In recent years the plasma proteomes of several different myelodysplastic syndrome (MDS) subgroups have been investigated and compared with those of healthy donors. However, the resulting data do not facilitate a direct and statistical comparison of the changes among the different MDS subgroups that would be useful for the selection and proposal of diagnostic biomarker candidates. The aim of this work was to identify plasma protein biomarker candidates for different MDS subgroups by reanalyzing the proteomic data of four MDS subgroups: refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia or refractory anemia with ringed sideroblasts (RA-RARS), refractory anemia with excess blasts subtype 1 (RAEB-1), and refractory anemia with excess blasts subtype 2 (RAEB-2). Reanalysis of a total of 47 MDS patients revealed biomarker candidates, with alpha-2-HS-glycoprotein and leucine-rich alpha-2-glycoprotein as the most promising candidates. Myelodysplastic syndrome (MDS) is a group of heterogeneous oncohematological bone marrow disorders characterized by peripheral blood cytopenias, ineffective hematopoiesis, bone marrow hypercellularity, and so forth . MDS claIn this work, the data from four previous proteomic studies of different MDS subgroups has been reanalyzed: refractory cytopenia with multilineage dysplasia , refractP values of all spots were computed by the software using one-way ANOVA analysis. Protein identification was performed for spots (proteins within the spots) that were not submitted for protein identification in the previous studies. An HCT ultra ion-trap mass spectrometer with nanoelectrospray ionization  coupled to a nanoLC system UltiMate 3000  was used to perform MS analysis. Mascot  was used for database searching (Swiss-Prot). Two unique peptides  were necessary to identify a protein. The procedure was described in detail previously 15], respWestern blotting of alpha-2-HS-glycoprotein showed several bands of approximately 50 kDa with a trend of decreasing intensity in advanced MDS subgroups. This result is in agreement with our electrophoretic data; however, it is apparent from the western blot that the representation of individual bands differs more substantially relative to total protein levels. The most obvious change can be observed for the bottom band, whose intensity increases substantially in the RAEB-1 subgroup. This supports the need for precise characterization of A2HSG posttranslational modifications and their quantification. Western blot analysis of leucine-rich alpha-2-glycoprotein revealed two bands of approximately 48 and 60 kDa. The lower 48 kDa band corresponds to that identified in 2D electrophoresis and shows the trend of increasing its intensity in advanced MDS subgroups. This is in agreement with the data obtained by 2D electrophoresis. As in the case of alpha-2-HS-glycoprotein, the modifications need to be characterized. Apolipoprotein A1 was shown to increase the spot volume in RA-RARS compared to RCMD and RAEB-2. It is clear from the western blot that the highest intensities were observed for both the RA-RARS and RAEB-1 subgroups. The fact that the changes were found for RA-RARS by 2D electrophoresis and not for RAEB-1 was most probably caused by the low number of RAEB-1 samples. No obvious changes were observed for albumin; therefore, we assume a minimal influence of the acute phase reaction on the results. Nevertheless, it has been recently shown that there are many other factors  that can strongly affect protein plasma levels . MoreoveIdentifying new biomarkers could have a significant impact on the clinical practice. While for diagnostics it is important to investigate the differences between the healthy (or nondisease) and patient cohorts, it is essential to identify alterations in the disease progression for prognosis and therapy monitoring. Considering MDS, it is important to describe the changes among different subgroups (as was the aim of our study), alterations related to the therapy , or specific changes during the disease progression. The changes related to MDS progression toward acute myeloid leukemia were investigated in work by Braoudaki et al.  in plasmIn conclusion, plasma protein biomarker candidates have been presented in this work with respect to different MDS subgroups. Alpha-2-HS-glycoprotein and leucine-rich alpha-2-glycoprotein appear to be the most promising candidates with regard to western blot observations, as well as our previous results detailing the differences in plasma proteome patterns between MDS subgroups and healthy donors. The presented results should be a catalyst for further MDS biomarker validation requiring precise protein posttranslational modification characterization, profiling of the changes in MDS subgroups, and extended statistical validation with large patient cohorts.


Microbial pathogens and pests of animals and plants secrete effector proteins into host cells, altering cellular physiology to the benefit of the invading parasite. Research in the past decade has delivered significant new insights into the molecular mechanisms of how these effector proteins function, with a particular focus on modulation of host immunity-related pathways. One host system that has emerged as a common target of effectors is the ubiquitination system in which substrate proteins are post-translationally modified by covalent conjugation with the small protein ubiquitin. This modification, typically via isopeptide bond formation through a lysine side chain of ubiquitin, can result in target degradation, relocalization, altered activity or affect protein–protein interactions. In this review, I focus primarily on how effector proteins from bacterial and filamentous pathogens of plants and pests perturb host ubiquitination pathways that ultimately include the 26S proteasome. The activities of these effectors, in how they affect ubiquitin pathways in plants, reveal how pathogens have evolved to identify and exploit weaknesses in this system that deliver increased pathogen fitness. Post-translational modification is a tool used by prokaryotic and eukaryotic cells to regulate protein function. These modifications enable diverse outcomes on target proteins. Addition/removal of small molecules  can directly regulate activity or promote protein/protein interactions. Addition of larger functional groups [e.g. hydrophobic groups  or sugars (glycosylation)] can define protein localization to a membrane or enhance stability. Post-translational modification also includes structural changes such as the formation of intramolecular disulphide or isopeptide bonds that promote protein stability.et al., et al., Attachment of other polypeptides, such as ubiquitin and the structurally related but sequence-diverse ubiquitin-like proteins , to substrate proteins modulates many biological processes from the cell cycle and cell division to apoptosis and the immune response and inflammation . In addition, proteins with a standard secretion signal and natural products can also perturb the host UPS.Pseudomonas syringae pv. tomato DC3000 is the causative agent of bacterial speck disease on tomato and Arabidopsis. The genome of P. syringae DC3000 encodes at least 28 type III secreted effector proteins  and predominantly catalyses Lys11-linked polyubiquitin chains in vitro using recombinant proteins , through a type IV secretion system, from a pathogen-encoded virulence plasmid into the plant genome. This activity is widely used for plant transformation with heterologous genes. One gene encoded on the virulence plasmid is VirF, an F-box motif-containing protein that interacts with A. tumefaciens VIP1 and VipE2 and targets them for degradation in the plant cell nucleus via a host SCF (Skp1-Cdc53-cullin-F-box) complex and the 26S proteasome  that when deleted cause a severe virulence defect  protein and HopM1 activity is specific for this particular ARF-GEF family member. The targeted degradation of AtMIN7 interferes with vesicle trafficking and the deposition of callose, the latter being a well-characterized read-out of surface-mediated plant immunity pathways. AtMIN7 is also involved in intracellular-mediated plant immunity pathways . Additional virulence associated targets of HopM1 activity have also been suggested , but also inhibit proteasome activity. These activities appear to be antagonistic. However, during infection, the action of these effectors may be spatially or temporally separated. XopJ and HopZ4 are localized to the plant cell plasma membrane and may only target a subset of the total proteasome complexes in the cell  and other immunity-related recognition events at the plasma membrane, and these all require CMPG1  in gland cells and these proteins contain a signal for secretion  library from the salivary glands of the pea aphid The host UPS is a target of multiple plant pathogen and pest effector proteins, suggesting it has a fundamental evolutionarily conserved role in pathogenesis and/or immunity and is an ‘Achilles heel’ of the host. It would not be surprising if novel perturbations of the UPS by pathogens and pests of plants continue to be discovered. It is important to understand the molecular mechanisms of these activities as they may present routes for the design of novel strategies to engineer increased plant resistance to pathogens and pests of relevance to agriculture.


