Danio rerio) is an excellent vertebrate model for in vivo imaging of biological phenomena at subcellular, cellular and systems levels. However, the optical accessibility of highly pigmented tissues, like the eyes, is limited even in this animal model. Typical strategies to improve the transparency of zebrafish larvae require the use of either highly toxic chemical compounds  or pigmentation mutant strains (e.g. casper mutant). To date none of these strategies produce normally behaving larvae that are transparent in both the body and the eyes. Here we present crystal, an optically clear zebrafish mutant obtained by combining different viable mutations affecting skin pigmentation. Compared to the previously described combinatorial mutant casper, the crystal mutant lacks pigmentation also in the retinal pigment epithelium, therefore enabling optical access to the eyes. Unlike PTU-treated animals, crystal larvae are able to perform visually guided behaviours, such as the optomotor response, as efficiently as wild type larvae. To validate the in vivo application of crystal larvae, we performed whole-brain light-sheet imaging and two-photon calcium imaging of neural activity in the retina. In conclusion, this novel combinatorial pigmentation mutant represents an ideal vertebrate tool for completely unobstructed structural and functional in vivo investigations of biological processes, particularly when imaging tissues inside or between the eyes.The larval zebrafish ( Pigment molecules, such as melanin, haemoglobin and myoglobin, are the main sources of visible light absorption in biological tissues1crystal, lacking the vast majority of skin pigmentation. Compared to the previously described combinatorial pigmentation mutant casper that has pigmented eyescrystal mutant constitutes a significant improvement for in vivo imaging of tissues inside or between the eyes.In order to understand complex biological phenomena, structural and functional information has to be extracted from intact animal systems at different spatial scales. Optical transparency of animals is a highly desirable feature to study these biological processes casper, which lacks all melanophores and iridophorescrystal) that not only lacks all melanophores and iridophores, but also has a non-pigmented RPE. This particular feature makes crystal larvae especially suited for imaging tissues inside or between the eyes while avoiding the use of chemical pigmentation blockers. Importantly, crystal mutants show no behavioural or viability deficits as compared to wild type animals. We also validate the in vivo application of this novel mutant by performing whole-brain light-sheet imaging and two-photon functional recordings of neural activity in the retinae of crystal larvae.In zebrafish, multiple populations of cells produce pigments that ultimately restrict the optical accessibility of tissues54610nacrew2/w2 mutant lacks all melanophores due to a mutation in the mitfa genenacre mutation does not affect the population of cells forming the RPE, this structure is still pigmented in this single mutant. 2) The albino  mutant is characterised by a general deficiency in the production of melanin due to a mutation in the slc45a2 generoy orbison (roya9/a9) mutant shows a complete lack of iridophoresnacrew2/w2;albb4/b4;roya9/a9; red boxes in crystal, lacks the vast majority of dark and reflective pigments normally present at the cutaneous level and, as a consequence, appears optically clear. The internal organs are clearly visible in adult (>3 months old) crystal mutants as opposed to wild type and single mutant fish crystal  lacks melanin in the RPE  do not exhibit any visible phenotype (data not shown). Overall, the crystal mutant constitutes a significant improvement in the optical accessibility of both larval and adult zebrafish, even superior to the previously described combinatorial mutant casperTo generate optically clear zebrafish lacking the vast majority of skin pigmentation without using chemical compounds inhibiting pigment formation, we undertook a combinatorial genetic approach. Three previously described viable mutations affecting different populations of chromatophores  were selbino albb/b4 mutanant fish . Compare the RPE . Consequ0 μM PTU , since Pin vivo imaging has to be characterised not only by optical clarity but also by normal functional and behavioural viability. The use of PTU to quickly obtain transparent zebrafish larvae is widespread across the research community. However, numerous studies reported severe morphological and behavioural side effects caused by the toxicity of PTU treatment (see Introduction). We thus wanted to compare the behavioural viability of crystal larvae to wild type larvae and to larvae raised in medium containing 200 μM PTU. As a representative visually guided behaviour we tested the ability of 5 dpf larvae to perform the optomotor response. During optomotor response assays, freely swimming zebrafish larvae respond to whole-field moving stimuli  by swimming in the same direction of stimulus motion  larvae, 4 dpf PTU-treated larvae (treatment from 1 dpf to 3 dpf) showed a large decrease in visual responses with direction- and orientation-selective neural responses being absent or severely impaired, respectively  linecrystal larvae, the excitation light can easily reach deep  regions of the brain , as opposed to nacre and casper mutants where a substantial portion of the nervous system (~18%) is not accessible through standard light-sheet imaging . Even though the optical accessibility of crystal larvae through light-sheet imaging is significantly higher than nacre and casper mutants, it is noteworthy that the spatial definition of brain regions between the eyes appears lower than other regions of the brain, likely due to scattering of excitation light by the lenses. Despite this fact, the light-sheet signal detected from these regions in crystal mutants is still many-fold higher than in the other animal groups  crystal larvae were immobilised in 2% low melting point agarose with one eye facing an LCD screen where square-wave gratings moving in 12 different directions were displayed (n = 8 larvae). Visually evoked calcium transients were recorded from amacrine and ganglion cells at 4 Hz using near-infrared (930 nm) two-photon laser excitation 2 5.0 mM HEPES, pH 7.6]. The combinatorial crystal mutant  was generated by sequentially crossbreeding three different single mutant strains, namely nacrew2/w2 Tg(elavl3:GCaMP6f)nacre mutant. Light-sheet imaging experiments were performed in nacre, casper and crystal mutants. Two-photon functional imaging experiments were performed in crystal larvae. To treat zebrafish larvae with PTU we followed standard proceduresTg(elavl3:GCaMP5G) larvae used for confocal functional imaging of visually evoked neural activity in the optic tectum were treated with 200 μM PTU from 24 hpf to 3 dpf and imaged at 4 dpf. This work was approved by the local Animal Welfare and Ethical Review Body (King’s College London), and was carried out in accordance with the Animals (Scientific Procedures) Act 1986, under license from the United Kingdom Home Office (PPL70/8057).Zebrafish were maintained at 28.5 °C on a 14 hr ON/10 hr OFF light cycle in Danieau solution  were calculated at each voxel. ΔF was used for population functional data , whereas %ΔF/F0 was used for manually defined regions of interest (ROIs). For each voxel or ROI the integral response over the epoch-interval was calculated to provide a single response metric of each presented direction of stimulus motion. The integral within each epoch window is a summary metric more resistant to saturation effects of the calcium probe than maximum signal change. A threshold for each voxel within an acquisition image sequence was determined from the variance of ΔF changes during the inter-epoch intervals and null condition, threshold = 5 × SDs. All voxels that were supra-threshold within at least two visual presentation epochs were regarded as visually responsive and subjected to further characterization.2. The DSI was defined as (Rpref−Rnull)/(Rpref + Rnull), where Rpref, the response to the preferred direction, was the integral response over the preferred direction epoch-interval. Rnull was similarly calculated as the integral response evoked by the direction opposite to the preferred direction. The OSI was defined as (Rpref−Rorth)/(Rpref + Rorth), where Rpref, the response to the preferred orientation, was the integral response over the preferred orientation epoch-interval. Rorth was similarly calculated as the integral response evoked by the orthogonal orientation. To minimize cross talk and over-fitting associated with DSI and OSI metrics, a stringent approach was undertaken. For a voxel to be regarded as direction-selective (DS) or orientation-selective (OS), mutually exclusive criteria were used: DS if DSI > 0.5 and OSI < 0.5; and OS if OSI > 0.5 and DSI < 0.5. In both cases, the goodness of fit (R2) for DSI and OSI, respectively, had to be >0.8; thus, the fitted curves explained at least 80% of the integral responses. A single von-Mises distribution was used to fit responses of DS voxels and estimate their preferred direction of motion angle from the centre of the fitted curve. The sum of two von-Mises (180° angular distance apart) was used to fit responses of OS voxels and estimate their preferred orientation of motion angles from the centres of the fitted curves. Circular variance was also calculated for comparison as an alternative metric of orientation selectivity (Circular variance < 0.4)To analyse the direction and orientation selectivity of visually responsive voxels direction- and orientation-selective indices (DSI and OSI)nacre, casper and crystal Tg(elavl3:GCaMP6f) larvae, we calculated the number of GCaMP6f+ voxels in each volumetric image by applying the adjust>threshold function followed by the analyse>histogram>list command in ImageJ3 = 1.086 × 10−1 μm3).To determine the brain volume imaged in 4 dpf Statistical test results are reported in Figures and Figure legends. Statistical analyses and tests were carried out using Prism 6 (GraphPad) or MATLAB R2014b (MathWorks). Before performing statistical tests, descriptive statistics  were used to choose the appropriate statistical test . The criterion for statistical significance was set at p < 0.05.How to cite this article: Antinucci, P. and Hindges, R. A crystal-clear zebrafish for in vivo imaging. Sci. Rep.6, 29490; doi: 10.1038/srep29490 (2016).


