO mice had increased transferrin receptor and Dmt1 mRNA levels.CP and HEPH are MCFs that are known to work in concert with FPN1 to increase the efficiency of cellular iron export8Cp/Heph KO mice cortex. Consistent with this, the cortex of the Heph/Cp KO mice had increased transferrin receptor and Dmt1 mRNA levels.This region is likely to be particularly important in conceiving iron that is not re-absorbed by the tubular cells of the cortex it induces part of cortex proximal tubular cells to up-regulated DMT1 protein levels to reabsorb urinary iron, which may explain why the DMT1 protein was increased in the in vivo, in polarized mouse intestinal enterocytes, HEPH is present both on the lateral surfaces of enterocytes and also in the apical supranuclear regionFpn1 mRNA and protein levels in the kidney did not vary significantly between mice of different genotypes, suggesting that FPN1 protein levels are insensitive to kidney iron accumulation. Nevertheless, the efficiency of export can still be enhanced by MCFs.A small percentage of TF-bound iron is filtered through the glomeruli of the kidney14Heph and Cp resulted in changes in the expression of other genes involved in iron metabolism. In the renal medulla, where ferritin protein expression and iron levels were significantly increased, Dmt1 + IRE and Tfrc mRNA levels, and DMT1 and TFR1 protein expression were significantly reduced. Recent studies suggest that DMT1 is localized to the late endosomal and lysosomal membranes of proximal tubule cells, where it facilitates the uptake of TF-bound iron16Dmt1 + IRE and Tfrc mRNA levels can respond to the local iron status of the renal medulla, presumably through IRP-IRE–mediated mechanisms2930et al.Dmt1 + IRE mRNA contains a single IRE in its 3′-untranslated region, similar to those in the Tfrc mRNA, and this element confers iron-dependent mRNA stabilizationHeph/Cp results in reduced iron export from renal cells, which in turn results in cellular iron overload and decreased Dmt1 and Tfrc expression.Double knockout of We propose that the role played by HEPH or CP protein in the kidney is similar to that of other cells types. Under physiological conditions, iron reabsorption is mediated through the uptake diferric TF by TFR1 and/or cubilin on the apical surface of the epithelial cells in the renal tubules1032Excess intracellular iron is toxic and prolonged exposure can lead to the generation of reactive oxygen species and tissue damage. Our demonstration that mice lacking both MCFs showed significant increase urinary iron excretion, proteinuria, as well as morphological abnormalities in the renal tubules suggests considerable structural and functional damage to the kidney. Together, these findings suggest that MCFs play an important role in protecting the kidney against injury.In summary, our work has provided further insights into iron transport pathways in the kidney and has shown that copper-dependent ferroxidases play an important role in the homeostasis of this essential organ.Heph KO and Cp KO mouse strains have been previously described834Heph−/− (Heph KO) mice were bred to Cp−/− (Cp KO) mice to generate Heph−/−/Cp−/− (Heph/Cp KO) mice. All mice were on the C57BL/6J genetic background and were allowed unlimited access to a standard rodent unpurified diet containing approximately 180 mg/kg iron provided by Jiangsu Province Collaborative Medical Bioengineering Co., LtdMale mice from each litter were euthanized at 6 months of age after anesthesia with an intra-peritoneal injection of 3% sodium pentobarbital. Blood was collected by cardiac puncture using potassium EDTA tubes  and the liver and kidney were removed. Whole blood was used for hematological analysis and centrifuged to provide plasma for iron measurement as previously describedFor immunostaining experiments, tissue was embedded in paraffin, sectioned and stained for ferric iron using Perls’ Prussian blue staining as previously describedDmt1 , Fpn1, Heph, Cp, and Tfrc in different parts of the kidney. All primers  according to the manufacturer’s instructions on an Applied Biosystems 7300 Real-Time PCR System instrument . Fluorescence curves were analyzed with the LightCycler software (version 3.5.3). Automated calculation of crossing points was carried out by the second-derivative maximum method. Each reaction showed a single specific peak in the corresponding melting curve.Total RNA was isolated from mouse tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Three micrograms of total RNA were reverse transcribed using a Transcriptor