MIR204 gene to be significantly associated with schizophrenia. This variant resulted in reduced expression of miR-204 in neuronal-like SH-SY5Y cells. Analysis of the consequences of the altered miR-204 expression on the transcriptome of these cells uncovered a new mode of action for miR-204, being the regulation of noncoding RNAs (ncRNAs), including several miRNAs, such as MIR296. Furthermore, pathway analysis showed downstream effects of miR-204 on neurotransmitter and ion channel related gene sets, potentially mediated by miRNAs regulated through miR-204.As regulators of gene expression, microRNAs (miRNAs) are likely to play an important role in the development of disease. In this study we present a large-scale strategy to identify miRNAs with a role in the regulation of neuronal processes. Thereby we found variant rs7861254 located near the  Their maturation process consists of two consecutive cleavage steps. The first step takes place in the nucleus, whereby the Microprocessor complex, consisting of ribonuclease Drosha and its cofactor DGCR8, frees the hairpin structured precursor miRNA (pre-miRNA) from the primary miRNA transcript (pri-miRNA) ,2. AfterIdentification of miRNAs involved in disease pathomechanisms is often performed at the RNA level, by comparing miRNA expression profiles in affected tissues between individuals with different phenotypes or in affected versus nonaffected tissue of patients ,13. The Here we describe a high throughput genetic approach for the identification of miRNAs regulating tissue-specific functions. This strategy is based on a large-scale targeted miRNA gene variant screening and association analysis with disease phenotypes related to the tissue of interest. Variants enriched in these phenotypes are then functionally investigated to study the wild type function of these miRNA genes. We applied this strategy to identify miRNAs with a function in neuronal pathways by variant discovery in brain expressed miRNA genes in patients with neurological disorders: schizophrenia and idiopathic generalized epilepsy (IGE). Two selected miRNA gene variants were functionally validated in the neuronal-like cell line SH-SY5Y.To study the function of miRNA genes in neurological diseases and to investigate which miRNA genes regulate neuronal processes, we created a Multiplex Amplification of Specific Targets for Resequencing (MASTR) assay ,18 for t-4; Bonferroni adjusted p = 0.04). Although the variant is located 107 nt outside the MIR204 hairpin, it is predicted to induce structural changes in the hairpin end and flanking regions, which can have an effect on its processing  and is located in MIR618, in the pre-miR-618 hairpin arm opposite of the mature miR-618 sequence. Based on the secondary structure prediction, we hypothesized that the variant allele may affect the distribution pattern of canonical and alternative mature miR-618 sequences (isomiRs), as it was predicted to induce a structural change at the site of Drosha cleavage  in replicates of wild type (MIR204WT) and mutant (MIR204SNP) cell lines by RT-qPCR and corrected expression for transduction efficiency. In a first set of triplicates of wild type and mutant cells, a lower expression of both mature miRNA sequences was measured in the mutant cells  analysis indicated that 15 genes are significantly differentially expressed in the mutant cells versus the wild type cells . GSEA was performed with Gene Ontology gene sets for molecular function, comparing MIR204WT to MIR204SNP cells Tables . 30 geneSince several ncRNAs are significantly upregulated in MIR204SNP cells, it is plausible to assume a more direct relationship between the RNA, DNA binding and ligase gene sets and miR-204 function and an indirect relationship with the neurotransmitter and ion channel gene sets, possibly via miR-296 or any of the other ncRNAs that are differentially expressed in the mutant cells.MIR296 is strongly upregulated, miR-296-5p and miR-296-3p are both significantly upregulated (9- and 12-fold) in MIR204SNP cells. Furthermore, consistent with the presence of other ncRNAs being upregulated according to the array results, five other mature miRNAs are deregulated , indicating that this might be a common function of MIR204.To evaluate the effect of miR-204 on mature miRNA expression, the cells were also subjected to small RNA sequencing. Differential miRNA expression analysis using DESeq2 [To assess whether the differentially expressed transcripts contain binding sites for miR-204-5p or miR-204-3p regulation, we predicted the presence of seed binding sites using the RNAhybrid software . BecauseMIR618, stable SH-SY5Y cells overexpressing wild type or mutant MIR618 were established and subjected to the same analyses as MIR204 cells. The expression level of miR-618 in the MIR618WT cells was too low to determine normalized relative quantities (NRQ) of miR-618 between MIR618WT and SNP cells by RT-qPCR. We reasoned this could either be due to very low or no expression of miR-618 in MIR618WT cells and/or due to the expression of an isomiR that cannot be detected by the assay used for RT-qPCR.To investigate the effect of the variant rs2682818 and the function of MIR618 cells failed to identify genes that were significantly differentially expressed in the mutant cells compared to the wild type cells  in MIR204 genotypes , indicating a MIR618 genotype-dependent specific event.Differential miRNA gene expression analysis on the small RNA sequencing data indicated that miR-618 was significantly differentially expressed with a 10-fold change. Because the secondary structure predictions indicated a potentially altered isomiR distribution, we investigated whether this was indeed the case. For this analysis, we used the raw read counts of all reads mapping to the miR-618 locus and grouped them in two classes: reads with the same 5' start as the canonical miRNA sequence as deposited in miRBase  termed . Given tGiven that miR-618 expression is upregulated in the MIR618SNP cells, the enriched gene sets are likely indirect targets of miR-618 since their expression is positively correlated with miR-618 expression. As no genes were significantly differently expressed after a 10-fold upregulation of miR-618, gene sets were only enriched in the SNP cells and the expression of miR-618 in MIR618WT cells was very low, this may indicate that miR-618 has no direct targets in this specific cell type, and that ectopic overexpression results in dysregulation of downstream pathways.MIR204 and for MIR618. The strategy can be adapted to identify miRNAs involved in a wide range of other organ- or tissue-specific functions, provided phenotypes are known, where the tissue or organ of interest is affected, and are available for genetic variant screening.We present a novel large-scale approach for the identification of miRNA genes involved in neuronal functions. The identification of relevant miRNA genes is based on an association study of variants in brain expressed miRNA genes in patient groups with neurological phenotypes and was performed here on schizophrenia and IGE. The genetic association of a miRNA variant with one of the phenotypes is employed as an indication for a miRNA to have a potential role in the regulation of neuronal pathways. This allowed us at the same time to identify genes with a potential function in neuronal processes and to identify variants that may be significantly associated with the studied phenotypes, such as the variant rs7861254 that is associated with schizophrenia. Functional validation of the selected miRNA genes was also performed on a large scale, implementing both genome-wide mRNA and miRNA profiling, to assess all possible targets of these genes and to assess whether the variants had any effect on the isomiR distribution pattern of the miRNA. This approach led to interesting and different findings for MIR204 is located within an intron of TRPM3, which encodes for a transient receptor potential cation channel that plays a role in detecting noxious heat and chemical stimuli in sensory neurons [MIR204. miR-204-5p was shown to have a tumor-suppressor function through the negative regulation of cell migration and invasion [TRPM3 both directly and indirectly in renal cell carcinoma cell lines, as part of a network involved in regulation of autophagy [ neurons . Severalinvasion –27. Its invasion –27. miR-utophagy . miR-204utophagy . In vivoutophagy . Previouutophagy . Transcrutophagy . Its rolutophagy ,32 and iutophagy . In addiutophagy   as miR-296-5p and -3p  are significantly upregulated in the MIR204SNP cells that have reduced miR-204 expression. Functional validation of miR-618 on the other hand did not identify any significantly differentially expressed target genes, when applying the same approaches. To the best of our knowledge, a direct regulatory role for miR-204 of other miRNAs has not been described yet, though it is consistent with the known function of miR-204 to target transcription factors as it characterizes miR-204 as a regulator of different classes of gene expression regulators in the cell.Next to mediating posttranscriptional regulation of mRNAs, miRNAs can target promoters of protein-coding target genes resulting in transcriptional activation or repression . It is tMIR296 expression at different levels indicates that the regulation occurs upstream of the pre-miRNA to mature miRNA processing and may take place either at pri-miRNA processing or at the level of transcription. For the other mature miRNAs that have different expression in MIR204SNP cells, the pri/pre expression is not significantly affected. Both a biological and a technical reason may exist. It could be that there are indeed no significant alterations at the pri/pre-miRNA level, pointing toward regulation at a different step compared to MIR296, e.g. Dicer processing or miRNA destabilization. On the other hand, it could be that the changes at the pri/pre level are not large enough to be picked up by the technology used. Further experiments are required to determine the direct or indirect nature of these interactions and at which step they take place.We did not find any transcription factors or other protein-coding genes to be significantly deregulated upon miR-204 expression manipulation and did find potential miR-204 binding sites in several miRNAs, pointing to a more direct role of miR-204 in regulation of other ncRNAs. Nevertheless, the possibility of intermediary targets, for instance those targets with altered protein expression but unaltered mRNA expression, cannot be ruled out completely. The upregulation of MIR618 is located within the first intron of the gene LIN7A. LIN7 proteins are present in pre- and post-synaptic protein complexes and are involved in regulating neurotransmitter release and transport of NMDA receptor vesicles [MIR618 has not been explored in detail yet. Overexpression of miR-618 in an anaplastic thyroid cancer cell line resulted in inhibition of proliferation, invasion and migration of these cells [MIR618 in lymphomagenesis [vesicles –42. The se cells . RIP-Chiagenesis . In the In this study, we found that the variant rs2682818 has a higher frequency in patients with IGE compared to control individuals . Validation in SH-SY5Y cells indicated that the homozygous variant genotype (CC) results in a significantly higher expression of miR-618 in SH-SY5Y cells compared to the AA genotype. In addition, the variant cells have a significantly lower frequency of 5’ change isomiRs than MIR618WT cells, which confirmed our structure prediction-based hypothesis that the variant may induce altered pre-miR-618 processing resulting in a changed isomiR distribution pattern. Nevertheless, the large expression change induced by the variant did not lead to expression changes of protein-coding or of other microRNA genes. The absence of deregulated miRNA genes shows that the miRNA-regulation mode of action is a finding specific to miR-204. On the other hand, the presence of many 5’ changed isomiRs and the lack of enriched gene sets with negative correlated expression despite a large change in expression of miR-618 between MIR618WT and SNP cells may indicate that miR-618 has no endogenous role in the studied cell type and that high ectopic expression of this miRNA results in side effects.MIR618 resulted in increased miR-618 expression and in a different distribution of miR-618 isomiRs. The variant rs7861254 near MIR204 is significantly associated with schizophrenia, identifying a new instance of a miRNA involved in schizophrenia etiology. By studying this variant, that reduced miR-204 expression, a new function of MIR204 became apparent: the regulation of other ncRNAs. Though the mechanism of miRNAs regulating expression of other miRNAs has been described before, it remains as yet an underexplored functionality of miRNAs. The discovery of a new schizophrenia related miRNA gene regulating neurotransmitter and ion channel gene sets and with a novel mode of action shows the utility of our large scale miRNA gene sequencing and functional analysis approach and indicates that it will be useful for the study of miRNAs in other tissues as well.With this study we demonstrated the different effects of two selected genetic variants on miRNA functionality. The variant rs2682818 in The study was approved by the Medical Ethical Committees of the universities of Umeå and Antwerp. Each individual signed an informed consent form for participation in the study.DNA of patients with schizophrenia, of patients with IGE and of control individuals was collected. The schizophrenia patients included in this study were recruited in Sweden (n = 186). Control individuals for this patient group were also recruited in Sweden (n = 1043), as part of the Betula study ,46. PatiA MASTR assay that amplifies 289 brain expressed miRNA genes was created (assay available upon request). This assay was subsequently used to screen the genomic DNA of patients and control individuals. Control DNA samples were pooled per seven samples prior to amplification, patient samples were amplified individually. Libraries were sequenced on MiSeq  using v3 chemistry. After performing filtering steps in GenomeComb  S1 FileS1 File, Variant and wild type miRNA genes were cloned by amplifying the miRNA gene and flanking sequences of at least 130 bp on either side from genomic DNA of patients heterozygous for the variant using sequence specific primers with attachment sites for Gateway cloning. Bead-purified PCR products with homozygous wild type or variant genotype were inserted into the pLenti6/EGFP-V5 destination vector via Gateway cloning as described by Almeida-Souza et al. . GeneratRT-qPCR (reverse transcription–quantitative PCR) was performed using TaqMan microRNA assays or Power SYBR Green PCR Master Mix (Life Technologies) on a ViiA7 Real-Time PCR System  to assess the expression of mature miRNAs and EGFP in each stable cell line. Data were normalized by geometric averaging of internal control genes . NRQ forMIR618 cells  and MIR204 cells  were submitted for analysis with the Human Transcriptome Array 2.0  and data normalization using RMA to AROS Applied Biotechnology (Denmark). Prefiltering was performed on the normalized data to exclude transcript clusters lacking a gene symbol or RNA accession number. Differential expression of genes between wild type and mutant cell lines was assessed by SAM v1.0 as implemented in MeV software v4.9.0 [200–400 ng total RNA aliquots of e v4.9.0 ,53. A twe v4.9.0 ,55 usinge v4.9.0 . For allMIR618 cells  and MIR204 cells  were submitted to Biogazelle (Belgium) for small RNA sequencing. Read QC, adaptor trimming, read mapping and annotation to miRBase 20 [MIR204 cells the independent filtering option was disabled. miRNAs with Benjamini-Hochberg adjusted p < 0.1 were called significant. For isomiR distribution analysis, isomiR read counts were normalized to the total read count of the mature miRNA  in each sample and counts were summed per functional group. IsomiRs were grouped per 5' start sequence: the 5' canonical group contains all isomiRs that have the same 5' end as the canonical miRNA listed in miRBase 20, isomiRs with 5' change are all other isomiRs. The two-tailed unpaired t-test with unequal variance was applied to test for differences in 5’ change isomiR frequencies between genotypes, p < 0.05 was taken as significance threshold.200–400 ng total RNA aliquots of RBase 20  was perfHybridization of miR-204-5p and miR-204-3p to transcripts was predicted using the RNAhybrid software v2.1.2 . For miRMASTR data has been deposited at the European Genome-phenome Archive, which is hosted by the EBI and the CRG, under accession numberEGAS00001001607. Microarray data is available at Gene Expression Omnibus with accession GSE69675. Small RNA sequencing data are available at Sequence Read Archive with study accession SRP059166.S1 FileThis file contains additional details of the variant screening strategy and additional results .(DOC)Click here for additional data file.S2 FileThis file contains additional details of the methods.(DOCX)Click here for additional data file.


