MicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored.We assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r = 0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar.The sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation. MicroRNAs(miRNAs) are an abundant group of small RNAs with length ranging from 18 to 25 nucleotides, averaging 22 nucleotides, and performing post-transcriptional regulation of the expression of genes involved in a wide variety of biological processes. The complex biogenesis of mature miRNAs has recently been reviewed , the majSample preparation is of major importance for NGS and assessing the quality of a library preparation by cloning validation before sequencing is necessary . DiffereThe cloning frequency of an individual miRNA should generally reflect its relative abundance in a sample, and the novel NGS methods offering a much richer source of sequence information should provide more accurate quantitative expression measurements . HoweverDifferent protocols of library preparation may influence the cloning frequency significantly. The adapter ligation efficiency can be affected by the 5'- and 3'-end nucleotides or the secondary structure of miRNAs, and the number of polymerase chain reaction cycles or gel isolation procedures may also influence the results. In this article we compared sequencing data of libraries constructed by the above-mentioned three different protocols, and validated some results by qRT-PCR using stem-loop primers . Bias ofWe assessed the quality of libraries by cloning and a Sanger sequencing approach. 211, 228 and 233 high quality reads were obtained for Hu sheep libraries constructed by SOLiD, Illumina V1 and Illumina V1.5 protocols, respectively. 221, 225 and 208 high quality reads were obtained for Dorper sheep using SOLiD, Illumina V1 and Illumina V1.5 protocols, respectively. The ratio of reads mapping to known microRNAs, rRNAs, and mRNAs, and the reads which have not been mapped to any known sequences, were compared between the different protocols , repeat regions, or are contained within the boundaries of protein coding genes for Dorper sheep and Hu sheep. The number of reads that were annotated as known microRNAs was 4,812,498 and 4,904,192 for Dorper sheep and Hu sheep respectively.The libraries constructed from Dorper sheep and Hu sheep, using the Illumina V1 protocol, were used for Illumina SBS sequencing. About 6 million raw reads were obtained for each library. Eighty-four percent of the Hu sheep reads mapped to known The length distribution of all cloning and SBS sequencing data was assessed Figure . They foThe sequence variations of miR-1 and miR-206 in our data were assessed by WebLogo tool , including the sequences mapping to mRNAs, repeats, or rRNA genes, and sequences that do not map to any known sequences Figure .The relative abundance of 9 miRNAs calculated by qRT-PCR was compared with the read counts of the same families in sequencing data. The muscle-specific miRNAs, miR-1 and miR-206, were the two most abundant miRNAs as analyzed by qRT-PCR, and confirmed by sequencing data, as well as by a previous report . SequencExcept for miR-486-5p, the miRNAs assessed showed a similar orientation of fold change using qRT-PCR or Illumina GA , and length distribution was observed for the different protocols of library construction. The SOLiD data differ from the Illumina data, due to distinct principles of adapter ligation in protocol. The adapter ligation principle based on 6 random nucleotides (N6) hybridization seemed to provide a more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- or 5'-ends, higher frequency of reads containing ESS, and higher frequency of reads which do not map to known miRNAs in sequencing data. The two nucleotides at the 3'-end of the majority of the reads of miR-1 in SOLiD cloning data were truncated, which may be due to RNA editing. We also found that the truncated sequence of miR-1 can form 3'-ESS while the original sequence can not Figure . The endWe finally assessed the relative abundance of 9 miRNAs by qRT-PCR. The principle of reverse transcription (RT)-PCR with stem-loop primer is based on hybridization as for the SOLiD protocol, which could explain the high correlation between qRT-PCR and SOLiD cloning data. The fold difference data between Hu sheep and Dorper sheep using qRT-PCR and SBS sequencing correlated significantly, and the fold difference data for miR-1 and miR-206 using SOLiD cloning were similar to data obtained with SBS sequencing and qRT-PCR, indicating that the methods using a hybridization principle may be more suitable for quantitative measurement of miRNA abundance. Moreover, qRT-PCR has been used prevalently for validation of microarray results ,14 and iTotal RNA from skeletal muscle tissues of Hu sheep and Dorper sheep were extracted using Trizol  according to the manufacturer's protocol. About 10 ug total RNA was treated with DNase I (NEB) and then purified by ethanol precipitation.In Illumina V1 protocol, isolated 18-30 nt small RNAs were first ligated with 5' adapter using T4 RNA ligase. Ligation products were gel-purified, then ligated with 3' adapter using T4 RNA ligase and purified. Small RNAs with adapters on both ends were used as templates for reverse transcription PCR to create cDNA constructs. The amplified cDNA constructs were subsequently purified and used for loading on an Illumina Cluster Station  of Hu sheep and Dorper sheep are shown. The miRNA ID and accession No of each miRNA family have been listed.Click here for filePrimer sequences for qRT-PCR. Sequences of RT stem-loop primers and forward/reverse primers of 9 miRNAs for qRT-PCR.Click here for fileThe qRT-PCR result and read counts for 9 miRNAs. Relative abundance for 9 miRNAs calculated by qRT-PCR are shown, with the read counts of sequencing data for these 9 miRNAs.Click here for file


They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation. NATs give rise to sense-antisense transcript pairs and the number of these identified has escalated greatly with the availability of DNA sequencing resources and public databases. Traditionally, NATs were identified by the alignment of full-length cDNAs or expressed sequence tags to genome sequences, but an alternative method for large-scale detection of sense-antisense transcript pairs involves the use of microarrays. In this study we developed a novel protocol to assay sense- and antisense-strand transcription on the 55 K Affymetrix GeneChip Wheat Genome Array, which is a 3' in vitro transcription (3'IVT) expression array. We selected five different tissue types for assay to enable maximum discovery, and used the 'Chinese Spring' wheat genotype because most of the wheat GeneChip probe sequences were based on its genomic sequence. This study is the first report of using a 3'IVT expression array to discover the expression of natural sense-antisense transcript pairs, and may be considered as proof-of-concept.Natural antisense transcripts (NATs) are transcripts of the opposite DNA strand to the sense-strand either at the same locus (By using alternative target preparation schemes, both the sense- and antisense-strand derived transcripts were labeled and hybridized to the Wheat GeneChip. Quality assurance verified that successful hybridization did occur in the antisense-strand assay. A stringent threshold for positive hybridization was applied, which resulted in the identification of 110 sense-antisense transcript pairs, as well as 80 potentially antisense-specific transcripts. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. For the annotated sense-antisense transcript pairs, analysis of the gene ontology terms showed a significant over-representation of transcripts involved in energy production. These included several representations of ATP synthase, photosystem proteins and RUBISCO, which indicated that photosynthesis is likely to be regulated by antisense transcripts.This study demonstrated the novel use of an adapted labeling protocol and a 3'IVT GeneChip array for large-scale identification of antisense transcription in wheat. The results show that antisense transcription is relatively abundant in wheat, and may affect the expression of valuable agronomic phenotypes. Future work should select potentially interesting transcript pairs for further functional characterization to determine biological activity. The first NATs were detected in viruses, followed by prokaryotes and then eukaryotes. For an excellent review of current NAT knowledge, please refer to Lapidot and Pilpel [A. thaliana [cis-encoded pairs. In wheat, antisense transcripts have been discovered from serial analysis of gene expression (SAGE) tags of developing grain [Natural antisense transcripts (NATs) are defined as transcripts of the opposite DNA strand to the sense-strand either at the same locus  mouse arrays to detect novel antisense transcription in mouse brain and kidney tissues. The results showed that the commercial expression arrays were sensitive enough to detect antisense transcription, but because it cannot be assumed that current commercial arrays contain incorrectly orientated probes, this type of study could not be repeated. Subsequently, Ge et al. [An alternative method for large-scale discovery of sense-antisense transcript pairs involves the use of microarrays. In the first study of this type, Yelin et al.  used a set al. . However [et al.  took adve et al.  developein vitro transcription of double-stranded cDNA to both amplify and label the target cRNA before hybridization. The wheat array currently provides the most comprehensive coverage of the wheat genome for a microarray and is a commonly used resource for transcript expression studies [In the current study, we sought to develop a protocol that could be used to assay sense- and antisense-strand transcription on the Affymetrix GeneChip Wheat Genome array, which is a 3'IVT expression array. The 3'IVT expression arrays rely on  studies ,20 and h studies . This stTotal RNA was extracted from five 'Chinese Spring' tissue types . In addition to maximizing discovery, these tissue types were also selected to align with the predominant tissues used for wheat EST sequencing efforts, including the International Wheat Genome Sequencing Consortium (IWGSC). All samples were of excellent quality as assessed by gel electrophoresis and spectrophotometry. The total RNA samples were mixed at equal concentrations before target preparation. The assay of sense-strand transcription followed the regular scheme as for all Affymetrix 3'IVT GeneChips . However, to assay antisense-strand transcription, the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay was used, which was designed specifically for use on Whole-Transcript Expression arrays. The WT target preparation method resulted in labeling the opposite strand to the 3'IVT assay and was therefore used in this study to assess antisense-strand transcription Figure . The mixaffyQCreport package [bioB, bioC, bioD and creX) produced similar signals from both assays although signals were slightly higher in the WT assay. The MAS 5.0 PMA calls and Robust Multi-array Average (RMA) summarized expression values were used to determine successful hybridization for each probe set. Because of differences in the two labeling methods, including starting amount of RNA and RNA amplification, the expression values of each array