Plasmodium vivax. Due to the potential for hemolytic reactions in G6PD-deficient patients, safe administration of these drugs requires assessing a person’s glucose-6-phosphate dehydrogenase (G6PD) status. Quantitative and qualitative phenotypic tests that assess overall G6PD activity within a blood sample are generally able to distinguish between G6PD-normal and deficient patients, not factoring in red blood cell (RBC) count and hemoglobin level. A cytofluorimetric assay  allows for screening of G6PD activity within individual RBCs and identifies heterozygous females. This method enables the discrimination of distinct cell populations based on their G6PD activity, estimating the actual proportion of deficient RBCs that are likely to hemolyze during drug treatment. As part of the study, we enhanced the G6PD classification method by analyzing data from the cytofluorimetric assay with a software tool developed at PATH. This software tool not only mitigates for variations in measurements generated by different flow cytometers, but also implements a metric for a more quantitative interpretation of cytochemical staining results. PATH developed the software tool by correlating enzyme activity , genotypic information, and complete blood cell count data with the distinct G6PD activity measured by flow cytometry. As an additional level of validation, the software tool is undergoing blind testing with several hundred subsequent samples collected from multiple international sites. PATH plans to make the software publicly available and hosted online to aid clinical and research studies in determining G6PD status.8-aminoquinoline drugs are critical to malaria elimination campaigns due to their unique ability to kill the dormant liver stage form of


Huntington's disease (HD) is a fatal neurodegenerative disorder with a characteristic phenotype including chorea and dystonia, uncoordinated fine movements, cognitive decline and psychiatric disturbances. Even though the clinical diagnosis of HD relies on the manifestation of motor abnormalities, the associated memory impairments have been growing in prominence. Indeed, cognitive deficits are evident along all the disease process even in the prodrome before any motor diagnosis is given.NTR signalling [per se. In this regard, in our recent studies we shed new light on the contribution of the hippocampal circuitry to synaptic and memory decline in HD. We demonstrated hippocampal dysfunction in a precise genetic HD mouse model that expresses endogenous levels of mutant huntingtin, HdhQ7/Q111 mice, manifested as alterations in spatial, recognition and associative memories.These clinical signs have been mainly attributed to corticostriatal dysfunction being deficits of neurotrophic support, caused by either reduced levels of brain-derived neurotrophic factor (BDNF)  or decregnalling , 3, one NTR since growing evidence indicate that p75NTR plays an antagonistic role in synaptic plasticity. We demonstrated up-regulation of p75NTR in the hippocampus of distinct HD mouse models and in human brain without evident changes in the cortex, which extends our previous data showing increased p75NTR expression in the HD striatum [NTR expression in hippocampal dysfunction we found preserved spatial, recognition and associative memories in new double-mutant mice expressing mutant huntingtin but “physiological” levels of p75NTR levels (Q7/Q111:p75+/−Hdhmice).To get insight into the molecular mechanisms underlying such defects, we focused on p75striatum . In agreNTR levels may mediate synaptic and memory deficits in HD is an intriguing question. On one hand, our results indicated that p75NTR directly or indirectly regulate the expression of different synaptic-related proteins previously implicated in HD synaptic and/or cognitive deficits, such as CBP, CamKII, GluA1 or BDNF since memory improvements in double mutant Q7/Q111Hdh:p75+/−mice correlated with a recovery of the expression and/or phosphorylation of these molecules. On the other, the loss of dendritic spines in CA1 pyramidal neurons exhibited by Q7/Q111Hdh mutant mice was also prevented by normalization of p75NTR levels. Altogether, these data suggest that synaptic and memory deficits in HD could be related with a reduction in proteins involved in synaptic function and in the number and complexity of hippocampal dendritic spines in agreement with a role of p75NTR as a negative regulator of dendritic spine dynamics and synaptic activity.How aberrant p75NTRcontributes to synaptic dysfunction and memory decline in HD by deregulation of RhoA activity, a small GTPase with complex effects on spines and thereby in synaptic plasticity [Q7/Q111:p75+/−Hdhmice suggesting a direct link between aberrant p75NTR activity, dendritic spine loss and aberrant RhoA activity. In agreement with our findings Plotkin and colleagues [NTR-RhoA signalling suggesting that early corticostriatal dysfunction in HD could also be attributable to a correctable defect in BDNF signalling. Altogether, this evidence strongly suggests that, in the early stages of the disease, p75NTR antagonism should be considered an effective therapeutic strategy for restoring BDNF neuroprotective and synaptic functions. However, remains to be investigated whether therapeutic approaches targeting p75NTR inhibition should also integrate TrkB activation to produce the most favourable benefits on motor and cognitive impairments.We further built on work showing that p75lleagues  have demNTR [NTR dysregulation is triggered by neuroinflammation at very early stages and whether modulation of the inflammatory response of microglia and astroglia in HD may contribute to antagonize the deleterious effects of aberrant p75NTR induced RhoA activity in synaptic plasticity. So far, our studies strongly implicates p75NTR-RhoA hippocampal dysfunction on cognitive decline in HD stressing the need to explore therapeutic strategies that target the identified pathways, not only in the striatum but also in other brain areas besides the basal ganglia which has been underestimated and are strongly involved in HD cognitive pathologyMoreover, we have recently demonstrated that fingolimod, a compound used as an immunomodulator in Multiple Sclerosis patients, restores hippocampal synaptic plasticity and improves memory function in a mouse model of Huntington disease acting through down-regulation of TNFα and p75NTR . These l


Arabidopsis roots since the late 1990s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labeling dividing stem cells with 5-ethynyl-2′-deoxyuridine (EdU), the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol’s staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol’s staining, provides a novel quantitative method to study the dynamics of stem cell behavior that govern homeostasis in the Arabidopsis columella root cap.The Lugol’s staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of  In multicellular organisms, a pool of stem cells is maintained throughout the life cycle to produce exactly and only as many cells as necessary for a specific tissue, an astonishing natural phenomenon known as stem cell homeostasis. Stem cells divide asymmetrically to produce two daughter cells of different fates. One of the daughters retains the self-renewal properties of the mother stem cell and keeps on dividing, whereas the other daughter is programmed to differentiate into a specialized mature cell. Asymmetrical division of stem cells and differentiation of mature cells are dynamically regulated so as to maintain the balance between self-renewal and lineage commitment in response to tissue requirements. In animals altered stem cell homeostasis has been linked to aging and carcinogenesis in various organs ; whereasTo reveal the mechanisms that regulate stem cell homeostasis, the dynamics of stem cell behaviors have been studied in various animal tissues where rapid cell renewal is needed. Because the intestine is one of the most rapidly regenerated tissues in the body, the intestinal crypt has become the archetypal system for quantitative understanding of stem cell dynamics and regulation . SeveralArabidopsis thaliana (Arabidopsis) consists of one layer of columella root cap stem cells resided below the mitotically less-active QC, one layer of differentiating columella root cap stem cell daughters and 3 layers of fully differentiated columella root cap cells  and wee1 (SALK_147968C) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). atm-1 is a kind gift from Lieven De Veylder’s lab  and allowed to grow vertically for 24 h. The seedlings were then fixed in freshly prepared fixative solution containing 3.7% (v/v) paraformaldehyde (Sigma) and 1% (v/v) Triton-X 100 (Sigma) in 1 × PBS solution (Vivantis) for 1 h in a vacuum chamber and washed twice with 3% (w/v) bovine serium albumin  in 1 × PBS solution. The seedlings were then incubated with 50 μl Click-iT® reaction cocktail  for 1 h protected from light at room temperature. The stained seedlings were then washed once with 3% (w/v) BSA in 1 × PBS solution and then stored in 1 × PBS solution protected from light until imaging.After 3 days of germination, the seedlings were transferred to on ½ MS medium with 0.8% agar supplemented with 10 μM EdU  and emission wavelengths between 655 and 750 nm were collected. Differential interference contrast (DIC) images of the seedlings were also captured in addition to the EdU stain.The roots of the seedlings were submerged in Lugol solution (Sigma) for 30 s. The roots were then mounted onto microscopic slides using clearing buffer  as the mounting medium. The seedlings were imaged using a DIC optic on a LEICA CTR5000 DIC microscope equipped with a Nikon DS-Ri1 Camera.wox5-1 mutants  was previously found to promote differentiation of columella root cap stem cells . HoweverS)-2′-deoxy-2′-fluoro-5-ethynyluridine (F-ara-EdU), an arabinofuranosyl-ethynyluracil derivative, has been described as being less toxic to animal and plant cells  they encountered and helped in improving some of the steps. XG contributed to optimization of fixation steps. JHH and JX analyzed the data and wrote the manuscript. All the authors commented and agreed on the 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.


Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne’s disease (JD), a chronic enteritis in ruminants that causes substantial economic loses to agriculture worldwide. Current diagnostic assays are hampered by low sensitivity and specificity that seriously complicate disease control; a new generation of diagnostic and prognostic assays are therefore urgently needed. Circulating microRNAs (miRNAs) have been shown to have significant potential as novel biomarkers for a range of human diseases, but their potential application in the veterinary sphere has been less well characterised. The aim of this study was therefore to apply RNA-sequencing approaches to serum from an experimental JD infection model as a route to identify novel diagnostic and prognostic miRNA biomarkers. Sera from experimental MAP-challenged calves (n = 6) and age-matched controls (n = 6) were used. We identified a subset of known miRNAs from bovine serum across all samples, with approximately 90 being at potentially functional abundance levels. The majority of known bovine miRNAs displayed multiple isomiRs that differed from the canonical sequences. Thirty novel miRNAs were identified after filtering and were found within sera from all animals tested. No significant differential miRNA expression was detected when comparing sera from MAP-challenged animals to their age-matched controls at six-month’s post-infection. However, comparing sera from pre-infection bleeds to six-month’s post-infection across all 12 animals did identify increased miR-205 (2-fold) and decreased miR-432 (2-fold) within both challenged and control groups, which suggests changes in circulating miRNA profiles due to ageing or development (P<0.00001). In conclusion our study has identified a range of novel miRNA in bovine serum, and shown the utility of small RNA sequencing approaches to explore the potential of miRNA as novel biomarkers for infectious disease in cattle. Mycobacterium avium subspecies paratuberculosis (MAP) infection [Johne’s disease is a chronic enteritis of ruminants that is caused by nfection . Clinicanfection . In herdnfection .H1 immune responses are characteristic of silent infections, with IFN-γ being the hallmark cytokine, and which can effectively contain mycobacterial infection [H1 responses gradually decline and TH2 responses become more dominant [H2 humoral response offers little protection against disease progression and faecal shedding of MAP commences [MAP infection stages can be sequentially classed as silent, subclinical, clinical and advanced . TH1 immnfection . This cedominant ,5. The Tommences . At thisγ based diagnostics have problems of specificity, as the crude MAP antigen preparations used in the whole-blood stimulations show cross-reactivity to antigens from other, environmental mycobacterial species. Similar specificity issues also arise with the available serological assays [Faecal culture is regarded as the “gold standard” for the diagnosis of MAP infection but requires lengthy incubation periods, and sensitivity can be low in early subclinical infections . Faecal l assays . In addil assays . OverallRecently, microRNAs (miRNAs) have attracted attention as potential prognostic and diagnostic biomarkers for numerous human pathologies . These smiRNA-sequencing can simultaneously identify and quantify the full repertoire of miRNAs in the circulation at any specific physiological state. A major hindrance with sequencing a cellular body fluids, however, is that their RNA concentrations are below the requirements of most commercial  library preparation kits. Nevertheless, there has been some recent notable successes with small biofluid volumes ,23, suggγ responses to MAP antigens (PPD-J) provided an immune reference in which to place the miRNA response. A data analysis pipeline was developed for bovine miRNA discovery, and a number of novel miRNAs were identified. Comparison of the miRNA repertoire between infected and control animals allowed an initial assessment of the potential of miRNAs as biomarkers of early silent stage MAP infection.Our hypothesis in this study was that MAP infection would cause significant changes in the miRNA profile of bovine serum and thus offer a novel diagnostic modality where MAP infection could be disclosed via miRNA profiles. To explore this, we applied RNA sequencing approaches to samples from an experimental MAP infection model. Small RNA-sequencing for miRNA detection on bovine serum has yet to be reported and thus a key aim of this study was to determine whether miRNAs could be sequenced from low serum volumes (≤ 1ml). In our infection model, 3–6 week old calves were infected with a high MAP-challenge dose, and the course of infection followed over the first 6 months of infection. This early period would typically represent the silent phase of infection, when faecal shedding and any serological response would be negative. Parallel whole blood IFN-Mycobacterium avium subspecies paratuberculosis (MAP) strain CIT003  was cultivated to mid-exponential phase in a modified Middlebrook 7H9 broth consisting of 0.47% Middlebrook 7H9 powder , 0.05% Casitone , 0.25% Glycerol , 10% Middlebrook OADC  and 2 μg/ml Mycobactin J  at 37°C with rolling at 4 rpm. Mycobacterial CFU numbers were estimated using both the “pelleted wet weight method” [9 CFU; this was used to infect thirty-five male Holstein-Friesian calves between three to six weeks of age, with the inoculum administrated orally on two consecutive days. Retrospective plate counts using Middlebrook 7H11 agar  supplemented with Mycobactin J revealed the viable challenge dose to be 3.8 × 109 CFU. In parallel to the challenged animals, a control group of twenty calves received a placebo consisting of sterile 7H9 broth. Blood samples were routinely collected from all animals in lithium heparin vacutainers . At intervals over the first six months of the experiment, cell mediated immunity was investigated using the BOVIGAM IFN-γ release assay  according to the manufacturer’s specification, with IFN-γ levels in whole blood samples being quantified following stimulations with MAP-derived PPD-J . Sera was prepared by centrifuging clotted blood samples from silicon-coated (red top) vacutainers  at 1200 × g for 20 mins. The collected sera were immediately stored at -80°C. Sera were routinely screened for screened for MAP-specific antibodies using ELISA (IDEXX). Faecal samples were collected from all animals prior to MAP-challenge and at intervals post-infection. These were cultured for 42 days using the TREK ESP para-JEM system (Thermo Scientific) according to the manufacturer’s recommendations. The animal work was approved by the Animal Research Ethics Committee of University College Dublin (AREC-P-12-61-Markey) and licensed by the Irish Government Department of Health and Children (B100-2828). method”  and optiμl eluates were mixed with 1/10 volumes of 3M sodium acetate pH 5.2 , 1 μl of glycogen  and 3 volumes of 100% ethanol  and stored overnight at -80°C. The samples were subsequently centrifuged at 16,000 × g for 25 mins at 4°C. The resulting pellets were washed with 70% ethanol, re-suspended in 6 μl of nuclease-free water and stored at -80°C.1 ml serum samples were incubated with 5 ml of QIAzol Lysis Reagent  for 10 mins at room temperature. RNA was subsequently extracted from the lysate using the miRNeasy Mini Kit  according to the manufacturer’s specifications. To precipitate the RNA samples, the 60 μl of the prepared RNA using the TruSeq Small RNA Sample Preparation Kit  with slight deviations from the manufacturer’s protocol. Fifteen cycles of PCR were performed, and the resulting amplicons were purified using an Agencourt AMPure XP kit . Briefly, 1.8× concentrations of AMPure beads were incubated with the library preparations for 15 mins. The bound PCR products were then washed twice with 80% ethanol, and subsequently re-suspended in 20 μl of nuclease-free water. The quality and quantity of each library preparation was determined using Agilent DNA 1000 chips  with an Agilent 2100 Bioanalyzer. Each construct was diluted to approximately 20 nM, and randomly assigned to one of two different pools. Electrophoresis using Novex 8% TBE Gels  was used to purify the pools and minimise adapter dimer contamination. The pools were each clustered in two lanes of an Illumina HiSeq 2500 Rapid Run Flow Cell (v1) and sequenced in a SE50bp format using Rapid SBS reagents .Libraries were constructed from 5 www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3445.Sequencing data is available in the ArrayExpress database  and the remaining reads used for analysis. An important aspect of the miRDeep2 results are the performance statistics which give an indication of appropriate score cut-offs by estimating false positives at each score level. Our results showed poor signal-to-noise even at high score levels likely owing to the relatively small percentage of mappable miRNAs in each sample. Therefore we chose a stringent score cut-off of 4  for the novel miRNAs and a cut-off of 0 for the known set. An additional reads-based filter was used to remove the remaining low abundance hits remaining from the miRDeep2 analysis. This filter removed all results with reads present in less than 50% of samples and mean normalised read count ≤150. Another algorithm, sRNAbench , was alsTo get an indication of read depth required for adequate miRNA discovery we took a single file from our dataset and ran both mirDeep2 and sRNAbench iteratively for increasing random subsets of the reads, ranging from 0.5 to 7 million reads, increasing by 1 million at each run.To ascertain the degree of contamination in each sample caused by degraded nucleic acid and other small RNA species, we applied a custom Python script to each sample. FastQC was used initially to determine the quality but no reads needed to be removed due to low quality. Corresponding