Lack of standardized care contributes to low survival in admitted out-of-hospital cardiac arrest (OHCA) patients. The objective of our study was to implement a Post Arrest Consult Team (PACT) and improve the quality of care for admitted OHCA patients.We conducted a prospective cohort study with concurrent controls from February 2011 to February 2013 in a network of 29 Toronto-area hospitals. The PACT was implemented in two hospitals and functioned as a consult service with a nurse and physician on- call 24/7. Patients from other network hospitals acted as concurrent controls. The PACT focused on four key processes of care: targeted temperature management (TTM); coronary angiography; avoidance of premature withdrawal of life-sustaining therapy (WLST <72 hours) on the basis of neuroprognostication; and electrophysiology assessment. We included nontraumatic OHCA patients who were >18 years old, survived at least 6 hours, and were comatose. We excluded patients with do-not-resuscitate orders, intracranial or other severe bleeding. We used generalized linear mixed models to assess whether PACT implementation was associated with higher odds of achieving each of the four targeted processes of care while adjusting for secular trends unrelated to the intervention.The primary analysis included 162 patients from two intervention hospitals and 892 from 27 control hospitals. Thirty- two percent of the patients were female and the mean age was 65.3 ± 16.5 years. Almost one-half (46%) of patients had a shockable initial cardiac arrest rhythm, 41% had bystander CPR, and 5% had an AED applied. PACT did not improve use of TTM , angiography for patients without ST-elevation myocardial infarction , or electrophysiology assessment  as compared with concurrent control hospitals. Patients in the intervention group were less likely to have life support withdrawn within 72 hours on the basis of neuroprognosis compared with patients in the concurrent control group .PACT was associated with reduced WLST <72 on the basis of neuroprognostication but did not improve other important post-cardiac arrest processes of care. Further work is underway to identify factors that influenced implementation. This will guide future consideration of the PACT model in other settings.


Background. Sunitinib is a tyrosine kinase inhibitor used in the treatment of metastatic renal cell carcinoma. The main difficulty related to the treatment is the development of drug resistance followed by rapid progression of the disease. We analyzed tumor tissue of sunitinib treated patients in order to find miRNAs associated with therapeutic response. Methods. A total of 79 patients with metastatic renal cell carcinoma were included in our study. miRNA profiling in tumor tissue samples was performed by TaqMan Low Density Arrays and a group of selected miRNAs  was further validated by qRT-PCR. Normalized data were subjected to ROC and Kaplan-Meier analysis. Results. We reported decreased tissue levels of miR-155 and miR-484 as significantly associated with increased time to progression . Conclusion. miR-155 and miR-484 are potentially connected with sunitinib resistance and failure of the therapy. miR-155 is a known oncogene with direct influence on neovascularization. Biological role of miR-484 has to be clarified. Stratification of patients based on miRNA analysis would allow more personalized approach in therapy of metastatic renal cell carcinoma. TKIs inhibit multiple receptor tyrosine kinases needed for the activation of intracellular signaling pathways controlling cell proliferation, survival, or angiogenesis. Almost all treated patients will eventually develop secondary resistance to TKIs . Other tl trials , could bEmerging evidence suggests that microRNAs (miRNAs) could be suitable biomarkers with diagnostic, prognostic, and predictive potential –6. TheseThe study protocol was approved by the local ethical committee and written informed consent was obtained from all patients. Metastatic RCC patients included in the study were from South Moravian region of Czech Republic with uniform exposure to the environmental factors. Hereditary cases of RCC were excluded from the study. Two cohorts of patients with mRCC treated with sunitinib in a standard regimen were set up retrospectively. The screening group included 16 patients from Masaryk Memorial Cancer Institute, Brno, Czech Republic (MMCI). Response to the treatment was assessed according to RECIST criteria after 9 months and patients were divided into two groups: (a) responders to the treatment  and (b) nonresponders with rapid progression. A group of candidate miRNAs was chosen and the expression was analyzed by qRT-PCR in the validation cohort of 63 mRCC patients from MMCI. Clinicopathological characteristics of both cohorts are summarized in Tumor tissue was provided as formalin-fixed paraffin embedded (FFPE) samples. Total RNA enriched with small RNA was isolated using mirVana miRNA Isolation Kit . Concentration and purity of the isolated RNA were determined spectrophotometrically using Nanodrop ND-1000 .miRNAs profiling was conducted using TaqMan Low Density Array (TLDA) technology. Megaplex miRNA RT primers set  and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) were used for reverse transcription. Reactions were carried out according to the manufacturer's protocol. 667 miRNAs were simultaneously quantified using ABI 7900 HT Instrument (Applied Biosystems).Gene-specific primers were used in reverse transcription according to the TaqMan MicroRNA Assay protocol (Applied Biosystems). qRT-PCR was performed on ABI 7500 HT Instrument (Applied Biosystems) using the Applied Biosystems 7500 Sequence Detection System. TaqMan (NoUmpErase UNG) Universal PCR Master Mix and specific primer and probe mix (Applied Biosystems) for each miRNA were used. PCR reactions were run in duplicates, and average threshold cycles and SD values were calculated.∗, which was uniformly expressed in all samples from screening cohort. Normalized miRNA expression data were evaluated using Bioconductor Limma differential expression analysis. P value lower than 0.01 was selected according to the potential of identified miRNAs to accurately discriminate responders and nonresponders in consequent HCL analysis. In validation phase of the study, average expression levels of miRNAs in RT-PCR quantification were normalized using miR-1233 as a reference gene. miR-1233 was selected according to our previous experience with normalization of renal cell carcinoma FFPE samples. Normalized expression data were evaluated by ROC analysis  and Kaplan-Meier analysis followed by log-rank test (GraphPad Prism 5.03). P values lower than 0.05 were considered statistically significant.Expression data from TLDA profiling were normalized using miR-625N = 8) or nonresponding (N = 8) group was performed. Limma analysis of normalized expression data identified 19 miRNAs differentially expressed .High-throughput miRNA analysis of tumor tissue of 16 patients treated with sunitinib belonging to either responding (xpressed . Six miRN = 63) by qRT-PCR. Normalized data were analyzed by ROC analysis and patients were separated into two groups according to the calculated criterion. Kaplan-Meier analysis revealed that lower level of miR-155 is associated with increased time to progression in patients on sunitinib treatment (The results obtained from the screening cohort were verified on the independent cohort (Our findings suggest a link between two miRNAs (miR-155 and miR-484) and disease progression in mRCC patients treated with sunitinib. Tyrosine kinase inhibitors inhibit tyrosine kinase domains of growth factor receptors, albeit their main activity is promoted by the inhibition of VEGF receptor cascade, which leads to the decrease in blood tumor perfusion and to the inhibition of neovascularization. Tumors of TKI treatment-refractory patients are able to escape from the VEGFR blockade , which iOur data imply that patients with higher tissue expression of miR-155 have decreased time to progression on sunitinib treatment and thus limited benefit from the therapy. However, we have detected a discrepancy between the results obtained from the screening and independent cohort. TLDA screening indicated that the nonresponders from the screening group have lower expression of miR-155 than the responders. Opposite result was achieved by qRT-PCR in the independent cohort (data not shown). We suppose that a bias might occur due to a small number of the specimens analyzed by TLDA, which is also significant limitation of our study.The expression of miR-484 in mRCC patients treated with sunitinib has already been noticed. Prior et al. described high tumor tissue levels of miR-484 as significantly associated with decreased TTP and overall survival . Our finResearch in ovarian cancer proved that miR-484 is excreted from tumor cells as a paracrine regulator of tumor microenvironment  and it iIndependent validation of our results in responders and nonresponders to the sunitinib treatment on larger cohorts of patients and functional analysis of miR-155/miR-484 regulatory involvement in VEGFR signaling might help to understand the underlying mechanism of sunitinib resistance and also prove the potential of these miRNAs to serve as a suitable predictive biomarkers in mRCC patients treated with sunitinib.