could not be validly compared. However, the PMA calls in combination with the expression values were used to determine positive hybridization to a particular probe set in each assay. This provided a qualitative measure of expression rather than quantitative, but for the purposes of this study which was to detect natural sense-antisense transcript pairs, this measure was satisfactory.Following hybridization and scanning, CEL files were analyzed to identify probe sets that showed successful hybridization in each of the 3'IVT and WT assay. The quality control metrics from the  package  of Bioco package  showed tbioB, bioC, bioD and creX) were used. Because these controls are spiked-in immediately before hybridization, they were expected to behave in the same way in both assays. The bioB control is spiked-in at the detection limit, while the others are spiked-in at staggered concentrations after bioB. Thus, the log2 expression value of bioB was considered the threshold for positive hybridization in each assay  values to detect successful hybridization in each assay. In both assays, a probe set must have firstly been called 'Present' with Wilcoxon rank sum test p-value < 0.01, and the RMA summarized log2 expression value must have been greater than 8.76 in the 3'IVT assay, and greater than 9.86 in the WT assay. This threshold cutoff identified 110 probe sets as positively hybridizing in both the 3'IVT and WT assays. In addition to the 110 probe sets, 8940 probe sets uniquely hybridized in the 3'IVT assay and 80 uniquely hybridized to the WT assay . Because the aim of this study was detect transcript pairs transcribed from both strands, we mainly focused on probe sets detected in both assays. These stringent detection criteria ensured that the probe sets left were highly expressed in both assays and could more reliably be considered as sense-antisense transcript pairs. In fact, the 80 antisense-specific probe sets could not necessarily be classified as antisense transcripts, because these may represent incorrectly orientated probes. Also, because the probes for a given transcript do not cover the entire sequence, there is a possibility for bias during hybridization. However, to form the basis of future studies these 80 probe sets were also given some attention.To determine a confident positive threshold for expression value in both assays, the expression values of spiked-in hybridization controls . Gene Ontology (GO) was based on the TIGR rice genome annotation such that if a unigene possessed a significant (<1e-10) BLASTx match to rice, as identified in HarvEST, the corresponding GO terms for the rice protein were used, if available. Of the 110 probe sets 76 could be annotated . An example of the electrophoresis results is shown in Figure This study reports on the first use of an Affymetrix GeneChip 3'IVT expression array for discovering both sense- and antisense-strand transcription. Through the adaptation of the Affymetrix WT assay, the antisense transcribed strand was successfully labeled and hybridized to the Wheat Genome Array, which allowed for the detection of natural sense-antisense transcript pairs. To our knowledge, the Wheat Genome Array does not contain any probes for known sense-antisense transcript pairs, thus the data from the hybridizations could not be standardized and/or normalized to a known sense-antisense transcript pair. Subsequently, a highly stringent data acceptance threshold was applied, based on PMA call and expression value cutoffs. This increased the confidence in detecting true antisense transcription. It is important to recognize the limitations of this study, which stem from the 'closed' nature of microarray systems. Because the Wheat Genome Array contains only known transcript sequences, the study is clearly limited to detection of transcript pairs that are present on the array. Further, the probes for each transcript are biased to the 3' end of transcripts and do not span the entire gene. Thus, because antisense-strand transcripts commonly have a different splice structure they may not be detected. Subsequently the 110 candidate sense-antisense transcript pairs and the 80 potentially antisense-specific transcripts that were identified are likely to under-represent the number of true transcript pairs. In future studies, custom microarrays containing probes for sense and antisense transcripts would be useful as different target preparation assays would not be required, but because we aimed to obtain a broad representation of the extent of antisense transcription we chose to use the most comprehensive Wheat Genome Array.The function of antisense-strand transcription is widely believed to regulate the expression of sense-strand transcripts at either transcription, mRNA maturation or translation . In factAn important observation in this study was the functional annotation of the sense-antisense transcript pairs, which indicated a significant over-representation of those involved in energy production, particularly photosynthesis. Additionally, many transcripts for ribosomal proteins involved in protein synthesis were identified. The abundance of antisense transcripts for these common plant processes may indicate that they are negatively regulated by antisense transcripts. Alternatively, the antisense transcripts could possibly be the result of ectopic expression. There is little data on large-scale antisense transcription profiling in plants to compare these results with, but a study in rice of leaf and seed tissue using Serial Analysis of Gene Expression (SAGE) identified sense-antisense transcript pairs and also found that the most abundant pairs were annotated as involved in energy production, including RUBISCO and a Photosystem I protein . The simet al. [The results of the strand-specific RT-PCR also showed that antisense transcription is likely to be tissue-specific. Only one of the RT-PCR results was not in complete agreement with the microarray result, which could be due to truncated antisense transcripts where the priming sites were absent. In their microarray study of human cell antisense transcription, Ge et al.  found thet al. [et al. [et al. [A recent study in wheat involving SAGE of developing grain also identified antisense transcripts , where tet al.  found th [et al.  of antis [et al. , which aThis study was exploratory and revealed that the method was successful in identifying sense-antisense transcript pairs using the commercial Wheat Genome Array. The next step from this study is to select potentially interesting antisense transcripts for further study. There were several transcript pairs belonging to functional categories including 'Cell death' and 'Transcription' that may be involved in the regulation of important biological processes, and the antisense-specific transcripts related to transcription are also of interest. An understanding of the role of antisense transcription as it relates to gene expression may be important for the expression of certain phenotypes of interest. Additionally, knowledge of natural antisense transcripts may also be important for altering gene expression through transgenic studies in plants. The abundance of antisense-strand transcripts in plants is supported by recent studies using 'open' transcriptomics systems including SAGE ,14 and MThis study demonstrated the novel use of an adapted labeling protocol and a 3'IVT Affymetrix GeneChip microarray for large-scale identification of antisense transcription in wheat, a crop of great economic importance. The results show that antisense transcription is relatively abundant in wheat, and may affect the expression of valuable agronomic phenotypes. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. Most of the identified sense-antisense transcript pairs were annotated as genes involved in energy production, indicating that photosynthesis is likely to be under regulation by antisense transcripts.® Plus RNA Purification Kit  with an on-column DNase treatment. Purified total RNA samples were quantified with a NanoDrop® ND-1000  spectrophotometer, and satisfactory purity was indicated by A260:280 ratios of 1.9–2.1 in 10 mM Tris-Hcl (pH 7.5). Integrity of total RNA samples was assessed by denaturing formaldehyde gel electrophoresis, where the presence of sharp 28S and 18S ribosomal RNA bands at an intensity ratio of ~2:1 (28S:18S) indicated good integrity. The five total RNA samples were then mixed at equal concentrations before analysis.The spring wheat genotype 'Chinese Spring' was selected for this study because the majority of GeneChip Wheat Genome Array  probe sequences were based on its DNA sequence. Five tissue types were selected for this study; i.Germinated seed , ii.Shoot , iii.Flag leaf , iv.Spike pre-anthesis , and v.Spike post-anthesis . For each tissue type except 'Germinated seed', six seeds were planted in 6-inch round pots using a potting mix . Seedlings were grown to the appropriate growth stages for tissue collection in a greenhouse with a stable temperature of 25 ± 2°C and a 16 h light/8 h dark cycle. All tissue samples were immediately frozen in liquid nitrogen, and total RNA was extracted from 1.0 g of each pooled tissue type using the TrizolT. aestivum) and are derived from the public content of the T. aestivum UniGene Build #38 . 1,215 probe sets are derived from ESTs of a diploid near relative of the A genome (T. monococcum), a further 539 represent ESTs of the tetraploid (A and B genomes) macaroni wheat species T. turgidum, and five are from ESTs of a diploid near relative of the D genome known as Aegilops tauschii. Probe sets consisted of pairs of 11 perfect match (PM) and mismatch (MM) 25-mer oligonucleotides designed from the 3' end of exemplar sequences, with nucleotide 13 as the MM. Array annotation information is available on the NetAffx data analysis center .The GeneChip Wheat Genome Array  is a 3'IVT array that includes 61,127 probe sets representing 55,052 transcripts for all 21 wheat chromosomes in the genome. 