First stand cDNA Synthesis Kit (Roche Applied Science) and qRT-PCR was used to measure the expression of  primers  were desHeph/Cp KO, Cp KO, and Heph KO mice (n = 4–5 per genotype) were homogenized in 1 X PBS containing 1% Triton X-100, 0.1% SDS, and a protease inhibitor cocktail . The total protein concentration was determined by the bicinchoninic acid (BCA) method . For most studies, samples containing 20–100 μg of protein were denatured by boiling for 5 minutes in 2 X SDS sample buffer. The proteins were separated by SDS–PAGE (7.5% acrylamide running gel) and transferred to nitrocellulose membranes. For FPN1 immunoblots, the samples were not heated. Blots were first incubated for 1 hour with blocking buffer (PBS containing 0.1% Tween-20 and 10% nonfat dry milk), and then incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: Anti-CP rabbit polyclonal antibody ; anti-HEPH rabbit polyclonal antibody raised against an N-terminus oligopeptide of hephaestin Kidney regions from 6 month old WT, Base-line urine was collected in all group mice. Total iron content in the urine of all group mice was measured using an atomic absorption spectrometer as previously described. Urinary protein concentration was measured using the bicinchoninic acid assay  and urine creatinine (CR) levels were measured using the creatinine assay kit  according to the manufacture’s instruction. Total urine iron content was normalized to urine CR and the urine protein excretion rate was expressed as the ratio of the urinary protein to urine CR.Heph, Cp, Fpn1, Dmt1 + IRE, Dmt1-IRE, and Tfrc) with WT control expression. Differences were considered statistically significant at P < 0.05. All statistical analyses were performed using GraphPad Prism 6 Software .Unless otherwise indicated, values are presented as means ± SEM. One-way ANOVA with Tukey’s test for multiple comparisons was used to compare means for mouse tissue iron concentrations  and protein levels  among the four groups. One-way ANOVA with Dunnett’s test for multiple comparisons was used to compare means for gene expression .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PRNP) genotype, which was either VRQ/VRQ or ARQ/ARQ, with or without additional polymorphisms at codon 141  of the ovine PRNP. This report describes the clinical, pathological and molecular phenotype of goat scrapie in those sheep that progressed to clinical end-stage.A study to investigate transmission of classical scrapie via goat milk was carried out in sheep: firstly, lambs were challenged orally with goat scrapie brain homogenate to confirm transmission of scrapie from goats to sheep. In the second study phase, milk from scrapie-infected goats was fed to lambs. Lambs were selected according to their prion protein gene (141 and one LL141) challenged with one of two scrapie brain homogenates, and six pairs of sheep  fed milk from six different goats, developed clinical disease, which was characterised by a pruritic  or a non-pruritic form . Immunohistochemical (IHC) examination revealed that the pattern of intra- and extracellular accumulation of disease-associated prion protein in the brain was also dependent on PRNP polymorphisms at codon 141, which was similar in VRQ and LL141 sheep but different from LF141 and FF141 sheep. The influence of codon 141 was also seen in discriminatory Western blot (WB), with LF141 and FF141 sheep showing a bovine spongiform encephalopathy-like profile (diminished reactivity with P4 antibody) on brain tissue. However, discriminatory WB in lymphoid tissues, and IHC pattern and profile both in lymphoid and brain tissue was consistent with classical scrapie in all sheep.Ten sheep  contains supplementary material, which is available to authorized users. PRNP) genotype on disease susceptibility was known.Classical scrapie is a transmissible spongiform encephalopathy (TSE) of sheep and goats that can be transmitted vertically from dam to offspring and horizontally between animals. Horizontal transmission was demonstrated not only between conspecifics but also from sheep to goats under natural conditions , 2, wher136RR154QQ171) whereas AA136RR154RR171 sheep are considered highly resistant [PRNP polymorphisms, such as phenylalanine (F) instead of leucine (L) at codon 141, which have been associated with atypical scrapie cases [136 Cheviot sheep with an F141 allele, which had been experimentally inoculated with classical scrapie [PRNP polymorphisms also affect classical scrapie susceptibility in goats. For example, lysine (K) instead of glutamine (Q) at