samples from two sequencing lanes were combined together by concatenating the files.The 3' adapter was trimmed and quality trimming was performed with q>20 using cutadapt.The cleaned reads were collapsed with the copy number of each unique read retained.bowtie-f-S-v 1—best–un <remaining><input collapsed fasta>><outfile>Collapsed reads were successively mapped with Bowtie  to multiThe total percentage of reads mapping to each class was calculated using the original copy numbers.We then applied the following steps for each sample:Bos taurus reference genome (UMD 3.1).The following RNA categories were used for mapping: known miRNA found from miRDeep2, tRNAs from the Genomic tRNA Database , rRNAs fsequence identityif the seed region was conservedif the miRNA is found inside a known gene, the gene type (e.g. protein coding) and whether located inside an exon or intronwhether there was base pair complementary of the seed in the 3' UTR of any enclosing geneA computational method to find conservation across mammal species was used to aid verification of novel miRNAs. This approach is somewhat similar to the one used by the authors of miRNAminer . A custoThe results were filtered to produce a list of likely conserved miRNAs (those with ≥90% identity in at least one other species) and the remainder considered to be non-conserved.EdgeR  was usedThe read count threshold used to filter the miRDeep results was arrived at by using the method of Koh et al.  to estimhttps://github.com/dmnfarrell/mirnaseqThe Python scripts used to perform all computational work described here are available at γ release assay was used to measure IFN-γ production in whole blood after stimulation with MAP-purified protein derivative (PPD-J). From the experimental animals, 20 MAP-challenged cattle and 20 control cattle were selected for IFN-γ analysis at two-months and six-months post-infection. At both intervals, control animals showed minimal responsiveness to PPD-J, while IFN-γ responses were variable across the challenged animals . This suggested that the animals were still in the silent phase of infection and consequently, assessing cell mediated immunity was deemed appropriate for measuring infection status. The BOVIGAM IFN- animals . Six chaBos taurus RNA annotations revealed that only a small percentage of reads in each sample (mean 4.3%) mapped to miRNAs. On average, 58% mapped to tRNA and ~7% to other non-coding RNA databases, while 19% of reads could not be reliably mapped at all  profiles across all samples were obtained from sRNAbench. The results were filtered to remove all low abundance isomiRs  that could be attributable to sequencing or alignment artefacts. As shown in previous studies  for manyOn average the dominant form accounted for 54% of all reads for a miRNA, but this varied greatly. Variant expression followed two main patterns as seen in other studies  those miAfter filtering-out low scoring and low frequency results, 30 putative novel miRNAs predicted by miRDeep2 were retained . The majDMBT1 gene along with three other novel miRNAs predicted by miRDeep2 but which did not meet the filtering criteria; these form a possible cluster of up to 4 miRNAs inside this gene. None of these miRNAs have so far been annotated in miRBase for any species. After filtering we only saw one probable false positive in the final list. This was 9_24602, found inside an existing predicted ncRNA Metazoan signal (Ensembl ID ENSBTAG00000048185). Further inspection of the read mappings indicated this may not be a real miRNA because of the inconsistent alignment of reads across the hairpin sequence.Of the putative novel miRNAs, several had interesting features and are detailed here. 20_11724 is contained inside the intronic region of the Drosha gene in all species and is >95% conserved in all species. This sequence is annotated as a Gencode predicted human novel miRNA (transcript ID ENSCSAT00000020128) but has not been observed experimentally before. 22_13473 was 99% conserved in all species and was found to be reverse complementary to the bta-miR-191 gene. 26_16197 is present in the The majority of candidates contained a seed that already exists in a known human miRNA but with the remainder of each sequence not well conserved. Some of the non-conserved hits are likely to be bovine specific. The predicted structures for the six top hits are shown in Both within group and between group comparisons were used to investigate differential miRNA expression. Stringency was ensured by using a combined FDR of <0.05 and a fold-change of 1.5 as cut-offs for significance. Examining differences between the 0 and 6 month time-point post-MAP infection identified two significantly differentially expressed miRNAs. Specifically at the latter interval, miR-205 was increased (2-fold) while miR-432 was decreased (2-fold). Control animals, however, also displayed similar miR-205 increases (2-fold) and miR-432 decreases (2-fold) at the 6 month time-point but were also accompanied by subtle increases (~1.5 fold) in expression for miR-27a, miR-92b, miR-10b, miR-143 and miR-126-5p . OverallSubsequently for each group, the reads counts of the eight differentially expressed miRNAs identified during the above pairwise comparisons  were plotted to gain further insight into their expression profiles . For allSmall RNA sequencing is currently the method of choice for identification of the entire repertoire of known and novel miRNAs in a given tissue type or specific disease state . There aet al. also reported a high percentage of fresh human plasma reads mapping to non-miRNA categories [It has been reported that large percentages of small RNA reads derived from mammalian sera typically either align to non-miRNA sequences or else cannot be reliably mapped –46. In ategories . An obviWe identified approximately 80–100 functionally relevant known miRNAs per sample, with the majority being orthologs of known human circulating miRNAs ,49. The Extensive isoform diversity for the majority of miRNAs was shown. Undoubtedly some of this variation is due to technical artefacts such as sequence mismatches of very short reads. However after low abundance filtering, multiple variants are still seen consistently across all samples. These consistent patterns of variant expression provide confidence that such variants are not simply due to degradation products . DistribVarious technical factors affect the outcome of miRNA-seq data analyses and it is not always obvious which algorithm is best suited to handling the data; for this reason we chose two approaches, namely sRNAbench and miRDeep2. For instance, sRNAbench was found to be more convenient for isomiR reporting. For several of the lower abundance known miRNAs we found large differences in total read count between sRNAbench and miRDeep2. However, both methods are however highly configurable  and the configuration of multiple profiling parameters could account for significant differences between the two programs.Tool selection will also have a significant impact on novel miRNA discovery. miRDeep2 was preferred for novel prediction because 1) its scoring model allows transparent screening of false positives and 2) it is designed to be applied to all samples in one run, thus independent samples are easily integrated for the whole study. We found sRNAbench somewhat more cumbersome in this regard. We note that miRDeep2 erroneously missed counting of bta-mir-1246 in the known list and assigned this as a novel miRNA; the reason for this is unclear. Therefore, care must be taken in parsing and filtering the novel candidates provided. However in general miRDeep2 proved to be a powerful ‘off the shelf’ tool for novel discovery once additional filtering was applied. Regardless of the data, it is strongly advised to use at least two tools for initial analysis both for consistency checking and since particular features of one tool may be more appropriate to the data in question .While the cancer and cardiovascular fields already have a significant body of literature dedicated to the potential of miRNAs as biomarkers, to date there have been relatively few published investigations of circulating miRNA biomarkers applied to infectious bacterial diseases. A few notable exceptions have explored the potential of miRNAs in tuberculosis cases –21,53 anγ responses in infected animals. While calves infected with MAP for only six months would not be expected to show pathology in the gastrointestinal tract, we hypothesised that the cellular responses to infection detected in peripheral blood may give rise to a miRNA profile that would be detectible in serum. A comparison of the serum miRNome of MAP-challenged IFN-γ responders to their age-matched unchallenged controls six months after infection did not identify a significant difference in miRNA expression. This lack of differential miRNA signature in infected animals is likely due to the insidious nature of the infection; at the time-points examined the animals would still be within the silent stage. Future analyses will focus on characterising serum miRNA profiles from cattle that are at a more advanced stage of infection, having developed a serological response to MAP indicative of a TH1 to TH2 switch and that have commenced faecal shedding.The lack of robust diagnostic and prognostic assays for MAP infection and resulting disease severely hampers the control and eradication of this infection in cattle. Circulating miRNAs hold promise as a novel diagnostic modality for the disclosure of MAP infection status. The presence of miRNA in peripheral blood is thought to arise due to a number of distinct mechanisms; for example, activated lymphocytes are known to release exosomes containing specific miRNAs , while aLongitudinal differences in miRNA expression were, however, evident and similar within both the challenged and control groups. Comparing the 0 to the six month time-points within each group revealed similar read count profiles for miR-205, miR-10b, miR-92b, miR-432, miR-27a, miR-127, miR-126 and miR-143. All these miRNAs were significantly differentially expressed within the control group but, only miR-205 and miR-432 were identified as significant in the MAP-infected group. These changes in the circulating miRNA profile are likely the result of developmental processes in the calves as all of these miRNAs have been shown to have cellular proliferation and development roles in various mammals –67. FurtIn summary we have used deep sequencing of small RNA fractions isolated from bovine serum to explore the utility of circulating miRNAs as potential biomarkers of infection and disease progression in MAP-infection. We have revealed a number of novel circulating miRNA in bovine serum, and identified miRNA whose abundance is linked to bovine development. While no robust miRNA signature of infection could be defined, this may be due to the silent nature of MAP infection at the early time point selected; future studies focussed on animals in the subclinical and clinical phases of infection may prove more revealing.S1 FigProportional mapping by Bowtie to each category of small RNA for each sample in the study. Bars are grouped by pool but not otherwise ordered.