59,356 probes sets represent modern hexaploid  bread wheat  primer. The double-stranded cDNA was cleaned up and used as template for in vitro transcription (IVT) in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for cRNA amplification and biotin labeling. The biotinylated cRNA target was then cleaned up, fragmented, and hybridized to the Wheat Genome Array. All hybridizations and data acquisition was performed at the Genomics Core Facility at Washington State University  according to standard Affymetrix protocols .The 3'IVT Wheat Genome Array detects sense strand transcription by generating antisense-orientated labeled complementary RNA (cRNA) from the original RNA sample that is then hybridized to the probes that are designed to hybridize to the antisense-orientated labeled cRNA. Although the system generates antisense-orientated labeled cRNA, the assayed strand for transcription is the sense strand. Thus, to assay sense strand transcription in the mixed Chinese Spring total RNA sample, the regular 3'IVT Affymetrix protocol was carried out  was used. This WT assay is intended for use on Affymetrix Whole Transcript expression arrays, which contain probes designed to hybridize with sense-orientated labeled cDNA. Because the probes of the 3'IVT Wheat Genome Array are designed to hybridize to antisense-orientated labeled cRNA derived from sense strand transcription, the sense-orientated labeled cDNA generated by the WT assay will not hybridize to the array unless it was derived from antisense transcription. Thus to discover antisense transcription in the mixed Chinese Spring total RNA sample, the target was prepared using the WT assay but hybridized to the 3'IVT Wheat Genome Array. Briefly, double-stranded cDNA was synthesized with random hexamers coupled to the T7 promoter, followed by IVT amplification with T7 RNA polymerase to produce cRNA. A second cycle cDNA synthesis was then performed using random primers for reverse transcription, which converted the cRNA into single-stranded cDNA in the same orientation as the original mRNA (sense-orientation). The single-stranded cDNA was then cleaned up, fragmented, and hybridized to the Wheat Genome Array according to standard Affymetrix protocol.To assay antisense strand transcription, the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay affyQCreport package [affy [p-value < 0.01 were considered 'present'.Using GeneChip Operating Software (GCOS) v.1.4 , image quality control was performed by inspecting raw intensity (DAT) files for scratches/smears and uniform performance of the B2 oligo around the border of each image. Data quality control was from raw data in CEL files using the  package  of Bioco package , which p package  package ge [affy  was usedge [affy ,28. Pre-ge [affy . Only th2 expression value greater than the bioB spiked-in hybridization control (>8.76 in the 3'IVT assay and >9.86 in the WT assay). Probe sets meeting these criteria were annotated using HarvEST (Affymetrix Wheat1 Chip version 1.52), which identified the corresponding unigene for each probe set and provided the current best BLASTX hit from the non-redundant (nr) database of NCBI, as well as the best BLASTX hits from rice and Arabidopsis thaliana TIGR databases . A database hit <1e-10 was considered as significant, otherwise the unigene was annotated as 'no homology'. Unigenes were assigned to functional categories based on Munich Information Center for Protein Sequences  classifications. All minimum information about microarray experiments (MIAME) guidelines were observed and GeneChip data was deposited into WheatPLEX [Probe sets called as present were also required to possess a summarized logheatPLEX  accessioheatPLEX  accessioTen probe sets that were found to be transcribed on both strands were selected for validation using strand-specific reverse transcription-PCR (RT-PCR) Table . The strTEC carried out the tissue collection, RNA extraction, RT-PCR, data analysis, and drafted the manuscript. MLS participated in the design of the study and performed the statistical analysis. TEC and XC conceived the study. XC participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.P < 0.01), >8.76 3'IVT assay log2 expression value and >9.86 WT assay log2 expression valueList of the 110 sense-antisense transcript pairs with Present calls  and >9.86 WT assay log2 expression valueList of the 80 potential antisense-specific transcripts with Present calls (. The data provided represents annotation and statistical values for the potential antisense-specific transcripts.Click here for file


A comprehensive analysis of the neuroanatomical expression profiles of 38 abundant conserved miRNAs in developing and adult zebrafish brain was performed. MicroRNA (miRNA) encoding genes are abundant in vertebrate genomes but very few have been studied in any detail. Bioinformatic tools allow prediction of miRNA targets and this information coupled with knowledge of miRNA expression profiles facilitates formulation of hypotheses of miRNA function. Although the central nervous system (CNS) is a prominent site of miRNA expression, virtually nothing is known about the spatial and temporal expression profiles of miRNAs in the brain. To provide an overview of the breadth of miRNA expression in the CNS, we performed a comprehensive analysis of the neuroanatomical expression profiles of 38 abundant conserved miRNAs in developing and adult zebrafish brain.miR-92b); expression associated with transition from proliferation to differentiation ; constitutive expression in mature neurons (miR-124 again); expression in both proliferative cells and their differentiated progeny ; regionally restricted expression ; and cell-type specific expression .Our results show miRNAs have a wide variety of different expression profiles in neural cells, including: expression in neuronal precursors and stem cells (for example, The data we present facilitate prediction of likely modes of miRNA function in the CNS and many miRNA expression profiles are consistent with the mutual exclusion mode of function in which there is spatial or temporal exclusion of miRNAs and their targets. However, some miRNAs, such as those with cell-type specific expression, are more likely to be co-expressed with their targets. Our data provide an important resource for future functional studies of miRNAs in the CNS. The expression of 30% or more animal genes is regulated by microRNAs (miRNAs) ,2. GenesTarget mRNAs are usually transcribed at low levels whenever their targeting miRNAs are expressed ,12. ThisDifferent experimental approaches indicate hundreds of miRNAs in vertebrate genomes -17. ManyGiven the known modes of action of miRNAs, knowledge of the temporal and spatial expression profiles of miRNA genes is an important initial step in elucidating their functions. Based on our previous miRNA expression analyses ,24, we sin situ hybridizations with locked nucleic acid (LNA) probes to 38 different miRNAs  at 3 and/or 5 days and/or 6 weeks ) and/or adult zebrafish ). Some of the miRNAs we analyzed belong to the same family or cluster and can differ in only one nucleotide located in, or outside, the 'seed' sequence . To examine if the LNA probes can discriminate between miRNAs having only one or more different nucleotides, we performed in situ hybridization for four miRNAs using one or two internal mismatches . We observe that in the case of let-7a, miR-92b and miR-153a, one mismatch strongly reduces the hybridization signal. Since there is still some staining left, we cannot exclude some cross-hybridization with other members of the respective miRNA families. In contrast, two mismatches in the miR-181a probe are sufficient to eliminate specific in situ hybridization signal, supporting the conclusion that probes with two or more different internal nucleotides detect signal from a single miRNA and not others with similar sequence [In order to survey the expression patterns of miRNAs in the brain, we performed sequence .We describe the range of different spatially and/or temporally restricted profiles of miRNA expression with illustrative examples below and more comprehensive neuroanatomical documentation in the figures, text and tables in Additional data files 1-27.With very few exceptions, the fish CNS is organized such that proliferative cells line the ventricles whereas differentiated neurons migrate away from this zone towards the basal, or pial surface of the brain. At larval (3 and 5 day post-fertilization (dpf)) stages, proliferative cells are present throughout the brain, including the periventricular telencephalic, thalamic and hypothalamic zones, tectal proliferative zone, cerebellar valvula and rhombic lip .miR-92b is expressed in proliferative zones throughout the 5 dpf embryonic zebrafish brain. Transcripts are detected in periventricular and adjacent cells of the ventral and dorsal subpallium . let-7a and let-7c, which differ in their sequence from let-7b by two and one nucleotide, respectively, located outside the seed region, appear to lack this retinal expression, although we cannot exclude that these LNA probes cross-hybridize to various let-7 family members in different brain areas .Like miR-92b in proliferating neural cells, miR-124, miR-138 and other miRNAs are expressed in differentiating cells of the larval brain. Among these, miR-124 is expressed in virtually all differentiating cells throughout the larval zebrafish brain and retina  shows a widespread but more restricted pattern of expression. Such patterns indicate expression is associated with differentiation with little specificity regarding the identity of the differentiating neural cells.In contrast to the restricted expression of miR-124 targets non-neural transcripts [miR-124 expression with the transition from neural progenitor to differentiated neuron. The full range of miR-124 targets is unknown and one might predict that in addition to non-neural transcripts, targets may also include genes associated with the neural progenitor state. In support of this, predicted targets [miR-124 include many 'early' neural genes, such as zic2a, pou5f1, otx2 and slit2  show expression in both proliferating and differentiating cells of the larval brain  but expression is maintained only in amacrine cells of the inner nuclear layer . For example, at 5 dpf, miR-137 expression is restricted to domains of the pallium . We observe that both are expressed in cells associated with the visual system, including retinal amacrine cells (INL) and ganglion cells , includTogether these results show that miRNA expression is frequently region or nucleus specific and can vary in the levels of expression from one group of cells to another.miR-218a is exclusively expressed in cranial motor nuclei  and spinal motor neurons . miR-182 and miR-96 show almost identical expression patterns to miR-183 , although miR-200a lacks the CNS and cranial ganglia expression sites common to miR-183, miR-182 and miR-96 .All three miRNAs are also expressed in neurons of the cranial ganglia . The expression of these miRNAs in peripheral sensory neural cells overlaps with miR-375 is exclusively expressed in the pituitary and a few scattered weakly labeled hypothalamic cells . Although expression of mouse miR-375 in the pituitary has not been ascertained, it is expressed in pancreatic beta cells [miR-375 has a similar function in both tissues.Finally, within the brain, ta cells  and appeIn conclusion, in the larval brain, miRNAs show widely divergent profiles of expression, varying from wide to very restricted expression either in particular brain subdivisions/areas or cell types.miRNAs preferentially target mRNAs with spatially or temporally complementary expression , raisingmiR-92b is expressed in periventricular cells and proliferative zones in the adult brain as it is in the larval brain. For instance, in both larva and adult, periventricular cells in the medial dorsal subpallium express miR-92b . Similarly showing conserved expression over time, miR-124 expression is excluded from periventricular cells and is detected in most differentiated cells throughout the adult brain as in the larval brain . For example, they are expressed in tectal cells  and dorsal periventricular hypothalamus (Hd) in Figure miR-181a and miR-181b but there is an additional copy of miR-181a on chromosome 22 and of miR-181b on chromosome 20 [osome 20 ,44.miR-222 and miR-221 share largely similar expression in the adult hypothalamus  but only miR-222 is expressed in the ventral intermediate hypothalamus at the larval stage . It is not obvious why there should be differences in miR-222 and miR-221 expression as they are present in the same cluster and one would predict that they are co-transcribed. There is a precedent for post-transcriptional regulation of miRNA