codon 222 is associated with increased resistance, and methionine (M) instead of isoleucine (I) at codon 142 is linked to an increased survival time [Three codons are considered important regarding scrapie susceptibility in sheep: codon 136, encoding alanine (A) or valine (V), codon 154, encoding arginine (R) or histidine (H) and codon 171, encoding glutamine (Q) or arginine (R). In Cheviot sheep, the most susceptible genotype is VV at codon 136, RR at codon 154 and QQ at codon 171 (VV142) to two of four VV136RR154QQ171 sheep by oral inoculation with placenta [A more recent study demonstrated transmission of classical scrapie from a goat  No 999/2001 and its amendments laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. More details on these goats are provided elsewhere [For the pilot study to determine susceptibility of sheep to goat scrapie, brain homogenates were prepared from two naturally infected Anglo-Nubian goats  accordinlsewhere , 13.PRNP genotypes VV136RR154QQ171  and AA136RR154QQ171  born from six ewes from our research flock that was established in England to provide classical scrapie-free sheep for research purposes [PRNP genotyping was performed as described previously [141 or F141 subsequently called LL141, LF141 or FF141). When the study started, recipient sheep were purely selected on the basis of their polymorphisms at codon 136 (VV or AA) to determine the most suitable genotype for a goat scrapie transmission study to sheep. As dam and sire of recipient AA sheep were not both homozygous LL141 or FF141, recipients were LL141, LF141 or FF141.Brain recipients were 10 Cheviot lambs with purposes . PRNP gew/v solution in physiological saline) of one of the two goat brain homogenates. Lambs were raised by their dams until weaning at approximately 10 weeks of age in three pens, two of which contained lambs challenged with either G1460 or G1451 brain and one housed a mixture of lambs challenged with G1460 or G1451 brain   from 6 mSheep were monitored twice daily by farm staff and were systematically examined for neurological signs first at 20 months of age, then at 31 months and then usually monthly or more frequently depending on clinical status, using a short examination protocol for detection of scrapie . A full Assessments were made blind without knowledge of the genotype or inoculum although the scrapie status was known once animals were mixed. Sheep were euthanased with intravenous injection of secobarbital and cinchocaine  at clinical end-point, which was reached when animals displayed progressive abnormalities in sensation  and movement .The brain and a range of lymphoid tissues were taken and processed for immunohistochemistry (IHC) and Western immunoblot (WB) examination.sc) profiles and total amount of PrPsc (see below). A naturally infected classical scrapie sheep, a sheep orally challenged with BSE brain homogenate and a sheep intracerebrally inoculated with CH1641 brain homogenate were used as controls; all three controls were AALL141. The robustness of the discriminatory IHC has been previously demonstrated [For IHC, tissue from the central nervous system (CNS) and lymphoid tissue were formalin-fixed, wax-embedded and examined with six different antibodies, which bind to different amino acid (aa) residues of ovine PrP: BG4 , 12B2 , 521.7 , 9A2 , BH1  and R145  and are used to distinguish classical scrapie from bovine spongiform encephalopathy (BSE) and CH1641-like strains , 21. Thenstrated .sc on a scale from 0 (none) to 3 (striking) in steps of 0.5 and including 0.2 for trace immunolabelling. Five different PrPsc patterns were distinguished: i) intraneuronal, ii) intraglial , iii) extracellular glia-associated , iv) grey matter neuropil-associated , and v) other types . The average value for each PrPsc pattern from the seven areas examined was used to create an individual PrPsc profile and, for grouping by genotype, the profiles constituted the mean of the individual sheep profiles within each genotype group.Lesion profiling was carried out as described previously : tissue sc (PrPres) was carried out on samples of the caudal medulla and medial retropharyngeal lymph node using antibodies Sha31 (aa 156–163) and P4 according to the BioRad TeSeE universal WB protocol . Parallel testing with the two specific monoclonal antibodies aids in the discrimination between classical BSE and scrapie where Sha31 detects PrPres in both cattle and sheep, whereas P4 is more selective for scrapie PrPres [142) brain sample, known UK bovine BSE brain sample and known ovine