Adolescence is a vulnerable time for the onset of depression. Recent evidence from adult studies suggests not only that negative attention biases are correlated with symptoms of depression, but that reducing negative attention biases through training can in turn reduce symptomology. The role and plasticity of attention biases in adolescent depression, however, remains unclear. This study examines the association between symptoms of depression and attention biases, and whether such biases are modifiable, in a community sample of adolescents. We report data from 105 adolescents aged 13–17 who completed a dot-probe measure of attention bias before and after a single session of visual search-based cognitive bias modification training. This is the first study to find a significant association between negative attention biases and increased symptoms of depression in a community sample of adolescents. Contrary to expectations, we were unable to manipulate attention biases using a previously successful cognitive bias modification task. There were no significant effects of the training on positive affect and only modest effects of the training, identified in post-hoc analyses, were observed on negative affect. Our data replicate those from the adult literature, which suggest that adolescent depression is a disorder associated with negative attention biases, although we were unable to modify attention biases in our study. We identify numerous parameters of our methodology which may explain these null training effects, and which could be addressed in future cognitive bias modification studies of adolescent depression. Adolescence is a time of increased vulnerability for depression. A prospective cohort study yielded one-year point prevalence estimates of episodes of major depressive disorder (MDD) that rose dramatically from around 2% in early adolescence (ages 13–15), to 15% in middle adolescence (ages 15–18) . While fProminent theories of adult depression have recently considered the association between heightened attention towards negative  stimuli and depression , with daReviews  demonstrN = 19 depressed participants)  . More recipants)  and angrcipants)  (versus cipants)  and facecipants)  in deprecipants) , with sucipants) . Few stucipants) , with oncipants) , and anocipants) . A finalcipants) . Of notecipants) . ModelliIf the study of attention biases and adolescent symptoms of depression is to inform treatment models, a crucial question is whether these biases can be manipulated such that they initiate affect changes. Novel experimental paradigms, referred to as Cognitive Bias Modification (CBM), have been developed to manipulate cognitive patterns  through repeated training. These paradigms could be useful for planning new interventions if induced attention biases enable changes in affect or depressive symptomology. The pioneering CBM-A paradigm is a modification of the dot-probe task , where tOne criticism of these two tasks is that they do not expose participants to training stimuli for long enough to facilitate attentional control . In addiN = 40) and the fact that the task failed to modify attention bias in the first place, limits the interpretation of these null-effects.Despite the relatively promising findings of previous CBM-A studies in relation to depression, one should remain cautious about their clinical potential, not least because at least one study failed to find positive effects on vulnerability for depression . In thisanxiety in children and adolescents  were associated with symptoms of depression in a sample of adolescents. Of note, we used a dot-probe stimulus duration of 500 ms (a) in order to facilitate comparison of the results with a previous study using the same paradigm and CBM-A procedure  and (b) negative  or neutral (control group) stimuli. We oversampled participants in the experimental group (N = 75) compared to the control group (N = 30) because of a priori expectations that there would be more variability in responsiveness to the experimental manipulation. Attention bias and affect ratings were measured again after training. Ethical approval for the study was provided by Oxford University Central University Research Ethics Committee (MSD/IDREC/C1/2010/56) and the study was therefore performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Participants aged 16–17 provided written informed consent and participants aged 13–15 provided written assent (written informed consent was provided by their parents).A community sample of 112 adolescents aged 13–17  = 0.76) were recruited through urban and rural secondary state schools and public advertisements in Oxfordshire, UK. The study session lasted 45 min. Adolescents recruited from schools were tested in groups at individual computers in a research laboratory at the University of Oxford. Adolescents recruited through public advertisements were tested individually in the same room and were given a £10 gift voucher for their participation. Participants first completed computerized measures of symptoms of depression, attention bias and affect. Participants were then randomly allocated to receive one of two CBM-A manipulations: learning to ignore emotionally N = 98) of the final sample. In community samples the CDI correlates highly with other self-report measures of low mood (sd = 6.6). Depression scores demonstrated high internal consistency (Cronbach’s α = 0.85). Since depression scores were non-normally distributed  a log10(x + 1) transformation was applied and transformed scores are reported hereafter.Symptoms of depression  were assessed in adolescents using the Children’s Depression Inventory (CDI)  and werelow mood . Althouglow mood . In the Attention bias was assessed using a modified version of the dot-probe task  see Fig. Stimuliα = 0.83) in adolescent samples (N = 103) of the final sample.Affect ratings were assessed using the Positive and Negative Affectivity Scale (PANAS) . The 20- samples . Affect Both training tasks involved identifying a distinguishing feature in a 4 × 4 grid, over six practice trials and a single block of 112 experimental trials  see Fig. In bothStimuli in the experimental training condition were modified from the original task to include 16 adolescent (instead of adult) faces selected from the NIMH Child Emotional Faces Picture Set (NIMH-ChEFS) . The facsd = 9.1). Dot-probe data from five participants (either pre- or post-training task data) were incomplete due to a technical error during runtime and these subjects’ complete data were therefore removed. There was no reason to believe there was a systematic pattern to the disruption and therefore the exclusion of these data is unlikely to have caused a bias in the final sample. Two participants in the control training condition were excluded because they obtained less than 65% accuracy on the task.Attention bias was calculated by subtracting mean RT (ms) to congruent trials from incongruent trials, such that positive scores represented a negative attention bias . Trials t-tests.Data were analyzed using SPSS. In order to assess whether participants with more symptoms of depression demonstrated a greater negative attention bias, a bivariate correlation analysis between (transformed) symptoms of depression and baseline attention bias was conducted. To test the main effect of the training manipulation  on attention bias change scores (post-training-bias minus baseline-bias score) and the interaction between training manipulation and (transformed) symptoms of depression (continuous variable), a repeated-measures custom model ANOVA was used. Two similar custom model ANOVA tests explored the effects of training manipulation and symptoms of depression on change scores for negative affect and positive affect respectively. Differences in baseline demographic characteristics and training performance between the experimental and control groups were assessed using chi-square and The final sample comprised 105 adolescents .R = 0.20, p = 0.05; N = 97; Pearson’s R = 0.41, p < 0.001) and positive  affect. However, there was no evidence of a significant association between baseline attention bias and negative affect , or between baseline attention bias and positive affect .A bivariate correlation (performed on data from the 98 (93%) participants who provided depressive symptom scores) revealed that symptoms of depression correlated significantly with baseline attention bias , symptoms of depression , baseline attention bias , positive affect , or negative affect  between the experimental training condition and the control condition  and took longer to correctly identify the target stimulus  than those who performed the experimental training task.There were no significant differences between the two training conditions in terms of participants’ age (N = 98 participants) revealed no main effect of condition on change in attention bias , suggesting that attention biases did not change as a function of whether individuals received experimental or control training. Neither was there a main effect of symptoms of depression  or a significant two-way interaction between condition and symptoms of depression .The ANOVA model  revealed no main effect of condition on change in negative affect . There was also no evidence of a main effect of symptoms of depression  on change in negative affect. A two-way interaction between condition and depressive symptoms was marginally significant , suggesting that the effect of the training task on change in negative affect varied as a function of participants’ symptoms of depression. In order to explore this interaction, a median-split variable was created based on (transformed) depressive symptom scores (median = 1.06)—and the effect of training condition on change in negative affect was investigated using paired-samples t-tests in each subsample separately. For participants with more symptoms of depression, those in the experimental condition showed a significant reduction in negative affect following the training , whereas depressed participants in the control condition showed no significant change . Participants with fewer symptoms of depression showed no significant change in negative affect in the experimental condition  or control condition .An ANOVA model  revealed no main effect on change in positive affect of condition , no main effect of symptoms of depression on change in positive affect , and no interaction between condition and symptoms of depression on change in positive affect .A similar ANOVA , which may be related to the low reliability of the dot-probe task. Although the dot-probe has been established as the standard measure of attention bias, developing new measures which more reliably measure attention bias in youth depression is important for future research in this field. Studies of attention bias in adult depression have recently used eye-tracking during passive viewing tasks. In a recent study of alcohol-related attention biases, eye-tracking was found to have superior internal reliability over traditional behavioural reaction-time measures . However, we also cannot exclude the possibility that the higher than expected error rate was due to our sample being relatively young. Of note, a recent study by Britton and colleagues  effect sizes, which may be even larger in clinical populations (N = 16), and when attention bias was unsuccessfully modified, there was no effect on emotional vulnerability (N = 10). Together these findings suggest that CBM-A paradigms do indeed have potential as clinical tools, but that more research is needed to understand why they are often ineffective in modifying attention biases in the first place.A critical clinical question is whether modifying attention biases has therapeutic potential for adolescents. CBM-A approaches were initially a topic of great excitement, with early studies demonstrating significant effects of training on negative mood and vulnerability for adult depression . CBM-A pulations  and are ulations  and youtulations  populatiulations  observedJust one study has measured the effects of a CBM-A task on vulnerability for depression in youth . Althougengagement with negative stimuli, the visual search CBM-A task trains engagement with positive stimuli and disengagement from negative stimuli. This may explain why De Voogd and colleagues (N = 53) benefited from training. However, there remained no evidence of an effect of training group on any of the three outcome measures , or of an interaction between training group and symptoms of depression . A fourth possible explanation for the lack of effects is that multiple training sessions may be needed to elicit robust effects on attention bias. The single-session nature of the CBM-A paradigm is another limitation of the current study.In our study we chose to use the visual search task because of its previous success in modifying attention biases and affect in adults and adolescents , and beclleagues  found eflleagues  have notlleagues . A secont-test analyses were unequal due to oversampling in the experimental group. Smaller sample sizes in high depressed (N = 18) and low depressed (N = 12) participants allocated to the control condition could explain the lack of training effects in these groups.Some evidence to support the beneficial effect of the CBM-A task is supported by the fact that the experimental training condition was associated with reduced negative affect (in participants with higher levels of depressive symptoms). However, whilst it is possible that this effect is mediated by a change in attention bias that was not detected by the dot-probe assessment task, changes in affect may also have been due to non-specific differences  between the two training conditions. Indeed, RT and accuracy data suggest that the experimental (faces) task was significantly easier to perform than the control (flowers) task, replicating findings from a recent study using the same CBM-A training tasks . FurtherOur study highlights the fragility of CBM-A data and the infancy of the attention bias literature in relation to adolescent depression. However, whilst remaining cautious about the clinical potential of CBM-A tasks for adolescent depression, the positive findings from CBM-A studies of adult depression, along with the finding that CBM-A paradigms generally modify emotional vulnerability when attention bias is successfully modified, suggests that exploration of the optimal paradigms and parameters needed for attention bias modification in adolescents is a worthy area of future research . There aAdolescence is a period of vulnerability for depression, yet little is known about the role of negative information processing in the onset of depressive symptoms during this developmental period. Data from an unselected sample of adolescents suggest that, as has been demonstrated in adult studies, negative attention biases are associated with increased symptoms of depression. In contrast to previous studies, we found no evidence that the visual-search CBM-A task could modify attention biases (as measured using the dot-probe task) or positive affect in our community sample of adolescents. Modest effects of the training on negative affect were observed for participants with more symptoms of depression, however these findings are interpreted with caution due to the post-hoc nature of analysis. Numerous complexities associated with measuring and modifying attention biases mean that further examination of these effects is needed before firm conclusions about the precise role of attention biases in adolescent depression, and their implications for clinical practice can be drawn.10.7717/peerj.1372/supp-1Dataset S1Click here for additional data file.