expression [In a similar manner, other miRNAs belonging to a particular cluster seem to largely share expression patterns but also have subtle differences in transcript localization. For instance, pression ,49, althmiR-219 is restricted to relatively few cells. For example, larval ventral thalamus  and ventromedial (VM) in Figure miR-219 is expressed in cells, possibly glia, associated with major tracts such as the lateral olfactory tract, post-optic commissure/optic chiasma and tract . Loss of expression sites is consistent with roles for miRNAs in the regulation of genes that are only transcribed during restricted developmental phases.Patterns of miRNA expression can change dramatically between larval and adult brain. For instance, in contrast to strong and widespread expression in some domains of the larval brain, adult expression of de novo expression in adults that may reflect expression in late differentiating cell types not present or not fully differentiated in larval stages. For instance, miR-34 shows conserved expression in the larval and adult hindbrain  and presumptive octavolateral area (OA) in Figure miR-222 expression is also largely conserved between larvae and adults . Similar to miR-34, miR-218a expression expands rostrally in the adult brain. In addition to conserved expression in motor nuclei  show expression in groups of dorsal lateral habenular cells of the adult brain. Thus, the adult expression of these three miRNAs may correspond to habenular neurons that have not yet formed or fully matured at 5 dpf. This is in contrast to miR-153a and miR-138, which are expressed in the habenulae in both larvae and adults  while miR-92b and miR-34b are expressed in ventricular cells adjacent to the mature habenular nuclei . This analysis of habenular miRNA profiles illustrates that specific brain nuclei can express combinations of different miRNAs, some throughout life and some associated with differentiation. One of best understood roles for any animal miRNAs is in the determination of left/right asymmetric fates for neurons in Caenorhabditis elegans [In addition to 4 Figure , several7 Figure  and miR- elegans  and it w elegans ,52.Our survey of miRNA expression has revealed enormous diversity in the range of expression profiles. Although one cannot make specific conclusions regarding function based upon expression pattern alone, expression profiles do allow one to make generalized predictions regarding miRNA roles. This is useful as despite their prevalence, virtually nothing is known regarding the function of most miRNAs that are expressed in the brain.miR-430 targets a large number of maternally deposited transcripts at the onset of zygotic transcription [A common feature of miRNA function is one of mutually exclusive expression of miRNAs and their target mRNAs ,12. In scription  and manycription ).miR-9 is broadly expressed in both proliferative and differentiated cells in many of its expression domains. However, there are sharp transitions between domains of expression and non-expression, with some structures, such as the habenular nuclei, devoid of expression. The absence of temporally restricted expression and presence of spatially restricted expression is consistent with the main targets of this miRNA being restricted to those regions lacking miR-9 expression.Many of the expression profiles we describe are consistent with the miRNAs being expressed at high levels at times or places complementary to their targets. For instance, miR-92b is downregulated in most mature neurons whereas, conversely, miR-124 is absent from proliferative cells and widely expressed in differentiated neurons. This profile is one of the best conserved miRNA patterns, as a similar restriction to mature neurons is seen for mouse and fly miR-124 [Many miRNAs show expression associated with a transition in the differentiation state of the expressing cells. For instance,  miR-124 . These pmiR-124, appear to show constitutive expression throughout the lifetime of the expressing neuron. One possible explanation for this is that such miRNAs continue to regulate some mRNAs that are expressed and required in the mature neurons and we consider this possibility in the next section.miRNAs that are induced when cells transition from one state to another are likely to target mRNAs that are expressed during the initial state and not required during the second state. One might predict this role to be required only for the period following the transition during which there is perdurance of mRNAs that were transcribed during the previous state. Major changes in the transcriptome that occur when cells transition between states are thought to be brought about by transcriptional mechanisms independent of miRNA function and so it seems likely that many target mRNAs will not be actively transcribed once a transition has occurred . HoweveAn additional possibility is that some of the miRNAs initially associated with transition to differentiation constantly survey the transcriptome for fluctuations in mRNA levels of genes that should not be actively transcribed in mature neurons. There are well-described mechanisms for repressing and silencing loci , but thmiR-124, miR-181, miR-222 and others in mature neurons is consistent with an initial role in the clearance of mRNAs from the neuronal precursor stage but later they may fulfill a different role in the surveillance of fluctuations in aberrant transcription from notionally 'silenced' loci. As the silencing of loci is generally efficient, one might conclude that for most of the time in most mature neurons, some miRNAs are doing very little indeed, consistent with the idea that miRNAs confer robustness to programs of gene expression [The constitutive expression of miRNAs such as pression ,12.We also find that neurons express multiple miRNAs and it is very likely that most miRNAs have many, perhaps as many as a few thousand, mRNA targets ,2,6,56. The mutual exclusion model of miRNA function may underlie most functions of animal miRNAs, but in some instances, miRNAs are expressed in the same cells at the same time as their target genes. Such a scenario has been termed an 'incoherent feedforward loop' as the induction of both miRNA and target mRNA would seem to be at crossed-purposes . In suchmiR-218a is predominantly expressed in most motor neurons. It is hard to reconcile this observation with any conclusion other than the miRNA is targeting genes that normally function within the expressing cells. There is a great deal of regulation of protein activity levels within cells but there are not many examples of miRNAs involved in such regulation. However, miR-134 is localized to dendrites and appears to modulate the levels of activity of a kinase that influences dendrite morphogenesis [A few of the miRNAs we examined show expression restricted to very few cells and usually expressing cells share some characteristics of function and/or form. For instance, ogenesis .miR-134 transcripts are localized to the dendritic compartments in which they function and miRNA activity is spatially/temporally regulated by extracellular signals [Given that some mRNAs are localized and translated locally within neurons , an  signals .We analyzed the expression of 38 conserved vertebrate miRNAs in zebrafish neural tissue from development to adulthood. This is the first study describing in detail miRNA brain expression and it shows several classes of expression profiles. It reveals a wide diversity in miRNA expression, ranging from single cell types to the majority of CNS cells and from transient to constitutive expression. Our survey of miRNA expression patterns suggests several modes of action within neural cells. The first is to function in neural stem cells/progenitors and the second is to facilitate the clearance of target mRNAs at spatial or temporal transitions. Subsequent to developmental transitions, miRNAs may constitutively survey target mRNAs to counteract stochastic fluctuations in aberrant transcription. Finally, cell-type specific miRNAs may modulate the spatial and/or temporal regulation of target mRNA translation within mature neural cells.In situ hybridization on zebrafish larvae was performed as described previously [in situ hybridization protocol for adult CNS tissue with some minor modifications. Adult brains were dissected and fixed overnight at 4°C in 4% paraformaldehyde. Proteinase K ) treatment was done twice for 30 minutes at 37°C with continuous shaking. The acetylation step in the in situ hybridization was often omitted since it did not change or improve signal. Post antibody washes were performed for 6 times for 30 minutes each at room temperature then overnight at 4°C and 6 further 15-minute washes at room temperature. Alkaline phosphatase enzymatic reaction was detected using the NBT-BCIP substrate and embryos were subsequently dehydrated to benzyl benzoate/benzyl alcohol. Preparations were subsequently sectioned (see below). miRNA probe sequences and annealing temperatures can be found in Table J in Additional data file 28.eviously ,36. We uin situ hybridization as described above and/or immuno-cytochemistry using rabbit anti-green fluorescent protein (GFP) antibody  at 1:1,000 and goat anti-rabbit Alexa 488 conjugated  at 1:1,000. When both methods were combined, alkaline phosphatase enzymatic reaction was performed using Fast Red  as substrate. Light microscopy images were acquired using a Nikon camera attached to a Leica or Nikon upright microscope and analyzed using Photoshop and Illustrator (Adobe) software. Confocal analysis was performed using a Leica TCS SP Confocal microscope using 25×/40× oil immersion objectives and a series of images were acquired at 0.8-2.5 μm intervals. Selected depths were projected by a combination of maximum intensity and opacity.Three days old Tg(huc:gfp)  or Tg) at 4°C. Next, specimens were transferred to a mold and embedded overnight in Technovit 8100 embedding medium (Kulzer) deprived of air at 4°C. Sections of 7 μm thickness were cut with a microtome ), stretched on water and mounted on glass slides. Sections were dried overnight. Counterstaining was done with 0.05% neutral red for 12 s, followed by extensive washing with water. Sections were preserved with Pertex and mounted under a coverslip. Penetration of all reagents even in adult brains was generally very good, although in some preparations deep tissue in the brain did not label well and readers should be cautious about interpretation regarding absence of expression in such cases.Whole larvae and brains stained by whole-mount Annotation of brain areas was made according to the atlases available for the embryonic and adult zebrafish ,64 and aNeuroanatomical documentation of the zebrafish brain, particularly at larval stages, is far from complete and so our annotation is not always definitive. However, for the sake of conciseness and readability, we generally do not use qualifiers such as 'presumptive'. Some of the miRNA expression patterns helped us annotate larval fish brain nuclei. The names of these nuclei are followed by a question mark and the cells belonging to them are delineated by dotted lines wherever possible.For predictions of possible mRNA targets of miRNAs, we used prediction programs and related resources available at ,40.CNS, central nervous system; dpf, days post-fertilization; GFP, green fluorescent protein; LNA