VRQ/VRQ non-neural mesenteric lymph node sample (for WB on lymphoid tissue) were included as positive controls for profile comparisons. All control samples were homogenates from a single naturally infected animal. All samples were tested once.WB examination for detection of the proteinase-resistant form of PrPe PrPres . Additiores concentrated according to the kit instructions and with the reagents supplied. As four samples produced weak profiles upon initial examination of caudal medulla tissue, these samples were processed in duplicate and the pellets combined (double pellets) for subsequent runs. Samples were heated for 4 min at 100 °C prior to loading on gels. A sample of 15 μl each was loaded in duplicate lanes onto pre-cast 12% bis-tris gels  and electrophoresed for 50 min at 200 V. The proteins were then transferred onto PVDF membranes (115 V for 60 min) and blocked (Bio-Rad blocking solution for Sha31 and milk powder solution for other antibodies) for 30 min at room temperature. They were probed with the kit primary antibody (Sha31) for 30 min at room temperature and additional antibodies  for 1 h at room temperature.From each sample, 0.35 g tissue was ribolysed, purified, treated with proteinase K  and PrPThe membranes were washed, incubated for 20 min in Bio-Rad secondary antibody (Sha31) or goat anti-mouse conjugate  at room temperature, washed again and the membranes were incubated with ECL substrate (Amersham) for 1 min. The signal was detected with the Fluor-S MultiImager (Bio-Rad).res signal emitted from the diglycosylated, monoglycosylated and unglycosylated protein bands was calculated by means of the volume analysis tool in the Quantity One software (Bio-Rad) for antibodies Sha31 and P4. The global background was subtracted and the ratio for the ovine scrapie control sample set at 1; the ratio for the test samples was then calculated relative to the ovine scrapie control sample. The cut off was set at 2.0 and any sample with a ratio below this was considered to be classical scrapie-like and any ratio above this was considered classical BSE-like.The total amount of PrPPRNP genotype, sex and survival time, of the 22 sheep that developed a TSE  are provided in Table PRNP genotype on survival time has already been discussed in the previous publication [Animal data, such as lication  and will141 . Of the four sheep with alopecia (one VV and three LL141 sheep), one LL141 sheep did not display a clear stereotypical response when scratched although the scratch response had been positive at the examination three days earlier. Scratch test scores ranged from 0.21 to 1.0 (median: 0.39) in VV sheep and 0 to 0.79 (median 0.5) in LL141 sheep. None of the LF141 or FF141 sheep displayed signs of pruritus and the scratch test score was 0 for all sheep with these polymorphisms. By contrast, the most frequent sign observed in LF141 or FF141 sheep was limb weakness  in combination with ataxia  whereas limb weakness was observed in only one LL141 sheep even though most of them  were ataxic . A head tremor was present in all six VV sheep but rare in AA sheep; other movement disorders were lip twitches  and spontaneous myoclonus (one LL141 sheep). Bruxism was audible in five of the six LL141 sheep and three of the six VV sheep but only in one FF141 sheep. Infrequent signs were cardiac arrhythmia , cataplexy-like collapse , inappetence (one LL141 sheep), disequilibrium when blindfolded (two VV sheep and one LL141 sheep) and othaematoma (one VV sheep). Table Signs of pruritus, characterised by a stereotypical response to scratching of the back (positive scratch test) and/ or alopecia with or without skin lesions suggestive of excessive rubbing, scratching or nibbling were displayed by all 12 sheep that were either VV or LLAdditional file 2:pruritic form of scrapie. The clip shows the clinical signs of ALRQ/ALRQ wether 1293, which was fed milk from scrapie-affected goat G1427 from 20 h after being born. This sheep, first shown at 1222 days of age and indicated briefly by a red circle to distinguish it from its pen mate, falls when turning. There is also excessive sinking in the hind limbs when it turns on the spot. Scratching of the back results in rising of the head and later lip licking (positive scratch test). Sixty days later, at 1282 days of age, there is evident hind limb ataxia, characterised by delayed hind foot placement and crossing of the hind limbs particularly visible when the animal turns. A positive scratch test can be elicited more easily by simply putting pressure on the spine. (MP4 