Chronic cough affects more than 70 percent of patients with Idiopathic Pulmonary Fibrosis and causes significant morbidity. Gastroesophageal reflux is the cause of some cases of chronic cough; and also has a postulated role in the aetiology of Idiopathic Pulmonary Fibrosis. A high prevalence of acid; and more recently non-acid, reflux has been observed in Idiopathic Pulmonary Fibrosis cohorts. Therefore, gastroesophageal reflux may be implicated in the pathogenesis of cough in Idiopathic Pulmonary Fibrosis.Eighteen subjects with Idiopathic Pulmonary Fibrosis underwent 24-hour oesophageal impedance and cough count monitoring after the careful exclusion of causes of chronic cough other than gastroesophageal reflux. All 18 were then treated with high dose acid suppression therapies. Fourteen subjects underwent repeat 24-hour oesophageal impedance and cough count monitoring after eight weeks.Total reflux and acid reflux frequencies were within the normal range in the majority of this cohort. The frequencies of non-acid and proximal reflux events were above the normal range. Following high dose acid suppression therapy there was a significant decrease in the number of acid reflux events (p = 0.02), but an increase in the number of non-acid reflux events (p = 0.01). There was no change in cough frequency (p = 0.70).This study confirms that non-acid reflux is prevalent; and that proximal oesophageal reflux occurs in the majority, of subjects with Idiopathic Pulmonary Fibrosis. It is the first study to investigate the effect of acid suppression therapy on gastroesophageal reflux and cough in patients with Idiopathic Pulmonary Fibrosis. The observation that cough frequency does not improve despite verifiable reductions in oesophageal acid exposure challenges the role of acid reflux in Idiopathic Pulmonary Fibrosis associated cough. The finding that non-acid reflux is increased following the use of acid suppression therapies cautions against the widespread use of acid suppression in patients with Idiopathic Pulmonary Fibrosis given the potential role for non-acid reflux in the pathogenesis of cough and Idiopathic Pulmonary Fibrosis itself.The study was registered with the Cardiff and Vale University Local Health Board Research and Development Committee (09/CMC/4619) and the South East Wales Ethics Committee (09/WSE04/57). Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrotic interstitial lung disease of unknown origin associated with the histological and/or radiological pattern of usual interstitial pneumonia . It typiThe mechanisms underlying the pathogenesis of IPF remain unknown. However, one hypothesis proposes that chronic aspiration of gastric contents may cause repeated injury to alveolar epithelium resulting in the pathological abnormalities of usual interstitial pneumonia . This hyGastroesophageal reflux has long been known to be an important cause of chronic cough . Indeed Despite the accumulating body of evidence linking IPF, cough and acid reflux, less is known about non-acid reflux in this context. A recent advance in the assessment of non-acid reflux is the technique of combined oesophageal pH and impedance monitoring. This provides a means of detecting and characterizing all reflux episodes regardless of pH . Using tCough reflex sensitivity to capsaicin is known to be increased in IPF suggesting there is up-regulation of sensory C-fibres in the airways in IPF . Cough rIn this context, the objective of the present study was to investigate the impact of high-dose acid suppressant therapy on GOR and cough in this condition.We recruited 18 consecutive non-smoking subjects meeting the American Thoracic Society/European Respiratory Society criteria for the diagnosis of IPF . SubjectAll acid suppression and prokinetic medication was withheld for at least one week prior to study assessment. The 18 subjects underwent oesophageal manometry immediately followed by 24-hour combined pH-impedance monitoring . Impedance detected reflux events were defined according to pH as follows: acid reflux, pH <4; weakly acid reflux, pH 4 – 6.9; non-acid reflux, pH ≥ 7 . Total rAll 18 subjects were then treated with open label high dose proton pump inhibitor therapy (omeprazole 40 mg twice daily or lansoprazole 30 mg twice daily) plus ranitidine 300 mg nocte for 8 weeks according to a protocol agreed a priori. Proton pump inhibition is endorsed by national and international guidelines as an empiric treatment for suspected GOR related cough ,24. AsymThe Wilcoxon signed-rank test was used to compare paired non-parametric continuous data. The Kruskal-Wallis test was used to compare differences between non-parametric categorical variables. Proportions were compared using Fishers Exact Test. Spearman’s rank order correlation was used to assess correlation coefficients between non-parametrically distributed variables. All tests performed were two-sided tests at the 0.05 level of significance (SPSS version 18).The study was approved by the South East Wales Ethics Committee (09/WSE04/57) and the Cardiff and Vale University Local Health Board Research and Development Committee (09/CMC/4619). Subjects provided written informed consent.The spectrum of GOR events recorded for individual subjects is shown in Figure Lower oesophageal sphincter pressure was within normal limits for 61% (11/18) of subjects. There was a significant negative correlation between the number of total reflux events and lower oesophageal sphincter pressure . Manometry studies showed 17% (3/18) of subjects had evidence of ineffective oesophageal motility. No other indices of abnormal oesophageal motility were detected.Following eight weeks treatment with high dose acid suppression therapy the majority of subjects had a decrease in the number of acid reflux events p = 0.02) and an increase in the number of non-acid reflux events (p = 0.01) Table   before 188; 338) and after  acid suppression therapy  is known to be rare in healthy individuals ,26.A potential mechanism for chronic cough in patients with IPF and GOR is enhancement of cough reflex sensitivity due to neuronal changes within the airways induced by gastroesophageal refluxate. It has been demonstrated that induced sputum from patients with IPF contains higher levels of the neurotrophins, nerve growth factor and brain-derived neurotrophic factor, compared with normal controls . FurtherGastric juice is a complex mixture of gastric acid, gastric enzymes such as pepsin; and bile acids and pancreatic enzymes from the duodenum. Hence, it may be of acid, neutral or alkaline pH. The pH of refluxate influences its biochemical activity and cellular toxicity. For example, animal studies have shown that within an acidic milieu pepsin potentiates the degree of acid-induced injury to oesophageal epithelial cells, but that at neutral pH pepsin is inactive and its oesophageal epithelial toxicity is ameliorated . The toxSupine reflux from the stomach into the oesophagus is rare in normal human subjects ,26 probaAs might be predicted, the present study objectively demonstrated a marked decrease in acid reflux following treatment with high-dose proton pump inhibitors (PPIs) and H2-receptor antagonists in the majority of patients with IPF. However, an unexpected finding was that this was consistently associated with an increase in non-acid reflux. Proton pump inhibitors are widely prescribed for acid suppression therapy in patients with IPF; therefore our findings that increased non-acid reflux may be a consequence of PPI therapy in such patients is an important observation.The effect of PPIs on duodeno-gastroesophageal reflux is complex. Whilst they reduce gastric volume, which should reduce reflux into the oesophagus, they also delay gastric emptying which might enhance reflux . It has The exclusion of confounding causes of cough and the invasive nature of the investigations performed has limited the size of the study cohort and also resulted in a high withdrawal rate for the second pH-impedance assessment. A combination of the small sample size and day to day variations in reflux frequency  may explDespite these limitations, the careful subject selection process means that relevant observations about the relationship between cough, gastroesophageal reflux and acid suppression therapy in this condition can be made. The invasive nature of the investigations also made attempts to recruit a suitable control group unrewarding. However, the normal ranges for impedance detected reflux parameters have been well defined in both North American  and EuroIn conclusion, proximal, non-acid reflux is prevalent in patients with IPF and may be implicated in both the pathogenesis of pulmonary fibrosis and sensitisation of the cough reflex in this condition. The lack of improvement in cough symptoms despite effective acid suppression therapy indicates that acid GOR reflux alone is not responsible for cough in the majority of patients with IPF. The finding that acid suppression consistently increases the frequency of non-acid reflux cautions against the widespread empirical use of acid suppression in IPF and GOR in the absence of objective pH or impedance testing. Our findings suggest that strategies for treating GOR in patients with IPF should target both acid and non-acid reflux, perhaps by including prokinetic therapies, rather than acid suppression therapy alone.GOR: Gastroesophageal reflux; IPF: Idiopathic pulmonary fibrosis; PPI: Proton pump inhibitor.The authors declare that they have no competing interests.CEK: conception and design of study; acquisition, analysis and interpretation of data; drafting, critical revision and final approval of manuscript. MJC: acquisition, analysis and interpretation of data; final approval of manuscript. GAT: acquisition, analysis and interpretation of data; drafting, critical revision and final approval of manuscript. NKH: conception and design of study; drafting, critical revision and final approval of manuscript. BHG: conception and design of study; acquisition, analysis and interpretation of data; drafting, critical revision and final approval of manuscript.CEK is a clinical research fellow and specialty trainee in respiratory medicine.MJC is a gastro-intestinal physiologist. GAT is a consultant in general and gastrointestinal medicine with an interest in gastroesophageal reflux. NKH is a consultant in general and respiratory medicine with an interest in interstitial lung disease. BHG is consultant in general and respiratory medicine with an interest in interstitial lung disease.