locked nucleic acid miRNA, microRNAs; nt, nucleotide; UTR, untranslated region. A full list of neuroanatomical abbreviations is provided at the end of additional data file 26.MK carried out the neuroanatomical analysis of the data, immunohistochemistry experiments and drafted the manuscript. WK carried out the in situ hybridization and sectioning experiments and participated in the design of the study and drafting of the manuscript. EB participated in the in situ hybridization and sectioning experiments. FR revised critically the manuscript. SW participated in the organization of the data and drafted the manuscript. RP conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.miR-7 expression in the zebrafish brain. Additional data file miR-9 expression in the 3 dpf zebrafish brain. Additional data file miR-9 expression in the 5 dpf zebrafish brain. Additional data file miR-9 expression in the adult zebrafish brain. Additional data file miR-34 expression in the zebrafish brain. Additional data file miR-92b expression in the zebrafish brain. Additional data file miR-124 expression in the zebrafish brain. Additional data file miR-128 expression in the zebrafish brain. Additional data file miR-135c expression in the zebrafish brain. Additional data file miR-137 expression in the zebrafish brain. Additional data file miR-138 expression in the zebrafish brain. Additional data file miR-153a expression in the zebrafish brain. Additional data file miR-181a expression in the zebrafish brain. Additional data file miR-181b expression in the zebrafish brain. Additional data file miR-183 expression in the zebrafish brain. Additional data file miR-200a expression in the zebrafish brain. Additional data file miR-218a expression in the zebrafish brain. Additional data file miR-219 expression in the zebrafish brain. Additional data file miR-221 expression in the zebrafish brain. Additional data file miR-222 expression in the zebrafish brain. Additional data file miR-375 expression in the zebrafish brain. Additional data file let-7a expression in the zebrafish brain. Additional data file let-7b expression in the zebrafish brain. Additional data file let-7c expression in the zebrafish brain. Additional data file let-7a, miR-92b, miR-153a and miR-181a.The following additional data are available with the online version of this paper. Additional data file miR-7 expression in the zebrafish brain.Click here for filemiR-9 expression in the 3 days post-fertilization (dpf) zebrafish brain.Click here for filemiR-9 expression in the 5 days post-fertilization (dpf) zebrafish brain.Click here for filemiR-9 expression in the adult zebrafish brain.Click here for filemiR-34 expression in the zebrafish brain.Click here for filemiR-92b expression in the zebrafish brain.Click here for filemiR-124 expression in the zebrafish brain.Click here for filemiR-128 expression in the zebrafish brain.Click here for filemiR-135c expression in the zebrafish brain.Click here for filemiR-137 expression in the zebrafish brain.Click here for filemiR-138 expression in the zebrafish brain.Click here for filemiR-153a expression in the zebrafish brain.Click here for filemiR-181a expression in the zebrafish brain.Click here for filemiR-181b expression in the zebrafish brain.Click here for filemiR-183 expression in the zebrafish brain.Click here for filemiR-200a expression in the zebrafish brain.Click here for filemiR-218a expression in the zebrafish brain.Click here for filemiR-219 expression in the zebrafish brain.Click here for filemiR-221 expression in the zebrafish brain.Click here for filemiR-222 expression in the zebrafish brain.Click here for filemiR-375 expression in the zebrafish brain.Click here for filelet-7a expression in the zebrafish brain.Click here for filelet-7b expression in the zebrafish brain.Click here for filelet-7c expression in the zebrafish brain.Click here for fileExpression of other miRNAs in the zebrafish brain.Click here for fileLegends of the figures presented in Additional data files 1-25 and 29 and the list of neuroanatomical abbreviations.Click here for fileTables A-I providing detailed information about the expression of each miRNA in different structures of the zebrafish central and/or peripheral nervous system.Click here for fileTables J-L, listing the miRNAs analyzed, the family/cluster they belong to and the mismatch locked nucleic acid (LNA) probe sequences used to test specificity of corresponding fully matching LNA probes.Click here for filelet-7a, miR-92b, miR-153a and miR-181a.Mismatch test for Click here for file


Optic nerve head drusen (ONHD) are white calcareous deposits, seen either superficially on the optic nerve head or buried within it. Diagnosis of ONHD is made by one or more ways: clinical exam, autofluorescence, ultrasound of the optic nerve, CT scan and/or visual field examination. The present study describes features of ONHD based on another diagnostic modality, the spectral-domain OCT .This is a retrospective case series of 5 patients with bilateral ONHD with a best-corrected visual acuity of 20/20 and no other posterior segment pathology. All the patients underwent fundus photography, fundus autofluorescence, B-scan ultrasonography, Spectralis OCT and Humphrey 30-2 threshold visual fields.All 5 patients had surface ONHD which were autofluorescent and echodense on B-scan ultrasonography. Spectralis OCT findings in the corresponding areas include 'scattered spots with high reflectivity' casting a shadow underneath. The reflectivity can be distinctly differentiated from the blood vessels on the optic nerve. Two patients had an arcuate scotoma on the Humphrey visual fields. No correlation was found between the changes on Spectralis OCT with that of visual field.Spectralis OCT is another useful ancillary investigation in the diagnosis of ONHD and we describe the features in the present study. Optic Nerve Head Drusen (ONHD) are white calcareous deposits that are generally asymptomatic and are bilateral in 66-85% of cases . They arThe diagnosis of ONHD is based upon a strong index of suspicion and confirmed by B-scan ultrasound of the posterior segment, which shows echodense structure with acoustic shadowing. CT scan may be required in the rare case when ultrasound is inconclusive -3. Time-Spectral-domain Optical Coherence Tomography (SD-OCT) by its ability to acquire 40000 A-scans per second provides high resolution images of the Optic nerve head . In thisThis is a retrospective case series report of 5 patients with ONHD in whom changes seen on spectral domain OCT (SD-OCT) were documented. Approval for this study was obtained from the University of Florida Institutional Review Board-03 (Reference number: 2008-003). Five patients with bilateral ONHD with best corrected visual acuity 20/20 and no other posterior segment pathology were included in this series. All the patients underwent a detailed ocular examination, posterior segment digital fundus photography , fundus angiography to document autofluorescence  and B-scan ultrasonography of the posterior segment .Optic nerve head scans, using the Spectralis OCT  were done on all the patients by an experienced operator using the raster lines protocol. The area scanned was 4 mm by 4 mm at 50 micron intervals. Cross-sectional images, one per second, were obtained with a longitudinal resolution of 7 μm. Patient's fixation was monitored by asking the patient to look at an internal fixation or by guiding through an infrared monitoring camera.The median age of the patients was 51 years (range 17-53 years). The optic nerve head of all the patients showed glistening surface deposits characteristic of drusen Figure , 2A, 3A,SD-OCT by its ability to acquire 100 times more number of A-scans as compared to Stratus OCT provides high resolution images of the cross section of the optic nerve head, a close approximation to the histopathological section -9. In ouVisual field changes described in cases of ONHD are characteristically arcuate scotomas corresponding to the nerve fibre layer defects arising out of mechanical compression due to the drusen . In our In summary, SD-OCT is a useful ancillary investigation in the diagnosis of optic nerve head drusen and in documenting the changes seen in retinal nerve fibre layer due to the compressive effects of the drusen.ONHD: Optic Nerve Head Drusen; SD-OCT: Spectral-domain Optical Coherence Tomography.The authors declare that they have no competing interests.RK, SG, KVC were involved in the conception and design of the study; RKM, LS was involved in the acquisition of data; RKM, SG, KVC and VSB were involved in the analysis of the data and preparation of the manuscript.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/10/11/prepub


Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes. Xylella fastidiosa is a gram-negative gamma-proteobacterium known to cause several economically important diseases in cultivated crops and many other plant species. The strain 9a5c (Xf-CVC) was the first plant pathogen whose genome was completely sequenced Xylella strains reveal interesting biological and evolutionary aspects regarding genome structure and gene content. Previous studies demonstrated that 98% of the Xf-PD genes are shared with Xf-CVC, with an average amino acid identity (considering only the coding regions) of 95.7%, and the main differences are from bacteriophage-derived regions. These bacteriophage-derived regions are responsible for chromosomal rearrangements and deletions in X. fastidiosa strains, thus playing a decisive role on the genome evolution of this plant pathogen 7 particles/ml in coastal sea water and even higher in some other habitats, such as freshwater ponds Recently published work demonstrate that virus particles, including bacteriophages, appear to be strikingly abundant, with a typical estimated concentration of 10attP, and a short sequence of bacterial DNA, the bacterial attachment site attB. Phage integrases all fall into a category of enzymes known as site-specific recombinases attB sequences and are grouped into two major families, based on their mode of catalysis: the tyrosine and the serine recombinases To accomplish integration, temperate bacteriophages encode a phage integrase enzyme that mediates recombination between short sequences of phage DNA, the phage attachment site X. fastidiosa strains, Xf-CVC, Xf-PD, Xf-OLS, and Xf-ALS, were compared with regard to their prophage content and respective predicted integrase genes. A total of 56 predicted integrases were identified, and network analysis and phylogenetic reconstructions support the existence of five major lineages related to known bacteriophages that infect gamma and beta-proteobacteria. By Bidirectional Best Hit (BBH) analysis (against 402 bacteriophage genomes), the integrases were all found to be associated mainly to phages containing structural genes of Caudovirales viruses. In silico gene expression analysis of Xf-CVC prophage-like regions reveals these prophages are probably actively transcribed, and this finding is supported by the presence of putative phage-like particles in Xylella cells both in planta  and in vitroX. fastidiosa strains.In this work, the genomes of four Identification and definition of prophage-like elements is not trivial task, but an empirical approach that needs a lot of insight X. fastidiosa genomes were scanned for the presence of predicted integrases associated