14151 kb)Additional file 3:non-pruritic form of scrapie. This clip presents the clinical signs of AFRQ/AFRQ ewe 1476 at 1464 days of age following oral challenge on its day of birth with brain from scrapie-affected goat G1451. Twitching of the upper lip (repetitive myoclonus of the lip muscle) is present. There is no evidence of pruritus because this is a fully fleeced sheep without obvious wool loss and it does not respond to scratching of the back (negative scratch test). There is lack of foot placement correction when the sheep is pushed sideways suggestive of proprioceptive deficits and it briefly sits down. Hind limb ataxia is evident with excessive sinking in the hind limbs. (MP4 13735 kb)sc immunolabelling in the brain of each sheep, which reflects the sum of all average scores for the different PrPsc patterns. Whilst there was little variation between sheep with an LF141  or FF141  PRNP genotype, more variation was seen in VV  and LL141 sheep  where the total brain PrPsc score in one sheep could be more than double the score in another sheep irrespective of whether they were challenged with brain or milk from the same donors . In lymphoid tissue, all antibodies detected the same levels of PrPsc in macrophages, which was consistent with classical scrapie.Discriminatory IHC using six different antibodies revealed that antibody detection in the different compartments in CNS tissue of AA sheep was dependent on polymorphisms at codon 141 of the ovine Individual scores are shown in Additional file PRNP polymorphisms at codon 141: PrPres was detected with this antibody in VV and LL141 sheep, similar to the classical scrapie ovine and caprine control samples, whereas reactivity was reduced in LF141 sheep and greatly reduced or absent in FF141 sheep, similar to the classical BSE control sample. Similar observations were made with two further N-terminal antibodies, 12B2 and SAF32. The Sha31/P4 antibody ratios were less than 2 for VV and LL141 sheep as seen with the classical scrapie control samples, but above 2 in LF141 and FF141 sheep. In fact, the ratio was above 5 in 4 of 7 LF141 sheep and in all FF141 sheep  exhibited the pruritic form, which is also the predominant form in sheep infected with the BSE agent and characterised by a positive scratch test (lip licking or head movements when the back is scratched), excessive rubbing and scratching resulting in wool loss [141 and FF141 sheep presented with the nervous form, characterised by ataxia and limb weakness without any signs of pruritus, regardless of donor goat, which is clinically unlike experimental BSE in sheep. These results provided further evidence that the clinical presentation is dependent on the PRNP genotype as suggested by studies in naturally infected sheep where strain as confounding factor could not be excluded [Clinical disease in sheep may vary according to breed, region and country, presenting either as pruritic or non-pruritic form . The resool loss . By contexcluded , 34.PRNP genotype at codon 141. PrPsc profiling by genotype revealed that the profiles did not differ between LF141 and FF141 sheep, which was characterised by the predominance of intraneuronal and intraglial PrPsc and an absence of extracellular glia-associated PrPsc, which was in contrast to LL141 sheep (either VV or AA) where glia-associated PrPsc was more prominent . Studies on the clinical progression in naturally affected sheep with scrapie suggested that head tremor, ataxia, teeth grinding, loss of body condition and loss of a menace response occurred more frequently with disease progression [sc scores, which had been challenged with milk or brain from the same donor, presented with more severe clinical signs: both brain-challenged VV sheep with a lower PrPsc score (1452 and 1425) were not ataxic and had a menace response unlike the other VV sheep (1424), which was ataxic, lost the menace response and had a considerably higher PrPsc score. Similarly, the severity of ataxia was considered less in an LL141 sheep with low PrPsc score (1294) compared to one with a high PrPsc score (1293), which also lost its appetite. However, two other VV sheep had almost identical signs (both ataxic with head tremor) despite considerable total PrPsc score differences and whilst one (1451) was also teeth grinding, the other (1419) displayed signs of disequilibrium, which suggested more severe gait abnormalities.There is evidence from studies in experimentally and naturally infected sheep with scrapie that there is poor association between the severity of