(TIF)Click here for additional data file.S2 FigRelationship between log normalised mean normalized read count and sample frequency (number of samples in which each miRNA is found). The majority of high abundance hits are found in over 80% of samples.(TIF)Click here for additional data file.S3 FigThe large numbers of reads>30 represent tRNA degradation product and the small peaks around 20 reads represent the miRNA content. Reads <18nt were removed for later miRNA analysis.(TIF)Click here for additional data file.S4 FigThresholds where the read count distribution of a pair biological replicates become similar can be estimated using the Komologorov-Smirnov statistic as a distance measure. This point can be defined as the initial minimum as increasing read count values are removed from each distribution. For our data these values ranged from 50–150 depending on samples. Data shown for here is for three pairs of replicates.(TIF)Click here for additional data file.S5 FigUnique log IsomiR copy number per miRNA versus log total abundance across all samples shows a linear relationship.(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(CSV)Click here for additional data file.


Yong  n = 26), typical Alzheimer’s disease (n = 17) and healthy control subjects (n = 14) completed centrally-presented tests of letter identification under six different flanking conditions  with two different target-flanker spacings . Patients with posterior cortical atrophy were significantly less accurate and slower to identify targets in the condensed than spaced condition even when the target letters were surrounded by flankers of a different category. Importantly, this spacing effect was observed for same, but not reverse, polarity flankers. The difference in accuracy between spaced and condensed stimuli was significantly associated with lower grey matter volume in the right collateral sulcus, in a region lying between the fusiform and lingual gyri. Detailed error analysis also revealed that similarity between the error response and the averaged target and flanker stimuli  was a significant predictor of error rate, more consistent with averaging than substitution accounts of crowding. Our findings suggest that crowding in posterior cortical atrophy can be regarded as a pre-attentive process that uses averaging to regularize the pathologically noisy representation of letter feature position in central vision. These results also help to clarify the cortical localization of feature integration components of crowding. More broadly, we suggest that posterior cortical atrophy provides a neurodegenerative disease model for exploring the basis of crowding. These data have significant implications for patients with, or who will go on to develop, dementia-related visual impairment, in whom acquired excessive crowding likely contributes to deficits in word, object, face and scene perception.Crowding is a breakdown in the ability to identify objects in clutter, and is a major constraint on object recognition. Crowding particularly impairs object perception in peripheral, amblyopic and possibly developing vision. Here we argue that crowding is also a critical factor limiting object perception in central vision of individuals with neurodegeneration of the occipital cortices. In the current study, individuals with posterior cortical atrophy ( Crowding is a form of inhibitory interaction that is present in spatial vision, involving the diminishing effect of nearby stimuli (‘flankers’) on the identification of a target stimulus . The occAlthough some theories of crowding suggest that it may arise from poor resolution of attention  or unfocOf these three classes of theory, the first  has been distinguished from the second and third (substitution and averaging) with the suggestion that crowding limits identification of target stimuli, whereas lateral masking limits both identification and detection  was available for 7/26 patients with PCA and 11/17 patients with typical Alzheimer’s disease (1-42 ≤ 450 pg/ml and/or tau/amyloid-β ratio >1). The healthy control subjects were matched to the PCA and typical Alzheimer’s disease groups on age and years of education, with the PCA and typical Alzheimer’s disease participants additionally matched for disease duration and Mini Mental State Examination score  under the following conditions (example stimuli are shown in n = 20) were alphabetic items presented in isolation. Letters were presented in random order for 6000 ms in a fixation box  at the centre of the screen.The target stimuli (In each of the following tasks (Tasks 2–6), target letter identification was probed under two spatial conditions, condensed and spaced.n = 24) were flanked on each side by a letter, forming a 3-letter non-word combination.Target letters (n = 24) were flanked on each side by a triangle presented at different orientations. Triangles were of equal height and line thickness to target letters.Target letters (n = 24) were flanked on each side by an Arabic numeral, chosen from a range between 2 and 9.Target letters (n = 24) were flanked on each side by black letters; presentation was as Task 2 except that items were presented on a grey background to match Task 6 (see below).Target letters (n = 24) were flanked on each side by white letters, all presented on a grey background.Target black letters (http://audacity.sourceforge.net).The edge-to-edge distance between the target letter and flankers was 0.1° of visual angle in the condensed condition and 1.0° in the spaced condition at a viewing distance of 50 cm; the height of stimuli (10.5 mm) corresponded to a visual angle of 1.20°. Participants were given one prompt for each trial where they named the flanker (‘Is that the letter in the middle?’); this prompt was intended to limit errors resulting from visual disorientation. The same combination of flankers was used for each target letter under both spatial conditions within each flanker condition. Alphabetic items occurred with equal frequency within each task. The stimuli were presented in blocks of six items, with blocks being administered in an ABBA design. All flanked stimuli were presented in the centre of the screen within a fixation box . All 26 patients with PCA completed Tasks 1, 2 and 4; 24 completed Task 3; and 22 completed Tasks 5 and 6. Naming latencies were manually determined from the onset of each letter using Audacity  words were matched for frequency, age of acquisition and concreteness and were administered in the same testing session as the crowding assessment. MMSE and disease duration were also used as behavioural covariates.Tests of early visual, visuoperceptual and visuospatial processing  were traSpacing (shape): difference in accuracy between spacing conditions (spaced-condensed) in Task 3 (shape flankers);Spacing (number): difference in accuracy between spacing conditions (spaced-condensed) in Task 4 (number flankers);Spacing (shapes/numbers): difference in accuracy between spacing conditions (spaced-condensed) in Tasks 3 and 4 combined;Polarity: difference in accuracy between Tasks 5 and 6 (reverse-same polarity);Polarity (condensed): difference in accuracy between Tasks 5 and 6 (condensed condition only).Scores on shape and number flankers (Tasks 3 and 4) were used as crowding indices, based on the rationale that errors with non-letter flankers were less likely to reflect attentional or executive deficits. Indices were based on raw score differences:n = 1397 (21.6%)]. Latency data greater than two standard deviations from the mean of each participant were considered outliers and removed [total n = 442 (8.73%)]. Prior to latency regression analysis, latency data were transformed using inverse transformation due to non-normal distribution of residuals. Naming latency data were only analysed for participants who made no errors or did not make enough errors to produce significant effects of spacing or flanker type on naming speed using logistic regression or chi-squared tests at the individual level .Latency data for erroneous responses, responses where participants had become overtly distracted from the task, or responses following prompts were removed from the analysis  could account for the spacing effect within the PCA or typical Alzheimer’s disease groups. None of the covariates could account for the overall group difference in naming accuracy between the PCA and typical Alzheimer’s disease groups. Similarly, none of the covariates could account for group differences when considering the condensed condition alone, however, visuoperceptual  to