to clusters of genes related to phages. Regions encompassing more than 10,000 bases and at least 80% of phage-related genes in their constitution were defined as prophage-like regions; the smaller ones were defined as prophage remnants. This strategy enabled the identification of 46 chromosome fragments in X. fastidiosa genomes predicted to be descended from ancestral invading bacteriophages  composition, it is possible to infer the candidates to be a probable complete or a defective prophage, for each strain . The prodnaA gene), some of them positioned near the putative terminus of replication . This particular distribution is suggestive that these prophage elements may represent recent acquisition in the X. fastidiosa genome probably relating to the moment in the cell cycle that insertion occurs as previously demonstrated for other prokaryotic genomes (20).Most of the prophage-like regions (60%) are localized between the position 900 kb and 1,800 kb of the chromosome  and the two candidate molecules (Xf-ALS and Xf-OLS), starting from the putative origin of replication, reveals at least 16 chromosomal regions in the four genomes that are translocated and/or inverted. The Xf-ALS strain presents 16 disruptions in its candidate molecule compared to the other three strains, followed by Xf-CVC strain with 14, and 13 chromosomal breaks in Xf-OLS and Xf-PD strains, suggesting that Xf-ALS strain is the most divergent in terms of genome structure and this divergence is directly associated to phage insertions. The association of phage related regions with breaks in chromosomal colinearity has been previously described when comparing Xf-CVC and Xf-PD genomes  genomes . Taken tThis analysis enables us to trace a possible evolutive scenario for each group of prophages-like elements. This analysis enables to hypothesize the most ancient insertions relative to the most recent ones. Firstly we report the elements inserted in the same genome context in different strains, indicating preferred sites of insertion; that, the ones related to common ancestral events.There is only one prophage insertion shared by the four strains with mostly the same gene content and genome borders: xpd6, xop10, cvc-r4 and al-r2. These regions possess the same upstream genome border located near an epsP synthase, and the downstream border near a tonB-dependent receptor, except for the xop10 element, where the downstream gene is located close to a methyltransferase. The cvc-r4 and al-r2 remnant regions appear to be degenerate regions that originated from a xop10-like ancestor. Region xpd6 appears to be a degenerate form of xop10, mainly by the presence of a frameshift in the xpd6 integrase, suggesting that xop10 might be the closest to the common ancestor of this group. The xpd6 and xop10 regions share 76.1% nucleotide identity and carry 49% of putative non-essential phage ORFs, 45% of hypothetical ORFs and only 6% of essential phage ORFs. The gene content of these regions includes a copy of virulence-associated protein I and a hicA/hicB toxin-anti-toxin system. Neither of these regions contains structural phage genes. The xpd6, cvc-r4 and xap10 elements have a tRNA-GLY in their constitution, suggesting a mechanism of acquisition of this tRNA by transduction.Another site of insertion is shared by three different strains, and it involves the remnants pd-r1, ol-r1, and the xap2 element. They are inserted between a fumarate hydratase and glucose inhibited division protein, in the vicinity of a tRNA-CYS. These three regions are most probably defective prophage in the process of genome decay, and pd-r1 and ol-r1 appear to be degenerated versions of xap2.In these two cases, the analysis strongly suggests an evolutionary mechanism of negative pressure in order to delete or fully inactivate these regions of the chromosome, in accordance with to previous studies in others prophage regions Acyrthosiphon pisumSeven prophage-like elements, xfp1, xfp2, xpd1, xop3, xap1, xap5 and xap6 are involved in large genome rearrangements and share at least 80% of nucleotide identity. These appear to be complete phages. Gene order and orientation is highly conserved among all the seven elements, the integrases being followed by non-structural  and structural genes with both classes separated by an endolysin gene. It is interesting to note that the DNA-packaging and head genes resemble in organization and sequence the lambda-like phages, and the baseplate, tail, and tail fibers genes  resemble the P2-like phages. This architecture suggests a hybrid phage (as previously observed for xfp1 and xfp2 Listeria innocua (derived from the phage Sfi11), and the phage AaΦ23 from Actinobacillus actinomycetemcomitans, generally associated to human oral infections Another class of related prophage regions display 80% nucleotide identity but it is restricted to the structural genes  The endolysin gene separates the non-structural genes from the structural ones, resembling in organization and sequence the prophage 4 that infect The fact that these elements are inserted at different genomic positions in the four strains, and that they share extensive sequence similarity suggest recent and independent acquisitions. This similarity also implies that these bacteriophages are frequent entities in the environment including the riparian vegetation, insect vector or any of the infected plants.Escherichia coli, Vibrio cholerae and Staphylococcus aureus by Boyd et al The hybrid origin of the prophage regions is probably the result of illegitimate recombination in the process of the horizontal genetic exchange, as observed in Mycobacteriophages by Pedulla et al From a total of 1,728 prophage-like genes in the four strains, 1,388 (80.5%) belong to 290 different best bidirectional hit (BBH) clusters, while 339 (19.5%) are not present in any BBH cluster. The latter group represents strain-specific prophage-like genes. In this specific group, 66 (19.5%) are ORFs with putative functions related to essential phage genes, and 28 (8.25%) to non-essential phages genes , while the remaining 245 ORFs (72.25%) are hypothetical or conserved hypothetical genes, representing an abundant number of ORFs that can be related to genomic differentiation.X. fastidiosa share 55% identity with orthologs found in the filamentous phage phiLf of Xanthomonas campestris pv. vesicatoria and phage phiSMA9 of Stenotrophomonas maltophilia, and with less than 30% identity to orthologs of X. campestris pv. campestris and RSM1 phage of Ralstonia solanacearum and R. pickettii genome. These organisms are necrogenic plant pathogens, except Stenotrophomonas, a human pathogen also able to colonize diverse plants, especially those in the Brassicaceae family Stenotrophomonas strains only recently after infection by a filamentous phage, which probably came after certain changes in its adsorption protein from plant-pathogens The most interesting cases of prophage-like ORFs in BBH clusters, and potentially related to bacterial pathogenicity, are the putative phage-related PI protein (Zonular occludens toxin- like protein) present in xop7, xap10 and xpd5 ; and the virulence-associated protein E, present in xfp5, xfp6, xpd8, xop6, xap8. The products of these ORFs may be related to interactions between the plant and the bacteria. Phage PI protein (zot) is required for phage assembly A group of toxin and anti-toxin proteins in prophage-like regions (higA/higB and relE/relB) was also found. These proteins are very common in plasmids, where they increase effective stability Xylella strain.Furthermore, the group of specific ORFs (belonging to no BBH cluster) related to non-essential phage functions have some interesting components: (a) virulence-associated protein in xfp3 (VapB-like), exclusive for the Xf-CVC strain; (b) modification methylase NspV and restriction NspV enzymes in xpd8; (c) the restriction enzyme NgoMIV and modification methylase NgoMIV in xap4; and (d) virulence-associated protein I in xop10, . All these ORFs may be involved in interactions between plant and bacteria (a and d), or between bacterial and phage genomes (b and c). On the other hand, these ORFs do not have BBH pairs against the 402 phage genomes, suggesting they are not necessary for phage biology, but with exclusive roles in each Xylella strains from symptomatic and asymptomatic plants isolated in South American (using as reference the 9a5c strain genome)  . High coXylella strains.Region xfp4 is present in low copy number in both symptomatic and non-symptomatic strains  and in equal copy numbers in the other strains. The principal feature of this region is the presence of three systems of toxin and anti-toxin genes. Despite the xfp4 appearing to be a defective prophage, this element apparently is not active at least in strains 56a, 9.12c and CV21. This supports the idea that this element is stable, under lysogenic state, being subject of genome decay or stabilization in the host by a selective negative pressure. These findings indicate that: (1) enrichment in the number of copies of ORFs in prophage-like regions compared to the core genome ORFs in different strains, (2) the different prophage-like regions have diverse hybridization profiles, and (3) a lytic cycle activity with the formation of new phage particles. Thus, element xfp4 may play an important, but different, role in different Previous microarray analyses of Xf-CVC strain 9a5c described the expression profile under stress conditions, particularly under heat shock conditions  more frequently than those that are down-regulated (34%). Anti-repressor genes are over-expressed (5 out of 6 genes), followed by genes involved in phage replication and structural genes, as well some integrases , while repressor genes are under-expressed. This suggests that under stress conditions the prophage-like regions are activated and may trigger induction of the lytic cycle, which ultimately results in the formation of virus-like particles (VLPs). Both switches from 25°C to 40°C and from 29°C to 40°C result in clear induction of gene expression of prophage genes which could be indicative that changes in the temperature in the orchard along the growing season of the plants could result in bursts of phage induction within the plant.Along the line, but in a distinct relation, the combined analysis of the DNA-DNA hybridization array It is also worth noting that genes not directly related to the phage structure, as anti-toxin and virulence-associated genes, are also induced under these conditions. For example, over-expression of the virulence protein present inside the xfp3 element, a protein that occurs exclusively in this element and strain, indicates a role in heat-shock conditions. Accordingly, the high number of hypothetical phage-related genes that are also differentially expressed suggests some important, yet unknown, roles for this class of genes.An extensive analysis of the 250-bp upstream and downstream regions of each prophage-like ORF  reveals that the prophages and remnant regions have an increased number of single nucleotide polymorphism and insertions and deletions, as well a reduced percentage of overall nucleotide identity when compared to the core genome . The nucXanthomonas genus have at least 35 types of tailed phages, where 25 (72%), 9 (25%) and 1 (3%) and are from Siphoviridae, Myoviridae and Podoviridae families respectively.All prophage-like