The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome.de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs.In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection.Our results suggest the use of ribo-depleted RNA in a The online version of this article (doi:10.1186/s12985-016-0539-x) contains supplementary material, which is available to authorized users. Next-generation sequencing (NGS) has proven to be a valuable tool for virus detection, discovery or diversity studies and has increased in popularity, while decreasing in cost. The percentage genome-wide coverage obtained, either through the mapping of reads or contigs (assembled reads) onto a reference genome, can serve as a form of virus detection. The confidence in a positive identification increases with greater coverage. Due to the variation in the number of reads associated with different genomic regions, an uneven coverage of the viral genome is often observed in RNA-Seq data. Variation in sequencing depth will, consequently, influence the percentage of genome coverage that can be obtained. It is therefore necessary to find the optimal amount of data needed to cover the complete or almost complete genome without generating an excess of sequence data. Therefore, the aim of this study was to illustrate the influence of sequencing depth on virus and viroid genome coverage, to provide a guideline for the number of reads required to offer maximum possible genome coverage.Closteroviridae, were selected. They were known variants of Grapevine leafroll-associated virus 3 (GLRaV-3) (variant group II) and Citrus tristeza virus (CTV) (genotype T3) that were graft-inoculated into Vitis vinifera (grapevine) and Citrus paradisi (grapefruit) plants, respectively. While the exact sequence information for the GLRaV-3 variant was available the genome of the specific CTV inoculum has not been sequenced. Along with the virus, the grapevine plants were additionally infected with Hop stunt viroid (HSVd) and the grapefruit plants with Citrus dwarfing viroid (CDVd). The viroid infections were first identified in the sequencing data. Phloem material was sampled from 12 grapevine and 6 grapefruit plants and total RNA was extracted, using a CTAB method adapted from Carra et al. [In this study two viruses from the family Citrus tristeza virus isolate T3 (Accession No. KC525952) and CDVd  reference genomes were used for citrus analysis. For the sRNA data a single mismatch per read was allowed while for the transcriptome data a single mismatch was allowed per seed of 20 nts. Following read-mapping, the genome coverage for each subset was determined by calculating the number of genome bases covered at least once. Heat maps were generated to show the variation in depth of coverage of genomic regions with increasing subset-size. The heat maps reflect the number of times (out of the 1000 replicates) a specific base was covered.To determine the minimum number of reads needed to cover a genome using read-mapping, subsets  were extracted randomly, using a custom Python script, from each complete NGS dataset to simulate different sequencing depths. A 1000 bootstrap replication of each subset-size was randomly selected from the complete dataset to assign a measure of accuracy to the estimation of the mean genome coverage. The subsets of reads were mapped onto the genome of interest using Bowtie  for sRNAThe results showed a low degree of virus genome coverage using transcriptome data generated from poly(A)-selected RNA for GLRaV-3 Fig. . At a maTranscriptome data generated from ribo-depleted RNA reached saturation from approximately 0.5 million reads  and covered ~98 % of the complete CTV genome Fig. . The mapThe difference in sRNA genome coverage between the two virus species Fig.  could beAs could be expected for their considerably smaller genome size, less reads were required for the two viroids, compared to the viruses, to reach genome coverage saturation  as was used for the read-mapping analysis. Each subset of reads was then subjected to de novo assembly using CLC Genomics Workbench 8. A bubble size of 50, a word size of 20 and a minimum contig length of 200 were selected as de novo assembly parameters. The contigs were mapped onto the reference virus and viroid genomes to determine the coverage thereof using a mismatch cost of 2 and a similarity fraction of 0.95.Reads were also de novo assembly of sRNA data than with poly(A)-selected data  [Resembling the results from the read-mapping analysis Fig. , a signiata Fig. . The polata Fig. . Except ata Fig. . The lowol (BWA) , 11 pipede novo assembly results for HSVd corresponded to that of CDVd . Some viruses or viroids may be more represented in the data depending on (i) the degree of infection, which may be host and/or virus dependent, (ii) the plant response to infection in the case of sRNAs and (iii) the nature of the genome in the case of different library types -selected RNA, ribo-depleted RNA libraries). This study however focussed on two non-polyadenylated single-stranded RNA viruses. The genomes of these closteroviruses represent some of the largest plant-infecting RNA virus genomes (~19 kb), which along with the viroid comparisons covered the wide range of genome sizes that could influence detection.de novo assembly of the reads and alignment-based similarity searches for virus identification. Specifically for closteroviruses detection, we recommend obtaining one million high-quality reads. Overall, the results presented in this study serve as a guideline for selecting the number of NGS reads needed to represent a full virus (or viroid) genome.We conclude that NGS data generated from ribo-depleted total RNA outcompeted data generated from poly(A)-selected RNA, potentially universally or at least for non-polyadenylated viruses such as closteroviruses. For some viruses, sRNA data may be equivalent to ribo-depleted total RNA data depending on the host response. For virus detection, we recommend sequencing ribo-depleted total RNA, followed by


Postural control during rapid movements may be impaired due to musculoskeletal pain. The purpose of this study was to investigate the effect of experimental knee-related muscle pain on the center of pressure (CoP) displacement in a reaction time task condition.Nine healthy males performed two reaction time tasks  before, during, and after experimental pain induced in the dominant side vastus medialis or the tibialis anterior muscles by hypertonic saline injections. The CoP displacement was extracted from the ipsilateral and contralateral side by two force plates and the net CoP displacement was calculated.Compared with non-painful sessions, tibialis anterior muscle pain during the peak and peak-to-peak displacement for the CoP during anticipatory postural adjustments (APAs) of the shoulder task reduced the peak-to-peak displacement of the net CoP in the medial-lateral direction (P<0.05). Tibialis anterior and vastus medialis muscle pain during shoulder flexion task reduced the anterior-posterior peak-to-peak displacement in the ipsilateral side (P<0.05).The central nervous system in healthy individuals was sufficiently robust in maintaining the APA characteristics during pain, although the displacement of net and ipsilateral CoP in the medial-lateral and anterior-posterior directions during unilateral fast shoulder movement was altered. The link between knee pain and motor control is clinically relevant since pain around the knee joint affects body control in adolescent , young aCompared with asymptomatic subjects, knee osteoarthritis (OA) patients showed reduced knee flexion moment during gait ,11. DuriAnticipatory postural adjustments (APAs), defined as the time window from -50 to +150ms from the prime mover onset, are generated by the central nervous system in a feed-forward manner to stabilise the body’s center of gravity ,17. DiffThe aim of this study was to investigate how experimental pain close to the knee joint affects APA responses in the CoP parameters. It was hypothesized that knee-related muscle pain will 1) increase the CoP displacement and velocity on the painful side, and 2) decrease the CoP displacement and velocity on non-painful side to compensate the increased CoP parameters from the painful side and, therefore, maintain balance stability.In this study nine healthy males were included without known musculoskeletal disorders . Given the gender variability in motor responses to pain, the present study only evaluated healthy male subjects, which restrict the generalization of the results to this population. Participants signed an informed consent prior participating in the study. The study was performed according to the Declaration of Helsinki and was approved by the local ethical committee .st baseline, (ii) non-painful session: isotonic saline injection, (iii) post-isotonic saline injection, (iv) 2nd baseline, (v) painful session: hypertonic saline injection, and (vi) post-hypertonic saline injection. Injections were given in the dominant side and defined as ipsilateral in the further description. One isotonic and one hypertonic saline injection were given per day in the same muscle . The order of the painful and non-painful injections was blinded for all participants.The subjects stood with preferred foot position on two force plates, with the arms relaxed along the side and looked at a circle  in front of them. The feet position was marked with tape to assure that the same position was used during all trials. The subjects performed, as quickly as possible, either a unilateral shoulder flexion  or bilateral heel lift movement after a go signal (sound cue) was presented. Before the go signal , a warning signal was delivered to the subjects to maintain a quiet standing posture before participants performed the tasks. The time of data collection from the warning signal to end of recording was 10 seconds in each trial. The participants performed 20 reaction tasks alternating between trials of unilateral shoulder joint flexion and bilateral ankle joints plantar flexion. The data collection was performed in two days with at least one week in between and consisted of 6 different sessions for each day: (i) 1Reflective markers (2 cm in diameter), tracked by eight infrared cameras  each sampled with 500 Hz, were attached bilaterally on the following palpable anatomical landmarks: iliac crest, medial condyle of femur, medial malleoli of the ankle, calcanei, and the first metatarsal head. On the dominant side, markers were attached to the lateral supra condylar prominence of the elbow, acromion and styloid process of the radius. The wrist movement during the unilateral shoulder flexion task was defined by the displacement of the marker placed on the styloid process of the dominant radius, and the heel movement during the bilateral heel lift task was defined by the markers’ displacement in both calcanei. The shoulder joint angle was defined by the two segments, dominant brachium , and trunk line (a line by the dominant acromion marker and the dominant iliac crest marker). The ankle joint angle was estimated by the angle between the shank  and foot  and sampled at 2 kHz. The CoP under the ipsilateral, contralateral foot, and the net CoP were calculated  after thMuscle pain was induced in the dominant side by intramuscular injections of sterile hypertonic . Injection of isotonic saline  in the same side was used as control (non-painful) condition. A 2 ml plastic syringe with a disposable needle  was used for the injections. The VM injection site was 5 cm proximal and 5 cm medial to the medial edge of the patella, and the TA injection site was 15 cm distally from the