confirm that patients were fixating centrally when making errors naming flanked target stimuli. These three patients demonstrated spacing effects on their naming accuracy and/or latency across flanker conditions. A head-mounted Eyelink II system was used to record gaze location at 250 Hz, with fixations and saccades parsed at standard velocity and acceleration thresholds (30°/s and 8000°/s2). Fixation data suggest that erroneous responses were made when participants were using central vision  showed significantly poorer accuracy for target letter identification with same rather than reverse-polarity flankers . This polarity effect only occurred for condensed flankers . Analysis of latency data (n = 10) found a significant effect of spacing  but not polarity (P > 0.9) on naming speed. In the typical Alzheimer’s disease group, there was no significant effect of polarity on naming speed (P > 0.8) or accuracy (P > 0.1); however, there was an interaction between spacing and polarity , with condensed flankers of same polarity having longer naming latencies. Although none of the control subjects made any errors, there was a trend towards longer naming latencies with same polarity flankers , although there was no interaction between spacing and polarity (P > 0.1).Patients with PCA  fell into three categories: (i) Type A (no response) in 29.9% of errors, the target remaining unidentified, which could result from participants being unable to either detect or identify the target; (ii) Type B (flanker identification), 36.3% error responses resulted from the participant providing the name of a flanker rather than the target (e.g. ZNH → Z); and (iii) Type C (letter not present in target/flanker array), in 33.8% error responses, the participant named a letter which was neither the target nor a flanker. The majority of these errors were suggestive of perceptual integration of flanker and target stimuli . This was despite accurate unflanked letter identification .n = 358): (i) Model 1 (target/flanker averaging): similarity between the error response and the averaged target and flanker stimuli was a significant predictor of error rate ; (ii) Model 2 : similarity between the error response and the individual flanker stimuli did not significantly predict error rate ; and (iii) Model 3 : similarity between the error response and the target or individual flanker stimuli did not predict error rate .An error analysis was carried out in order to assess the extent to which averaging of target and flanker features  predicteP < 0.05): a more pronounced crowding effect for letters surrounded by shapes and numbers was associated with reduced grey matter volume in this region  despite similar accuracy  in naming letters presented in isolation (Task 1). This detrimental effect of closely-positioned flankers upon letter identification ability was associated with significant reductions in grey matter volume in the right collateral sulcus.post hoc evaluation of fixation patterns during trials on which crowding errors were made indicate that at least these participants were using central vision, hence demonstrating that performance does not merely reflect normal peripheral crowding.Before considering the implications of this neurodegenerative perspective on crowding, it is first necessary to rule out various alternative explanations for the observed pattern of performance. First, the crowding effect in PCA could not be attributable to a generic ‘dementia effect’ as individuals with typical Alzheimer’s disease showed no difference compared to healthy controls in their accuracy on any of the flanked letter identification tasks, and estimates of disease severity could not account for the magnitude of the spacing effect. Second, attentional dyslexia is an unlikely candidate explanation as it involves deficits in the recognition of multiple, concurrently presented stimuli of the same category, for example letters presented with other letters . In TaskThe types of errors made by the patients with PCA have implications for accounts of crowding. Error responses were classified into three categories: no response (Type A), naming of a flanker (Type B) and responses not present in the target/flanker array (Type C). Different accounts of crowding would predict a differential weighting of errors towards these three types. Classic lateral masking  would prFurthermore, in the current study we attempted not only to establish the frequency of error types on our central flanked letter identification task, but also to distinguish between averaging and substitution accounts. This was achieved by testing the visual similarity between individual error responses (Type B and C) and (i) the averaged image of the flankers and target (to test the averaging account); or (ii) the images of the individual flankers and target items (to test the substitution account). Analysis revealed that error identity was only significantly predicted by the averaged flanker/target images. These findings are most compatible with compulsory averaging accounts of crowding  position in the periphery  might suggest which regions are implicated in at least one stage of crowding. Reverse polarity has been shown to segregate information via on and off pathways at the level of bipolar cells in the outer retina, which continue to stay relatively distinct until reaching the early visual cortex . AlthougEstablishing a model of acquired excessive crowding in individuals with neurodegenerative disease may offer an opportunity to test mechanistic accounts of crowding through future pathological studies. For example, integration and averaging models of crowding  will permit a novel, dynamic perspective upon the phenomenon and its interaction with basic and higher-order visual processes.Both quantitative (spacing and polarity effects) and qualitative (error types) aspects of flanked letter identification performance, and the cortical localization of the spacing effect, indicate that individuals with PCA commonly develop excessive crowding in central vision. Just as crowding in the normal periphery operates irrespective of object type (e.g. faces:


Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy.We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry.We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival .In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies. Liver cirrhosis constitutes the most severe stadium of chronic liver injury caused by various pathogenic factors and affects approximately 10% of world’s population  with simde novo from serine and palmitoyl-CoA through the action of various enzymes via dihydro(dh)Cer, through hydrolysis of sphingomyelin by sphingomyelinases and through degradation of complex glucosphingolipids [de novo Cer synthesis, has been shown to inhibit both hepatitis B virus (HBV) [de novo very long chain Cer’s (C22-C24), has demonstrated increased rates of hepatocellular apoptosis and a severe hepatopathy in these animals with a significant impairment of liver homeostasis [Ceramide (Cer) is the structural and functional hub of sphingolipid metabolism with pleiotropic effects on cellular and molecular physiology of both cell-autonomous systems as well as complex organisms . Cer is golipids . Though golipids ,11 as wegolipids ,13. In tgolipids , diabetegolipids , metabolgolipids , Alzheimgolipids  and acutgolipids . Furthergolipids –21 and agolipids . Additious (HBV)  as well us (HBV) . Moreoveeostasis ,26. In tRecently, we were the first to report, that long (C16-C20) and very long  chain Cer’s accumulate in the serum of patients with NAFLD as compared to healthy individuals and patients infected with HCV, whereas particularly C24Cer was significantly diminished in the serum of HCV patients compared both to healthy individuals as well as to NAFLD patients . MoreoveIn the current study we assessed possible correlations of various serum SL parameters with severity of cirrhosis, rate of hepatic decompensation and overall survival in a prospective series of 244 patients with newly or already diagnosed cirrhosis who were treated as in- or out-patients in our department from March 2009 until June 2011 as previously described . Inclusihttp://www.R-project.org software version 3.0.2). Statistical comparisons were carried out using the non-parametric Kruskal-Wallis test with post-hoc tests as well as Mann Whitney-U or the stratified van Elteren tests to determine differences among patient groups. Bonferroni correction was calculated for adjustment of multiple testing if appropriate. Kolmogoroff-Smirnoff’s delta was used to test for Gaussian distributions. The data in the box plots are expressed as Means ± Standard Error unless otherwise specified. Time-dependent receiver operating characteristic (ROC) curves and area under the curve (AUC) values were calculated by the R-software for the evaluation of the predictive accuracy of C24Cer regarding overall survival. For assessment of independent predictors of survival, a time-dependent multivariate analysis was computed by the free software R. Only patients with complete data for the predicting covariates were included in multivariable analysis. Survival curves were calculated with the Cox regression model provided by BiAS. The primary end point was overall survival. The level of significance is set at α = 0.05 representing the 95% confidence interval (CI). Statistically significant differences are indicated in the corresponding Figs: “*” = p<0.05, “**” = p<0.01, “***” = p<0.001.Calculations were made with GraphPad Prism v5.01 for Windows , BiAS software for Windows  and the free software R , hepatic encephalopathy (HE), hepatorenal syndrome (HRS) and variceal bleeding and was observed in 73.7% of all patients. A small group of 29 (11.9%) patients was diagnosed with hepatocellular carcinoma. Overall 50 (20.4%) patients died within the study duration. Biochemical, clinical