ORFs with putative functions related to structural phage genes  are grouped in BBH clusters. The phage family represented the most in these BBH pair groups of BBH is the Siphoviridae family (51%), followed for Myoviridae (32%) and Podoviridae (10%) families (7% of the BBH pairs are from unclassified Caudoviridae phages). The most important and studied phage within the Siphoviridae family is phage lambda, widely found in the chromosome of enterobacteria, where it plays diverse biological roles, most of them related to acquisition of virulence genes by the bacteria through LGT in planta were observed by transmission electron microscopy  full-length gene; (2) ORF with a frameshift (FS) or a stop codon in the frame (SCF); or (3) small fragments (less than 150 residues). There are 33 full-length integrases: ten in the Xf-ALS, nine in the Xf-OLS, and seven each in the Xf-CVC and Xf-PD strains. Most of these are associated with the largest prophage-like regions and the genomic islands (giCVC and giPD). There are ten integrases with FS/SCF three each in the Xf- PD and Xf-OLS, and two each in the Xf-CVC and Xf-ALS strains, all associated to prophage-like regions with length less than 20 kb. Finally, there are 13 integrase fragments, with five each in the Xf-CVC and Xf-ALS, two in the Xf-PD, and one in the Xf-OLS strains, most of them associated with phage remnants.Integrases are useful markers for identifying prophages and potential indicatives of LGT events in bacterial genomes se genes . The intThere are three main relationships between the integrases and these prophage elements: (1) all the  complete and the largest prophage elements carry full-length integrases, (2) truncated integrases (with SCF/FS) are present in probable defective and smaller prophages and (3) fragments of integrases are found mainly in phage-remnants, while non-remnant regions always bear another full-length integrase when a fragment is present. These results suggest the existence of a selective negative pressure associated with the integrase inactivation with further genome decay of the most ancient prophage elements.From the alignment with model tyrosine-recombinases, the conserved active residues R [212], K [225], H [308], R [311], H [333] and Y [342]  by pairwise sequence diversity, and are presented in a spring-embedded layout . This neXylella phage integrases share at least 60% of identity, xop5 shares a maximum of 45% of identity with the other ones. The main feature of xop5 prophage is the presence of a Tfp pilus assembly protein in the region related to putative structural phages genes. However, this element does not have any well-characterized structural phage gene. This suggests an ancient and defective prophage element, but exclusively in the Ann1 strain genome.To further evaluate such diversity, the integrase sequences were aligned against 186 integrases present inside phage particles  and agaiPseudomonas, Actinobacillus and Nitrosomonas (Burkholderia ssp. (a beta-proteobacterium) and Archaea species (Methanobacterium and Methanothermobacter).Integrase group A comprises the largest number of integrases, all of them related to probable complete prophages. This integrase group is related to prophages from few beta- and gamma-proteobacteria species, such as osomonas , revealiosomonas , being rEscherichia, Yersinia, Marinomonas and several Xanthomonas species). This is probably a restricted group of integrases, emerging late in the evolutionary scale , all from Siphoviridae viruses.Group D comprises eight integrases (including one from a remnant phage region), and it is related phylogenetically to a broader class of prophages infecting Gram-positive and Proteobacteria species . InteresXylella integrases over a broad range of tree branches. Four integrases  branched together, along with integrases of prophages infecting several species of beta- and gamma-proteobacteria  are related to a large group of integrases from prophages of several beta- and gamma-proteobacteria, including several  species . Moreove species . It was Xylella integrases are able to do generalized and specialized transduction, suggesting an import role in the genomic evolution of their hosts. Collectively, these results suggest a broad evolutionary history for each group of integrases identified in the Xylella genomes. They are related to several groups of prophages, infecting different groups of bacteria, including the closely related groups of beta- and gamma-proteobacteria, as well distantly related groups, such as Firmicutes and Actinobacteria. There is a large diversity of putative X. fastidiosa prophages related to Siphoviridae, Podoviridae or Myoviridae viruses. Moreover, the fact that X. fastidiosa prophage-like elements are related to several groups of phages, may be potentially misleading and be due to wrong models of viral taxonomical classification (currently based on features encoded by a minority of their genes), as long debated in the literature. For example, Lawrence and colleagues Most of the phages associated with Xylella 72% of these sites are associated with tRNAs  associated with prophage elements. These fragments are probably relics of an insertion with disruption of the ancestral tRNA without the reconstitution. With respect to tRNA-Gly, present inside prophage elements cvc-r4, xpd6, xop10 and xap3 and not directly involved as site of insertion, it may be a product of phage-mediated LGT to bacteria. This is supported by comparative analysis showing that at least 81 (19%) of a total of 430 phage genomes analyzed bear a tRNA in their genomes and have no direct viral function . They arXylella.It is interesting to note that the largest (in length) prophage-like regions, and probable complete and active prophages, are associated with tRNAs with higher numbers of copies in the chromosome . This indicates that they are preferred sites of insertion and markers for genome rearrangement of recent phage acquisitions. On another hand, none of the inherently unsuitable tRNAs described previously  X. fastidiosa genome organization and differentiation. The data presented in this work clearly demonstrate the role of the phages in the diversification and speciation of X. fastidiosa, both in a short time-scale promoting local and global rearrangements and activation/inactivation of host genes, and in a large evolutionary time-scale promoting speciation within the group by the acquisition of novel “cargo” components. This is highlighted by the diverse and common insertions for the diverse prophages elements, indicating a differential impact of common and diverse prophages in the Xylella genomes. Accordingly, it is still unknown if these prophages are functional or if they represent ancient insertions and are stalled in the bacterial chromosomes. Moreover, there is also evidence supporting that the phage activity perhaps is still in process: (1) higher levels of expression of phage-related genes, including those related to induction of the lytic cycle under stress conditions; and (2) direct observation of putative phage-like particles associated with Xylella cells in vitroin planta by transmission electron microscopy.This is the first extensive study showing that the prophage-like elements have a role or function in the process of X. fastidiosa genomes, the prophages are also capable of carrying some “cargo” genes with function not directly related to the phage propagation. In fact, the no assignment of a known phage function may be solely due to our current lack of knowledge concerning these genes. This is supported by the fact that most of these genes are strain specific, and thus they may be associated to specialization of the phage to the host, suggesting an important role in the generation of new variants of bacterial strains.Besides being responsible for abrupt large-scale alterations in the structure and organization of Xylella integrases are mostly related to lambda-like phage integrases. These phages are widely known as genetic mosaics, and some are able to perform generalized transduction that may confer drastic changes in their hosts. These findings suggest a combined model of evolution to Xylella integrases and their elements, in which site specific and illegitimate recombination take place, and the mosaic architecture of the prophage elements represent a creative process in order to generate genetic variation, driving forces to the evolution of this genera.On the other hand, Xylella strains genomes. Also, the data support that phage regions are transcriptionally active and may be producing virus-like particles; however, the association between expression and disease development remains to be demonstrated. Also, whether the enrichment of phage related ORFs is associated with gene duplication or virus-like particle formation is unknown. These observations open avenues to functional studies that may result in alternative strategies of disease control.Taken together, these results helped to determine the role and diversity of each prophage-like region, disclosing the mechanism and integration sites of the integrases associated to these regions and their influence in the differentiation of Potential ORFs with gene products assigned as integrases were identified by keyword and protein domain searches with BLAST program −5.Two-dimensional distance-constrained, spring-embedded and cluster-based phage-integrase network layouts were constructed with InterView program All previously described prophages in the Xf-CVC and Xf-PD genomes Functional annotation of ORFs within potential phage regions was carried out by using the SABIA package X. fastidiosa prophage-like elements and 402 phage genomes deposited in GenBank (http://www.ncbi.nlm.nih.gov/genomes/static/phg.html)). All the comparative analyses were based on the Bidirectional Best Hit (BBH) methodology −5. Comparisons between the prophage-like full-length elements were carried out with the M-GCAT program http://www.perl.com), by the methodology proposed by Souza R.C. (unpublished data).SABIA Comparative software http://www.php.net) were written in order to generate the prophage gene maps . Further information and all supplementary material are available on the project website: http://gracilaria.ib.usp.br/integraseDB.Scripts in PERL and PHP (http://www.ncbi.nlm.nih.gov/geo) site http://www.r-project.org]). Differentially expressed transcripts or differentially gene contents were identified by using the Significance Analysis of Microarray (SAM) method http://blasto.iq.usp.br/~tkoide/Xylella/Heat_shock).Meta-analyses of independent microarray datasets were performed in order to study the gene expression pattern of prophage-like elements in CVC strain in different heat shock conditions. Microarray data were extracted from series GSE3044, GSE4161, GSE4960, GSE6619 and GSE8493 Figure S1Xylella phage integrases primary sequence (groups A to E). Alignment done with CLUSTALX 2.0 program and manually adjusted. Red boxes, the conserved catalytic residues. Only part of each alignment, containing the conserved residues, is shown.Multiple alignment of (0.15 MB DOC)Click here for additional data file.Figure S2Distribution of tRNAs in 81 phage genomes (out of 430 available in the NCBI database) (detection by tRNAscan-SE). Frequency is given in relation to the number total of tRNAs identified in all genomes.(0.03 MB DOC)Click here for additional data file.