clinical signs and the level of PrPres  or PrPsche brain –39. Thisgression , 40. Thesc profiles in LL141 sheep challenged with milk from different donors may be the results of possible strain differences although it does not explain why the same phenomenon was not observed in LF141 sheep unless the wild-type alleles (LL141) produce greater phenotypic variability.The differences in the PrPsc profiles in VV and LL141 sheep challenged with brain and milk from the same goat respectively may be to some extent explained by the different survival time and clinical severity of the sheep, which may alter the proportional localisation of PrPsc immunolabelling so that sheep with longer survival times and more severe clinical signs have proportionally more intraneuronal and intraglial and less glia-associated immunolabelling, possibly as a result of progressive removal and internalization of glia-associated PrPsc as the disease progresses . However, this does not quite explain the profile differences seen in VV sheep where the profiles were similar in sheep with the shortest and longest survival time  is dependent on tissue, cell type and location within the tissue (intra- or extracellular). Extracellular PrPsc, which is not truncated, should be detected similarly with all antibodies whereas intracellular PrPsc is truncated and therefore some antibodies (raised against the globular and C domains) should detect it while others (raised against the N terminus) may not. Whilst discriminatory IHC applied to lymphoid tissue agreed with the molecular profile obtained from lymphoid tissue, which identified the samples as consistent with classical scrapie, tests applied to CNS tissue produced a more varied result, which was dependent on PRNP polymorphisms at codon 141. This particularly affected the detection by N-terminal antibodies, such as 12B2 or P4. The magnitude of PrPsc immunolabelling with the 12B2 antibody was reduced in sheep carrying the F141 allele compared to LL141 sheep, which was particularly striking for extracellular and intraglial PrPsc. Similarly, P4 antibody reactivity in the WB was reduced in brains from LF141 and particularly FF141 sheep. This antibody is used for the discriminatory WB to distinguish classical scrapie from BSE because brains from classical bovine BSE and experimental classical ovine BSE cases show no and greatly reduced signal, respectively [141 sheep, including VV sheep, were classified as classical scrapie because PrPres was detected with antibody P4, and the Sha31/P4 antibody ratio was as expected for classical scrapie isolates, whereas LF141 sheep produced an intermediate BSE-like profile with reduced detection with P4, and FF141 sheep produced a BSE-like profile with little or no PrPres detection with P4 and the Sha31/P4 antibody ratio was unlike classical scrapie for both genotypes.Discriminatory IHC using six different antibodies with different affinity to PrP141 allotype towards the N terminus from the P4 epitope, thus resulting in PrPres detection by both Sha31 and P4. In contrast, proteinase K appeared to cleave the F141 PrP protein towards the C terminus from the P4 epitope, possibly around the 141 position, thus resulting in detection by Sha31  LL141 sheep challenged with the same isolate produced a profile consistent with classical scrapie; ii) testing of lymphoid tissue produced a molecular and pathological phenotype consistent with classical scrapie; iii) the discriminatory IHC was unlike BSE and iv) the lack of pruritus in LF141 and FF141 was inconsistent with the clinical phenotype reported for classical BSE in sheep [The WB results highlight the difficulty in interpreting the discriminatory WB by examination of brain in cross-species transmission experiments because LFin sheep . There iPRNP polymorphisms, particularly at codon 141, which has to be taken into account when investigating strain characteristics by laboratory methods.In summary, this study provided evidence that the clinical, pathological and molecular phenotype of classical scrapie transmitted from goats to sheep is affected by Additional file 1:study design. This file provides a graphical overview of the design of the pilot and milk transmission studies. (PDF 197 kb)Additional file 4:sc scores to create the brain PrPsc profiles as well as the scores for central nervous and lymphoid tissues obtained on the discriminatory IHC. (XLSX 38 kb)clinical and pathological data. This file provides more detail about the clinical presentation, including scratch test score, and shows the individual PrP