and demographic characteristics of the patients are shown in Long chain (C16-C20) and very long chain ceramides  are the most abundant SL metabolites in the serum of healthy individuals  as well In own previous studies we identified C24Cer being significantly decreased in chronic HCV infection as compared to healthy individuals and patients with NAFLD  as well Decreased serum concentrations of long and very long chain Cer’s associated with the presentation of major clinical complications defining hepatic decompensation such as ascites, SBP, HE and HRS . ParticuMELD and Child-Pugh score, constitute the “gold standards” to predict mortality rates in patients with cirrhosis ,6. SinceWhen overall survival was evaluated in patients with low C24Cer , a significant decrease of cumulative survival was identified as compared to patients with higher C24Cer values (p<0.001) comparable to the cumulative survival observed according to high (≥15) and low (<15) MELD values p<0.001, . Though,We have previously observed a deregulation of bioactive SL’s with major impact on hepatic pathophysiology, particularly Cer concentrations, in the serum of patients with chronic liver disease as compared to healthy individuals  as well Our current analysis focused on the serum concentrations of very long chain Cer’s and long chain Cer’s in patients with cirrhosis since on the one hand many of the enzymes producing very long chain Cer’s, as for instance Cer synthase 2, are mainly expressed in the liver  and on tFurther, our findings show that low serum C24Cer is significantly associated with the rate of hepatic decompensation  and overInterestingly, we additionally observed that clinical presentation of ascites showed a significant inverse correlation solely to the levels of C24Cer and dhC24Cer and not to other (dh)Cer species . The occHowever, the highly interesting novel associations identified by our analysis raise the question about the underlying mechanism of C24Cer decrease in cirrhosis and the biological significance of this phenomenon. Numerous studies have attributed critical second messenger functions to Cer including the orchestration of cellular responses upon various stimuli and the induction of apoptosis, senescence and autophagy ,35. Yet,Although offering promising results our study had some limitations. As a clinical association study it cannot proof a causal relationship between SL metabolism and cirrhosis. Since serum Cer’s are bound to lipoproteins such as low density lipoprotein (LDL), high density lipoprotein (HDL) and very low density lipoprotein (VLDL) it cannot be excluded that the observed differences are due to changes of the mentioned lipoproteins in patients with cirrhosis. Unfortunately, serum levels of these lipoproteins were not available in our patients. Most of the included patients showed a hepatic decompensation at time of inclusion and follow-up data regarding clinical recompensation or anew decompensation were unfortunately not available. Despite our effort to provide a comprehensive multivariable analysis regarding prediction of overall survival, data on prevalence of diabetes mellitus, metabolic syndrome and insulin resistance were lacking in our analysis. Nevertheless, our observations offer a suggestive functional link between SL’s and cirrhosis and thus a significant advance over previous studies since they demonstrate significant variations in serum SL’s according to different severity, decompensation and overall survival of cirrhosis.In conclusion, our study identified, to our knowledge for the first time, very long chain Cer’s and particularly C24Cer to be associated with hepatic decompensation and to correlate independently with overall survival of end-stage liver disease. Particularly, an updated MELD score by inclusion of C24Cer, showed an increased predictive accuracy regarding overall survival in patients with decompensated cirrhosis. Certainly further studies are needed in order to elucidate the underlying mechanism as well as the role of serum SL’s and particularly C24Cer in the non-invasive mortality prediction in patients with cirrhosis.S1 File(DOCX)Click here for additional data file.


Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers  relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings. The enzyme-linked immunosorbent assay (ELISA) is used in clinical and laboratory settings to accurately and reproducibly quantify soluble proteins2Owing to their versatility and reliability, ELISAs have been used to detect HIV/AIDS681012Many diseases, for example acute graft versus host disease (GVHD), cannot be diagnosed with sufficient specificity and sensitivity using single biomarkers891415Many multiplex assays rely on the application of a cocktail of detection antibodies. After extensive optimization, such cocktails can often provide satisfactory results17181920TM, can theoretically achieve up to 100-plex. In reality, however, capture and detection antibody cross-reactions limit LuminexTM-based protein detection to the initial screening of biomarkers, rather than clinical biomarker verification27Previously, several other approaches were developed to mitigate antibody cross-reactions for improved multiplex protein detection2324Here, we eliminate the problem of detection antibody cross-reactions in multiplex ELISA by using aqueous two-phase systems (ATPSs) composed of the phase-separation-promoting polymers polyethylene glycol (PEG) and dextran (DEX)2729ATPS-ELISA is performed using the same steps as conventional sandwich ELISA, except that ATPS-ELISA uses detection antibody solutions that are deposited in DEX droplets over the capture antibody spots  by microAs shown in The impact of optical crosstalk was not significant in our system, as assessed by signal area intensity plots and background measurements . These pCustom plates molded from polystyrene were used to facilitate capture antibody and detection antibody alignment in our multiplex assays. The plates were designed to prevent movement of the DEX droplets after pipetting and to prevent inadvertent misalignment of the capture and detection antibody spots. Prior to fabricating our final plate, we tested a range of antibody well diameters (0.5 mm to 2 mm) with different edge-to-edge spacing (0.3 mm to 0.6 mm) and a depth of ~110 µm. Using the smallest well diameter and spacing, it was possible to deposit a 7 × 7 array (49-plex) of detection antibodies, as well as 4 × 4 (16-plex) and 3 × 3 (9-plex) detection antibody arrays . Figure Based on the number of biomarkers selected for our study, we designed our final plate with four antibody wells 4-plex;  that werWe analyzed plasma samples from three patient groups: healthy controls (n = 20), allogeneic bone marrow transplant patients who did not manifest symptoms of acute GVHD  and allogeneic bone marrow transplant patients who had been diagnosed with acute GVHD . The biomarkers and samples we used were used previously as part of several larger studies that analyzed GVHD status using serial singleplex ELISA914162), we would expect there to be greater signal per unit volume (10 µL) of antigen compared to the 384-well plate reservoirs used for single ELISA (>23 mm2). This is reflected in our standard curve data, which show that the ATPS-ELISAs enter linear ranges for signal detection at lower antigen concentrations than the conventional single ELISAs. These observations are consistent with the observations of others that suggest that smaller spot sizes relative to the assay volumes produce superior LoD and LDR values31The ATPS-ELISA procedure, allowed us to accurately assess the GVHD status of the samples . For exaBland-Altman analysis was used to evaluate the agreement between ATPS-ELISA and standard single ELISA . We obseIt is also important to emphasize that the multiplex biomarker panel was critical for obtaining high sensitivity (correct classification of patients with GVHD) and high specificity (correct classification of patients without the disease). For example, one of the GVHD– patients had high levels of HGF (6615 pg/mL). Based only on the levels of this biomarker, this patient could have been diagnosed with acute GVHD and could have potentially received unnecessary treatment. However, with the inclusion of the three other markers measurements that were all low, this patient can be considered GVHD– and avoid unnecessary treatments that may cause dangerous side effects. In other cases, a single biomarker did not reach the diagnostic threshold for GVHD in spite of the patient's GVHD+ status. In these cases the information obtained from the other three biomarkers in the panel that are all elevated can be used to correctly assess the disease status of the patient.We demonstrated that our system can utilize chemiluminescence to determine the levels of biomarkers without antibody cross-reactions that can cause false positive and false negative signals using heavily cross-reacting antibodies as a test example , 2 and 31414Our assay offers several advantages over conventional single biomarker ELISAs including the use of small plasma volumes , cost savings (less antibody consumption) and use of readily available plate materials and detection systems. For example, performing similar experiments using the individual wells of a conventional 384-well plate would require at least 40 µL of sample (~10 µL/well) to fill the 4 wells required for each of the 4-biomarkers tested. In contrast, our detection antibody patterning technology uses only 10 µL of sample total, since the 4 antibody spots are patterned within a low profile well of 6.5 mm diameter (an intermediate diameter between 96 and 384 format wells). We used 384 well format plates for our comparison to singleplex ELISA. Although the smaller well size allowed us to use 10 µL sample volumes for both the ATPS and regular ELISA assay formats, we did observe higher