The Stabilin receptors are a two-member family in the type H class of scavenger receptors. These dynamic receptors bind and internalize multiple ligands from the cell surface for the purpose of clearing extracellular material including some synthetic drugs and for sensing the external environment of the cell. Stabilin-1 was the first receptor to be cloned, though the biological activity of Hyaluronic Acid Receptor for Endocytosis (HARE)/Stabilin-2 was observed about 10 years prior to the cloning of Stabilin-1. Stabilin-1 has a more diverse expression profile among the tissues than HARE/Stabilin-2. This review will focus on HARE/Stabilin-2 and its interactions with hyaluronan, heparin, and phosphorothioate antisense oligonucleotides and what is known about how this receptor participates in signaling upon ligand binding. The discovery of the cloned Stabilin receptors began with Stabilin-1 in the early 1990s. A monoclonal antibody raised against whole human spleen homogenate produced a monoclonal antibody (MS-1) against an antigen that was initially identified as a large protein that is preferentially expressed in the sinusoidal endothelia of spleen, lymph node, liver, and adrenal cortex  in cultuThe location of expression determines functionality, especially with receptors that bind to multiple ligands. One of the first studies for rat 175HARE expression was performed by Zhou et al. in which crude tissue extracts were produced in Nonidet NP-40 from spleen, lung, small intestine, large intestine, muscle, kidney, heart, brain, and liver. The protein was immunopurified by a monoclonal antibody bound to sepharose beads against rat Stabilin-2 and then separated on a 5% acrylamide gel for SDS-PAGE and probed with mAb-235 against rat 175HARE and radiolabeled HA. These results indicated that Stabilin-2 was only expressed in liver and spleen tissues and the ratio of the isoforms varied between these two tissues .http://gtexportal.org/home are also helpful in determining Stabilin expression throughout many tissues, but results should be interpreted cautiously as transcriptomic data is fully correlative for protein expression [One year after the initial publication, Politz et al. used Northern blots to confirm the expression of both human Stabilin-1 and Stabilin-2. This expanded the known tissues for these two receptors where Stabilin-2 expression was strong and verified in liver, spleen, lymph node with much lighter expression in heart, placenta, skeletal muscle, pancreas, thymus, and bone marrow . Stabilipression ,20.N-acetylglucosamine and these disaccharides are linked by β1-4 glycosidic bonds. HA is a necessary extracellular matrix molecule essential for a wide variety of functions including joint health and lubrication, ocular integrity, skin hydration, fertility, cancer metastasis, and many more physiological functions [Hyaluronan (HA) is a very long polymer containing disaccharides composed of glucuronic acid β1-3 linked to unctions ,23,24,25unctions . It is tunctions . Tissue unctions . Furtherunctions . Both StTo investigate the HA–Stabilin-2 interaction more closely and in detail, we expressed the human 190HARE  in Flp-In HEK 293 cells . Using aThe X-Link domain of Stabilin-2 has the highest homology to another well-known hyalectin, tumor necrosis factor inducible gene 6 protein (TSG-6), a protein whose expression is induced by inflammatory mediators, growth factors, ovulation inducers, and matrix remodeling ,35,36. IN-acetylglucoaminidase, and hyaluronidases for polymer degradation. The HA polymer is degraded into monosaccharides that are transferred to the cytosol where glucuronic acid is converted by isomerases for metabolism. The N-acetylglucosamine is also transferred to the cytoplasm and phosphorylated then deacetylated for the eventual formation of acetate, lactate, and fructose [Data derived from cell models suggests that the degradation of internalized HA does occur, though this is highly dependent on the cell type. For example, SK-Hep-1 cells are of endothelial origin , but arefructose ,43,44.N-acetylglucosamine and glucuronic (epimer: iduronic) acid. These two sugars may be modified in the Golgi of the cell to give rise to up to 32 different disaccharide combinations resulting in more than 100 trillion unique heparin polymers [Heparin is a complex polysaccharide made up of two base sugars, polymers . The heppolymers . Heparinpolymers . Heparinpolymers . Three fpolymers . Regardless of the route of administration, heparin clearance from circulation is dependent on the liver and kidneys. Longer polymers tend to be taken up by the liver for degradation and then undergo filtration by the kidney. It was discovered that the SECs of the liver are the primary site for heparin clearance from blood ,51. The O sulfate increases binding affinity at least fivefold [O sulfate is for overall metabolism and uptake in the liver, we subcutaneously injected mice with physiological doses of dodecamers (12mers) with or without a single 3-O sulfation residue . The 3-O sulfate routed the polymers through the liver for endocytosis and degradation and also increased the half-life of the 12mer in blood. Without the 3-O sulfation, the polymer had a far higher chance of getting immediately filtered by the kidneys [O modification along the polymer affected affinity to Stabilin-2 and liver, increased the half-life in blood, and, possibly, allowed interaction of heparin with other blood components. It is clear that the Stabilin-2 receptor directly affects the half-life of systemic UFH and LMWH. As previously mentioned, heparin is a very heterogeneous molecule with respect to both size and modification. Currently, therapeutic heparin is a natural product that comes from swine gut mucosa from which it is purified and refined ,55. The fivefold . To dete kidneys . From thThe precise location of where heparin binds with the Stabilin-2 receptor is not known and the primary sequence of the amino acids does not reveal an obvious cluster of positively charged amino acids that are common for many heparin binding proteins. Since both Stabilin-2 and 190HARE bind with heparin with the same affinity , the putHeparin does not interfere with HA binding on Stabilin-2. In the TSG-6 model, the X-Link domain may bind either HA or heparin, but not both together at two different binding sites within the module . In contN-acetylgalactosamine for targeting the liver parenchyma [Antisense oligonucleotides are chemically synthesized oligonucleotides that bind RNA through Watson–Crick base pairing and mediate its destruction via endogenous enzymes such as Dicer and RNase H ,69. Thesrenchyma  and the renchyma . Other drenchyma .Only a few receptors are known to internalize phosphorothioate-based (PS) ASOs and they are the asialoglyoprotein receptor (ASGPR) , scavengsecreted ecto-domain of the 190HARE (s190HARE) in an enzyme-linked immunosorbent assay (ELISA)-like assay at 140 nM. The results show that as the number of flanking residues decrease in number, so does the competition with the 20mer ASO. Using a fluorescence polarization assay, we determined that a full PS oligo has the highest affinity for s190HARE which decreases as PO residues are substituted in. Modifications of the 2′ ribose has very little effect on Stabilin binding and the nucleotide sequence has no effect on binding. Single-stranded oligos have a much higher avidity for the Stabilins than double-stranded oligos. It is thought that the double-stranded oligo is more rigid and interactions between the oligo and the Stabilins need to be flexible to accommodate the interactions of the anionic sulfur atom with the protein surface [Since ASOs may be synthesized with a wide variety of chemistries, we asked which chemistries are the best ligands for the Stabilins. In Miller et al., we used an iodinated single-stranded 5-10-5 MOE fully PS gapmer as the probe and competed it with a 200-fold concentration of a variety of ASOs of different lengths and chemistries . The non surface ,78. Know-/- mice accumulated a significantly lower amount of ASOs in their liver and spleen. What was surprising in this work was that efficacy of ASO-mediated knockdown in the liver of WT and Stab2-/- mice was similar, leading to the idea that initial uptake is front-loading the nonproductive pathway and that low amounts of ASOs are very effective at mRNA suppression. ASO accumulation was primarily in the SECs, though the gene of interest targeted by the ASO was in the hepatocytes. More work needs to be done on how the architecture of the liver with differential scavenger receptor expression affects the distribution and efficacy of ASOs.An independent genome-wide association study using a hybrid mouse diversity panel confirmed that Stabilin-2 and vesicle-associated membrane protein 3 (Vamp3) are closely associated with hepatic ASO deposition in more than 100 genetically unique strains of mice . Throughmalat-1 in 190HARE cells [The Stabilin receptors are only one piece of the endocytosis machinery involved with PS-ASO uptake. Since there is a weak correlation between uptake and efficacy in the GWAS study, it is hypothesized that there are several ASO endocytosis mechanisms and each of these pathways have varying levels of ASO efficacy. Using a screen for several common markers for early and late endocytosis-related proteins, we found that Rab5C, and not Rab5A or Rab5B, is essential for productive uptake of an ASO targeting RE cells . “ProducRE cells . Even th2497, Ser2537, Tyr2519, and Tyr2531) with the NetPhos 2.0 program.Although the ecto-domains of Stabilin-1 and Stabilin-2 exhibit high homology, the cytoplasmic domains (consisting of 4% of the receptor) are very diverse and show no homology. To date, few signaling studies have been performed for Stabilin-1 , therefo2519 the most severely affected. Double- and triple-deletion mutants were made to zero in on the significance of the redundancy of these motifs and a triple mutant lacking M1,M2, and M4 while retaining Tyr2519 reduced endocytosis by 42%, while the same triple mutant that was lacking Tyr2519 had a reduction of HA uptake of 95% when compared to WT [2519 to alanine had only a 5% reduction in endocytosis of HA, suggesting that efficient ligand uptake, receptor routing through the cell, ligand delivery to lysosomes, and recycling of the receptor to the membrane surface is a coordinated effort utilized by all four motifs of 190HARE [Endocytic receptors often contain one or more specific domains that facilitate entry of the receptor in the forming endosome. The 190HARE cytoplasmic domain contains five endocytic domains with two that significantly overlap, technically forming a single motif adjacent to the transmembrane domain  and induced activation of both NF-kB and ERK1/2 is the NPLY motif in which the Tyron of M3 . With thon of M3 , this reon of M3 ,89. Thison of M3 . In addion of M3 . The mecThe Stabilins are an enigmatic receptor family in which their entire coordinated physiological functions are still not well understood. How does a receptor bind multiple ligands and know how to propagate signal transduction based on the ligand? What is the relationship of the isoforms of Stabilin-2 to each other and with Stabilin-1 when expressed within the same cell? Though there have been multiple splice variants of Stabilin-2 discovered on the transcript level , are the