corner of patella. Previous studies applying experimental pain in the VM ,37 and Tside , painful muscle , and saline (isotonic and hypertonic) was applied in the baseline session data to evaluate the CoP asymmetry between feet. The effect of pain was assessed with a three-way RM-ANOVA with factors: saline, time , and painful muscle on the following parameters: movement reaction time , relative CoP negative and positive time-to-peak, AP and ML CoP displacement and the velocity. The CoP parameters for ipsilateral, contralateral, and net CoP were normalised (%) by baseline values before analysis. The Newman-Keul’s (NK) post-hoc test was applied when the main RM-ANOVA indicated significant difference. Significance was accepted at P < 0.05.All results are reported as mean ± standard error of the mean (SEM). Maximum VAS scores during the trials were assessed using a paired t-test comparing control and pain sessions. A three-way repeated measures analysis of variance (RM-ANOVA) with The maximum VAS score after the VM injections was 0.3 ± 0.1 cm  and 4.7 ± 0.8 cm . The maximum VAS score after the TA injections was 0.4 ± 0.1 cm  and 4.7 ± 0.6 cm . The maximum VAS score after hypertonic saline for the both VM and TA injections demonstrated a significantly higher score than the control injections (t-test: P < 0.01).The average onset of target movement from the go-signal (reaction time) is shown in The time-to-peak relative to the target movement onset  and peakThe CoP peak-to-peak displacement  and velosaline and time was found for the normalised ipsilateral CoP displacement in the AP direction where it was reduced during pain when compared with the non-painful condition  when controlling balance, but also the final neuromuscular output represented by the net CoP . PreviouExperimental pain close to the knee joint affected gait , forwardExperimental pain has different effects on each task as observed in CoP displacement, indicating that the APA strategies are different in each of the tasks evaluated. For example, TA muscles have a great participation in the APA generation during unilateral shoulder flexion to counterbalance the perturbation caused by the fast arm movement , howeverIt was surprising that VM pain did not affect the displacement of net CoP in the unilateral shoulder flexion task since previous studies reported impaired postural control due to experimental pain on the quadriceps femoris muscle . One posPain did not affect any postural CoP parameter during bilateral heel lift task, although it has been shown previously that such paradigm affect the onset activity of different postural muscles . When coThese results indicate that during experimental muscle pain close to the knee joint, the central nervous system in healthy individuals is robust in maintaining the APAs motor strategy similar to what is observed in pain free conditions. Although similar experimental pain models have shown good validity in clinical settings, translation of these results to real chronic pain patients should be done with caution since muscle structures are usually not the source of pain in this population. Additionally, in chronic pain patients, other factors that were not accounted for in our experimental model also affect balance of APAs, for example as age ,46–53, gS1 TableThe excel archive contains 13 sheets (one for pain scores and 12 for CoP variables) with respective data for all 9 subjects . For the “Pain-score” sheet, pain scores are reported for both type of saline injections (isotonic and hypertonic) in the vastus-medialis (VM) muscle and tibialis-anterior (TA) muscle. Center of pressure displacement (named “DIS” in the respective sheets) and velocity (named “VEL” in the respective sheets) are reported for both directions [medial-lateral (ML) and anterior-posterior (AP)] for the ipsilateral (IPI), contralateral (CON), and net (NET) components during shoulder flexion and bilateral heel lift tasks.(XLSX)Click here for additional data file.


Recently, we have proposed a method for on-the-fly system matrix computation where the tube-of-response (TOR) is approximated as a cylinder with constant density (TORCD) and the cubic voxels are replaced by spheres. We could show that with this model the PET image quality can be notably improved compared to the vendor provided image reconstruction of our Philips Ingenuity-TF PET/MR. In this work we address the question whether image quality can be further improved by using a variable density TOR (TOR-VD). The radial variability of TOR-VD was modelled by a Kaiser-Bessel function. Free parameters of this density model were used to optimize image properties regarding resolution, noise, and Gibbs artifacts. Additional, a TOR-VD model accounting for position dependent effects along the TOR caused by the finite solid angles of the detectors is under investigation. Phantom measurement were performed with a Philips Ingenuity-TF PET/MR scanner. Listmode data were reconstructed using TOR-CD and TORVD, respectively on two different grids with cubic voxel size of 2 mm and 4 mm. Image quality was assessed with resolution-noise curves and investigation of the radial position dependence of the spatial resolution. For 2 mm voxels, TOR-VD consistently yields a slight improvement of the investigated image quality measures compared to TOR-CD. For 4 mm voxels both models lead essentially to the same results. These findings can be understood as a consequence of the relative size of voxel and TOR. For typical whole body studies (4 mm voxel size) a variable TOR does not improve image quality beyond what is achievable with a constant density TOR. For smaller voxel size the image quality can indeed be somewhat improved with a variable TOR but at the expense of drastically increased computation time.


Further investigations are required to isolate the active constituents of these plants. The Gwich’in, which belong to the Athapaskan language group of Native Americans live in the borderland between Alaska and Canada. We analyzed 29 medicinal plants of this tribe for their growth inhibitory activity against CCRF-CEM T-cell acute lymphoblastic leukemia (T-ALL) cells. The anti-leukemic activity of these plants has not been investigated as yet. Considering the poor cure rates of some ALL forms, there might be a great potential for medicinal plants as resource for natural products to treat T-ALL. We found that the hexane extracts of three plants revealed considerable growth inhibition on CCRF-CEM cells. The 50% inhibition concentrations (IC


These techniques have challenges such as low contrast between soil pore space and roots, long X-ray imaging time, and low spatial resolution. In this study, the use of synchrotron (SR) based phase contrast X-ray imaging (PCI) has been demonstrated as a minimally invasive technique for imaging plants. Above ground plant parts and roots of 10 day old canola and wheat seedlings grown in sandy clay loam soil were successfully scanned and reconstructed. Results confirmed that SR-PCI can deliver good quality images to study dynamic and real time processes such as cavitation and water-refilling in plants. The advantages of SR-PCI, effect of X-ray energy, and effective pixel size to study plant samples have been demonstrated. The use of contrast agents to monitor physiological processes in plants was also investigated and discussed.Minimally invasive investigation of plant parts  has good potential to elucidate the dynamics of plant growth, morphology, physiology, and root-rhizosphere interactions. Laboratory based absorption X-ray imaging and computed tomography (CT) systems are extensively used for  Internal structural visualization and quantification of plant parts are essential to fully understand the structure-function relationship and dynamic processes in plants. Knowledge of internal structures of plants is crucial for better understanding of morphological diversity, physiology, function, and evolution. Internal structural characterization of plants on the micron to nanometre scales is usually achieved by analyzing excised cross sections using optical light microscopy (LM), transmission electron microscopy (TEM), scanning electron microscopy (SEM) or confocal laser scanning microscopy (CLSM). These techniques are limited by sampling area, accurate sample location, number of analyzed samples, and extensive sample preparation protocols, all of which can make data interpretation more difficult. The dynamic processes like water or nutrient flow and long term continuous monitoring of plants to understand physiological changes or responses to abiotic, biotic, or nutritional stresses are more easily studied with less invasive techniques.The light and electron microscopy techniques produce good quality images at high spatial resolution. However, they are largely restricted to two dimensional 2D) imaging. SEM allows visualization and study of surface characteristics of plants or plant structuresD imaging342), selective wavelength, and partial coherence compared to X-rays from laboratory X-ray machines. High flux density simultaneously reduces exposure times and improves the signal to noise ratio (SNR). Wavelength tunability allows the use of the optimal wavelength for the plant component being imaged (in situ roots or above ground soft tissues are better imaged with different wavelengths). The partial coherence of the SR X-ray beam has unique advantages to image low density tissues that have weak absorption or interactions with X-rays10The use of X-rays to study opaque and thick agricultural samples started in the 1920s and 3D visualization of structures using X-rays—computed tomography (CT) was realized and demonstrated in 1973Some of the common SR based X-ray imaging modalities are absorption imaging, phase contrast imaging (PCI), and diffraction enhanced imaging (DEI). X-rays typically interact with samples by being absorbed or scattered. Absorption X-ray imaging is based on sample density differences whereas PCI is based on the refraction of X-rays. The X-ray absorption and phase contrast (PC) images reveal morphological features of dry plant samples at sub-micrometer spatial scale1215in vivo; 3) determine the optimum effective pixel size required for plant imaging using PCI; 4) determine the best soil type to image plant roots in vivo; and 5) explore the use of contrast agents to monitor physiological and dynamic processes in live plants.The main requirements for PCI are highly parallel or coherent X-ray beam and large sample to detector distance compared to absorption imaging20222325The experimental setup used for acquiring projection 2D) images using absorption and PCI modalities at the biomedical imaging and therapy beamline-bending magnet (BMIT-BM beamline) is shown in D images The goal is to compare SR-PCI with SR-absorption and laboratory absorption measurement for above and below ground plant samples. In the below ground samples (soil-root system), the difference between SR-PCI and SR-absorption image was not obvious and therefore compared SR-PCI of soil-root samples with laboratory absorption measurements. The optimum X-ray energies required for above and below ground plant imaging were determined by recording a series of images at different X-ray energies  and the As SR X-ray flux is high, the goal is to record plant images with minimum exposure possible. The effective pixel size of X-ray images acquired using PCI depends on detector resolution and sample to detector distanceTo determine the best soil type for soil-root imaging of plants using SR-PCI, canola and wheat seedlings grown in an 8 mm diameter flacon tube using sandy clay loam (representative of Saskatchewan soil) and