background and slightly worse ROC when using 384 well plates, likely due to a difference in the capture antibody area to sample volume ratio and possibly also due to the deep well geometry of conventional 384 well plates that can increase pipetting errors and make it more difficult to wash completely.The ATPS-ELISA also provides advantages over miniaturized single ELISA systems, such as Droparray, which use non-aqueous solutions  to cover the aqueous dropletsTM, ATPS-ELISAs can be performed with a broad range of detection antibodies, including polyclonal antibodies and cross-reacting antibodies. LuminexTM, on the other hand, must be carefully tested when introducing new antibody reagents, since it is not specifically designed to suppress antibody cross-reactivity. Moreover, our assay does not require specialized analyzers or sample handling devices. Instead, our assays can be read on common readers using plates that can be easily fabricated in many laboratories from bulk polystyrene by a hot-embossing-based rapid prototyping process. We envision such plates to become available for broader dissemination by injection molding. Finally, ATPS-ELISAs are highly scalable. By depositing antibody reagents in microdroplets, we prevent antibody cross-reactions and obtain multiplexed assays with comparable results to conventional singleplex ELISAs.In contrast to commercially available multiplex biomarker analysis systems, such as LuminexWhile many potential biomarkers have been discovered, there are multiple barriers  to biomarker verification, qualification and FDA approval. One technological barrier has been the lack of multiplex immunoassays that are efficient in usage of precious samples (consume less per run), robust in providing quantitative data without an increase of background or cross-reactions, rapidly customizable and free from reliance on difficult to access reagents, plates and hardware32We also remove the bottleneck for clinical translation in several ways. First, multiplex signals become more robust due to lack of cross-reactions. Second, the validation procedure becomes easier because each biomarker in the panel is arrayed (or patterned) independently of the others. Therefore, if reagents change or one assay does not work, the remaining biomarkers are still valid. Finally, the increased variety of antibodies that can be used with ATPS-ELISA reduces the time and cost associated with the development and validation of new panels of multiplex immunoassays.In the context of acute GVHD diagnosis and biomarker qualification, it is important to use a panel of multiple biomarkersThe ability to identify high-risk patients using biomarker panels early in their transplant course, before the development of GVHD, may permit more stringent monitoring and preemptive interventions. Another avenue for using these biomarker panels is post-transplant monitoring of the response once the treatment has startedThe compatibility of ATPS-ELISA with plasma suggests that our assay will also be compatible with other bio-fluids, such as urine, cerebral spinal fluid, salivary fluid and cell lysates. In addition, our test uses less sample, which is important for large retrospective studies where the volume of samples are often limited due to limited storage space. The judicious use of patient samples will also be relevant to pediatrics, were the volumes of samples are limited due to the size of children. Further studies with more rigorous standards for assay validation will enable translation to clinical biomarker qualification and determination of the clinical cutoffs for our assay3839In conclusion, ATPS-ELISA provides multiplex biomarker detection for validation of new biomarker panels and diagnosis of complex diseases, such as GVHD, where custom multiplex immunoassays are required. The ATPS-ELISA technology prevents cross-reactions among antibody reagents, reducing the risk of false positive and false negative detection of disease markers. This strategy is compatible with standard immunoassay reagents, workflows and equipment, allowing it to be easily implemented in clinical and laboratory settings.ATPSs were created from aqueous solutions of 20 wt% DEX 500,000 (Pharmacosmos) and 20 wt% PEG 35,000. ELISA kits for human sTNFR1 (cat # DY225), human HGF (cat # DY294), human ST2 (cat # DY523) and human trappin-2/elafin (cat # DY1747) were purchased from R&D Systems. SuperSignal ELISA Femto Substrate (cat # 37074) was purchased from Thermo Scientific. Identical reagents, concentrations and incubation times were used for both the ATPS-ELISA and single ELISAs according to the manufacturer's recommendations.The partition coefficients for the various biotinylated detection antibodies were determined by dot blot on polyvinylidene fluoride (PVDF) membranes. Briefly, the antibodies were diluted 1:500 and thoroughly mixed in ATPSs consisting of 10% PEG 35,000 and 10% DEX 500,000. The tubes were then centrifuged at 400 rcf for 15 min to cause complete phase separation of the PEG and DEX. The PEG and DEX fractions were collected and placed in separate tubes at 4°C. The PVDF membranes were prepared by soaking in methanol for 15 s, followed by soaking in water for 1 min and PBS for 5 min. The PVDF membranes were transferred onto PBS-moistened chromatography paper and spots containing 0.5 µL of the separated PEG and DEX samples were pipetted directly onto the membranes. The membranes were then blocked in 1% BSA for 1 h and washed 4 times in PBS containing 0.05% Tween-20. The membranes containing the PEG- and DEX-antibody spots were incubated in streptavidin-conjugated HRP for 1 h and washed 4 times with PBS. Chemiluminescence signals were developed using SuperSignal ELISA Femto Maximum Sensitivity Substrate  and detected using a Fluorchem M imager (Protein Simple).Polystyrene plates were spotted with capture antibodies reconstituted in PBS (4 µL per spot). The plates were stored at 4°C overnight in sealed containers containing PBS-soaked Kimwipes to limit evaporation. The next day, the plates were washed five times with wash buffer  and blocked with 3% casein for 1 h. After blocking, the plates were washed five times. The rapid and thorough washing performed in this step (and later after the detection antibodies were applied) ensured that the antibodies in solution would not be able to produce measurable cross-reactions with neighboring antibody spots. Next, the plates were incubated with the sample solutions (10 µL each for ATPS-ELISA) for 2 h. Following antigen application, plates were washed five times and incubated for 2 h with detection antibodies in either the traditional ELISA or ATPS-ELISA format. For conventional singleplex sandwich ELISA, detection antibodies were bath applied according to the manufacturer's specifications. For ATPS-ELISA, the plate was first filled with a solution of PBS containing PEG and 0.1% casein. DEX droplets containing the appropriate biotinylated detection antibodies (4 µL in volume) were then pipetted into the PEG over the corresponding capture antibody spots. In addition to providing phase-separation and confinement of the DEX droplets, the relatively large volume of PEG used to cover the plate prevented evaporation of the detection antibody solutions. The 4-plex antibody solutions were applied using a Matrix adjustable multipipettor (Thermo). An Ultimus I pneumatic pump (Nordson EFD) connected to a glass capillary needle containing the DEX/antibody solution was used to fill smaller wells . The details of this system and its operation are described elsewhere41To optimize the plate design and demonstrate the capabilities of our multiplex system we fabricated a polystyrene ELISA plate using PDMS hot embossing. Xurography (razor writing) of ~110 µm-thick vinyl tape was used to generate tape templates for the larger sample wells and the smaller detection antibody wellsHeparinized plasma samples were collected from patients who received allogeneic bone marrow transplantation at the University of Michigan between 2000 and 2010. Plasma samples were collected under protocols approved by the University of Michigan Institutional Review Board and stored at the University of Michigan. The protein standard buffer consisted of PBS containing 10% healthy pooled plasma and 1% FBS, while sample buffer was PBS containing 1% FBS. Initial spike-and-recovery experiments indicated that signal intensities varied depending on patient plasma buffer and the sample matrix (plasma). To control for matrix effects, FBS was added to the PBS sample buffer such that appropriate signal intensities were achieve when control plasma was spiked with a known concentration of recombinant biomarker protein.All plots and statistical analyses were carried out in Sigmaplot with Sigmastat (Systat Software). Standard curves were generated over a wide range of concentrations to ensure that there were no hooking effects at high concentrations and to assess the level of non-specific background signal at low concentrations. The values for single ELISA (collected using a chemiluminescence plate reader) were scaled to produce values of the same order of magnitude as the ATPS-ELISA values (collected using the Fluorochem M western reader). The standard curves were fit using a four parameter logistic function. The limit of detection (LoD) was determined from the equation LoD = LoB + 1.645 (SDlow concentration sample), where SD is the standard deviation and LoB is the limit of blankJ.P.F. performed experiments, analyzed data, prepared the figures and wrote the manuscript. J.B.W. performed experiments, assisted with data analysis and figure preparation and helped write the manuscript. A.B.S. assisted with experiments and helped write the manuscript. M.T. assisted with experiments. S.P. and S.T. oversaw the project and the preparation of the manuscript. All authors reviewed the manuscript.Supplemental Information