planting mix were recorded at the optimized X-ray energy of 24 keV. Contrast agents have been used in SR-PCI for medical applications282920In another study, movement of water through canola seedling stem was observed . Two 10 in vivo, under a wide range of imaging parameters to optimize data quality. In this study, we selected major crops, wheat and canola, to provide an appropriate experimental setup for SR-PCI using the BMIT-BM beamline and to compare PCI with absorption imaging . However, high phase signals from the edges of plant parts in the projection images (at distances higher than 60 cm) created unwanted artefacts during CT reconstruction as shown by Pratt In this study, X-ray images of soil-root system recorded using SR-PCI were compared with a laboratory X-ray imaging system. In a previously reported work, the optimal X-ray energy for root detection using SR-PCI is determined to be 25 keVIn general, optimal effective pixel size required should be determined for each specific sample type in combination with specific SR technique being used. Little is known about effective pixel size required for plant imaging and the effect of pixel size on the quality of X-ray images. Although different resolution detectors are available, the selection of detector depends on the purpose of the study where the normal tendency is to use the highest resolution possible. Image pixel sizes from 10s of micron to several hundred nanometers have been used to study plant samples using PCI3140in situ in soil. The root system may in fact hold the key to fully understanding plant growth and development, and to develop crop varieties with improved yield by optimizing nutrient use efficiency4342The importance of root systems for water and nutrient uptake by plants has motivated researchers to seek a better understanding of root system development Several studies have attempted to reconstruct 3D images of plant roots in natural soil with a variety of results464347High resolution X-ray CT imaging can be used to monitor plant structures and physiological processes such as xylem network architecture and real-time water uptake26483049The phase contrast images can be recorded before and just after the absorption edges of elements to highlight the elemental distributions in samples, also called K-edge subtraction technique50The results from the water rehydration study clearly show the water refilling of canola stem cavitation when exposed to iodine and water . The PCI5226planta in great detail.The computed tomographic implementation of emerging X-ray transmission imaging modalities, such as SR absorption and PCI present important applications to biology and medicine555657585960in vivo is the plant movement due to environmental factors. Therefore, lots of published works on above ground plant are done on excised or dry plant parts such as cut bamboo or dry leaves25in vivo  the plant is exposed to, and hence plant tissues may be damaged. The radiation damage from SR imaging is still lower than conventional systems due to fast imaging and use of monochromatic beams at the SR limits the exposure of plants to unwanted low energy X-ray beams . For a broadband X-ray source, the low energy X-rays typically do not contribute significantly to the image, while providing the majority of the absorbed dose to sample. The unwanted X-ray beams that are not useful for acquiring images may contribute more to the sample damageWe have developed, for the first time at the Canadian Light Source, novel methodologies for using SR-PCI for better understanding of complex internal plant structures and dynamic processes. The SR-PCI technique is a minimally invasive technique that could be used successfully to reconstruct internal structure of plants in 3D using micron- to nano-scale resolutions. The technique is very useful to understand dynamic processes by monitoring the plant states at different conditions of abiotic, biotic, or nutritional stresses. Furthermore, it is expected that the relatively low radiation dose absorbed by the samples will, in future, allow longitudinal studies in which the same living plant can be imaged in a series of time separated exposures. The image qualities presented in this work are better than some of the earlier reported works. The beam parameters used in this work can be improved by dedicated beamlines optimized for plant research. The work illustrates proper selection of soil types, effective pixel size, X-ray energy, and sample to detector distances are required for high quality plant images.Brassica napus, cultivar Fortune RR), and wheat plants  used in the experiments were grown under controlled conditions using a plant growth cabinet (18–23 °C) with 14-h photoperiods (512 μmol.m−2s−1). The seedlings were transported to the beamline for imaging a few hours prior to beamtime and were kept in a well-lit room until the data was collected. Wheat spikes of Sumai3 and Muchmore were excised from plants in the growth chambers 4 hours prior to beamtime and were kept at room temperature inside air-tight freezer bags. The seedlings were grown using soil-less planting mix  for above ground imaging experiments. For soil-root X-ray imaging, soil-less planting mix and sandy clay loamThe canola  at the Canadian Light Source65To determine the advantages of synchrotron based SR-PCI for soil-root imaging, a 10 day-old canola plant first imaged by SR-PCI was then scanned using a laboratory X-ray machine , as the difference of absorption and phase contrast images collected using the synchrotron SR-PCI did not show clear differences compared to above ground plant parts. The image contrast based on the histogram distribution, phase signal, visual image quality, and data acquisition time between SR-PCI and laboratory X-ray imaging were compared.TM 240, Ioversol Injection 51%). Plant parts were then imaged using X-rays at or just above the absorption energy of iodine at 33.4 keV.Iodine is used as a contrast agent in medical imaging to improve the contrast in images. The use of iodine to improve the contrast in plant vessels such as xylem to study plant physiology and process dynamics in live plants was determined in this study. The SR-PCI of plants were first recorded at an X-ray energy of 18 keV and then the plants with roots (with soil washed out) or wheat spikes were inserted into a solution of water and organic iodine 1:1 (v/v)  solution of water and organic iodine and plants were kept under a 60 W incandescent light to increase the transpiration rate. The canola stem was imaged at the same X-ray energy every 10 min for about an hour and once every 15 minutes after the first hour.The projection X-ray images were first normalized using flat and dark signal images . The 3D reconstruction of images was carried out using NRecon software  and 3D data analysis (segmentation etc.) was accomplished using Avizo standard software .How to cite this article: Karunakaran, C. et al. Factors influencing real time internal structural visualization and dynamic process monitoring in plants using synchrotron-based phase contrast X-ray imaging. Sci. Rep.5, 12119; doi: 10.1038/srep12119 (2015).


To identify ciliopathy-causing genes in a very large cohort of patients with symptoms consistent with cilia dysfunction.1,056 index cases with nephronophthisis-related ciliopathies were screened for mutations in all genes encoding components of IFT-B and 572 unrelated individuals with early onset retinal dystrophies or multisystemic ciliopathies were subjected to targeted ciliome resequencing.IFT81 mutations were identified in two consanguineous sporadic cases. The first individual harbored a splice site change predicted to result in an inframe exon skipping; the second carried a 4 bp deletion resulting in a loss-of-stop with extension of the deduced protein by 10 amino acids. The spectrum of IFT81-related disease expression included nephronophthisis, retinal dystrophy, cerebellar atrophy, and polydactyly. Fibroblasts from one affected individual showed no difference to control cells with regard to IFT81 localization or binding to IFT25, but a statistically significant decrease in ciliated cell abundance was noted. GLI2 expression and ciliary localization were impaired suggesting altered sonic hedgehog signaling.Homozygosity for Mutations in all components of IFT-A complex have been reported to cause ciliopathy phenotypes. In contrast, only two peripheral IFT-B members, IFT172 and IFT80, were known to be involved in these conditions. The identification of mutations in the IFT-B core protein IFT81 in two unrelated patients out of 1268 individuals with ciliopathy further elucidate the role of this complex in human disease and show that defects in the IFT-B core are an exceedingly rare finding supporting the view that it is indispensable for ciliary assembly in development.


Newcastle disease is still a serious disease of poultry especially in backyard free-range production systems despite the availability of cross protective vaccines. Healthy-looking poultry from live bird markets have been suspected as a major source of disease spread although limited studies have been conducted to ascertain the presence of the virulent strains in the markets and to understand how they are related to outbreak strains.This study evaluated the occurrence of Newcastle disease virus in samples collected from poultry in live bird markets across Uganda. The isolates were pathoyped using standard methods (mean death time (MDT), intracelebral pathogenicity index (ICPI), and sequencing of the fusion protein cleavage site motif) and also phylogenetically analysed after sequencing of the full fusion and hemagglutin-neuraminidase genes. The isolates were classified into genotypes and subgenotypes based on the full fusion protein gene classification system and compared with other strains in the region and world-wide.Virulent avian paramyxovirus type I (APMV-1) (Newcastle disease virus) was isolated in healthy-looking poultry in live bird markets. The viruses belonged to a new subgenotype, Vd, in genotype V, and clustered together with Tanzania and Kenya strains. They harbored low genetic diversity.The occurrence of virulent AMPV-1 strains in live bird markets may serve as sources of Newcastle disease outbreaks in non-commercial farms. Newcastle disease is a highly contagious disease affecting chickens and other poultry species and wild birds. It devastates unvaccinated flocks in periodic outbreaks resulting in up to 70-100% mortality, which is a big loss to the households. It is caused by RNA viruses of the Avian Paramyxovirus type 1 (APMV-1),   into the allantoic cavities of 9- to 11-day-old embryonating specific-pathogen-free eggs. The eggs were incubated at 37°C and candled twice daily (early morning and late afternoon) for 7 days, and the time of embryo mortality was recorded. The MDT for a minimum lethal dose was interpreted as the mean time in hours for the embryo death in the highest dilution at which all eggs died. The isolates were classified as: velogenic viruses if they killed the embryos in fewer than 60 hours; mesogenic if they killed the embryos in 60 to 90 hours; avirulent viruses if they failed to kill the embryos at all. The selected isolates were also tested for virulence in one-day old specific-pathogen-free chicks by intra-cerebral injection of virus. The results were converted into an intra-cerebral pathogenicity index (ICPI) that varied from zero to a maximum of 2.0 for velogenic viruses as described . The isolates with identical sequences were not deposited in the GenBank instead they were represented by one sequence and they have been indicated in the phylogenetic tree legends.