Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as  Escherichia coli, Salmonella spp., and Campylobacter spp. are persistent and widespread problems in food production and preparation [E. coli outbreaks and are considered one of the top commodities for food-borne disease outbreaks [Food-borne pathogens including paration –2, and tparation . Leafy vutbreaks . Flies mutbreaks –5. Howevutbreaks , but it Musca domestica L.), are closely associated with animal waste, from which they can acquire a variety of potentially pathogenic protozoa, viruses, and bacteria [E. coli from livestock to children [Filth flies, including the house fly  were from an established UC Riverside colony maintained on butternut squash fruit at 25 ± 2°C, 40% RH and 14L:10D photoperiod in an insectary room. Pesticide-free butternut squash grown in the Agricultural Operations fields at UC Riverside were harvested, thoroughly washed with a dilute solution of micro90 detergent, rinsed with water, dried for 1–2 days, and stored at 15.5°C until use (up to 1 year). Pea aphids (Acyrthosiphon pisum Harris) were established at UC Riverside in 2009 from a colony maintained at Oklahoma State University. Pea aphids were reared in a greenhouse on faba bean plants (Vicia faba L.) and maintained under ambient daylight conditions.House flies were collected by sweep net from a southern California dairy facility in 2010 with the owner’s permission and subsequently maintained as a colony in an insectary at constant conditions of 25 ± 2°C, 40% relative humidity (RH), and 12L:12D photoperiod. Immature flies were fed a standard fly rearing medium , and aduBioassays were conducted in a room maintained at 25 ± 2°C, 40% RH, and 14L:10D photoperiod in the UC Riverside Insectary building. The ventilation system in the room provided a slight negative pressure with air pulled through a centrally positioned ceiling vent creating airflow throughout the room. Prior to initiating bioassays, the entire room was scrubbed with unscented soap and water, and floor drains were sealed to reduce outside odors. Eight screen-cages  were set up on wire shelving with a 57-watt incandescent bulb within an 20.3-cm aluminum reflector housing placed 25 cm above each cage to provide light that was evenly distributed across each cage. Each cage contained two 2-L glass beakers to hold test materials. Beakers were washed with micro90 detergent and rinsed thoroughly with deionized water to remove odors prior to use in bioassays. For each bioassay period, treatment and control materials were randomly assigned to a beaker position (left or right side of the cage) in cage #1, and the position of the treatment and control material was then alternated in cages #2–8 to minimize within-cage position effects. The beakers were placed approximately 10 cm apart from each other and covered with mesh netting held in place with a rubber band to prohibit fly access to material held within the beaker. White sticky cards  were folded into a tent shape, with the sticky surface on the inside, and fixed above each beaker with a metal wire .Flies (3–5 d-old) were removed by aspirator from colony cages, sorted on a chill table  into 8 groups of 50 female flies , and each group was placed into a release chamber  covered by a plastic lid with many small holes for air exchange and a 2.5 cm diameter hole blocked with a cork that could be easily removed allowing flies to exit. The release chamber contained a 30 mL plastic cup  with water-soaked paper towel to provide moisture for the flies. Flies were starved in the bioassay room for 40–44 hr before use. The release chamber was used to slow the release of flies into the bioassay cage because early trials showed that a rapid release of flies into the assay cage resulted in substantial undirected flight and similar capture of flies on both treatment and control sticky cards within the first few minutes of the assay start time. An assay period thus began by placing a release chamber positioned equidistant from the treatment and control beakers into each of the 8 assay cages, followed by removal of the cork to allow starved flies to individually emerge from the release chambers during the 24 hr bioassay period, after which the number of flies captured on each sticky card was recorded.Prior to use in an assay, clean butternut squash fruit were introduced into the citrus mealybug colony for at least 4 wk resulting in infestation of squash fruit by large numbers of nymph and adult mealybugs. This exposure period ensured sufficient production and deposition of mealybug honeydew onto the squash. Faba bean plants 30–45 cm tall were infested with ~ 50 pea aphids of different life stages and then held in the colony room for an additional exposure period of 2–3 wk to allow for sufficient production and deposition of aphid honeydew onto leaf surfaces. Uninfested faba bean plants were grown in a separate colony room under similar conditions and for the same 2–3 wk period. Following the aphid exposure period, both infested and uninfested faba bean plants were cut near the base of the stem for placement into beakers within the bioassay cages.The following bioassay comparisons were made using colony insects: (1) Infested squash were compared to an empty beaker during 4 separate bioassay periods to evaluate variation in fly response by cage and by assay period; (2) Mealybug-infested squash were compared to uninfested squash during two bioassay periods, with both infested and uninfested squash incubated in the citrus mealybug colony room for the same 4 wk exposure period; (3) Uninfested squash were compared to empty beakers to evaluate fly attraction to the squash alone; (4) Uninfested squash punctured 30× with a needle to simulate insect feeding damage and then incubated in the mealybug colony room for 2 wks until signs of decay were obvious were compared to intact, uninfested squash to evaluate whether house flies were attracted to damaged and decaying fruit alone; 5) Pea aphid-infested faba bean plants were compared to uninfested faba bean plants . Four caCitrus sinensis Osbeck) and marsh grapefruit (Citrus paradisi Macfadyen) leaves infested with whiteflies (Family: Aleyrodidae) were collected from a single site in Riverside, CA. These two types of citrus plants were maintained as separate samples for fungal isolation but were combined together in equal amounts by mass for house fly attraction bioassays. Similarly, cottony cushion scale (Icerya purchasi Maskell) and citrus mealybugs on honey mandarin orange trees (Citrus reticulata Blanco) were collected from a single site at the UC Riverside Agricultural Operations citrus groves and maintained as separate samples for fungal isolation but were combined together for bioassay. Eucalyptus leaves  infested with lerp psyllids (Glycaspis brimblecombei Moore) were collected from a eucalyptus grove on the UC Riverside campus. Lerp psyllid honeydew was also collected on leaves from a red ironbark eucalyptus tree  in Riverside, CA. Data from one cage was not included in analyses due to flies accessing the beaker materials. A second collection of foliage from the same red ironbark eucalyptus with lerp psyllid honeydew received ~20 mL of water in the collection bag and was incubated at 60% relative humidity and 25°C in the insectary room for one day before evaluation in the bioassay. Crepe myrtle (Lagerstroemia sp.) cuttings infested with an unidentified aphid species were collected from a residential yard with the owner’s permission in Riverside, CA. Because plant material and honeydew dries out quickly after collection, attraction of house flies to each of these field-collected honeydews was evaluated within 48 hr of collecting material from the field. If available, uninfested material from the same plant or a nearby plant of the same species was used in control beakers, otherwise control beakers contained nothing (Plant stems and leaves with accumulated honeydew and without honeydew (control) were collected using latex or nitrile gloves to avoid contamination with human skin odors. All honeydew-contaminated and control samples were pruned from the plant with sterile shears, stored in oven bags  to maintain odors, and placed on ice for transport back to the laboratory for bioassays. Transport of plant material and honeydew was approved by the California Department of Food and Agriculture (permit #2776). Navel orange ( nothing .2O), where wash volume depended upon the volume of the field sample. Similar methods were used for host plant materials from citrus mealybug and pea aphid colonies, except that 8 week-old butternut squash infested with citrus mealybugs and clean butternut squash controls were gently washed with 30 mL of dH2O and the runoff was collected. Following their use in bioassays and for fungal collection, all plant material and honeydew residues were autoclaved before disposal.For each honeydew-contaminated and uncontaminated (control) sample taken from the field, a small portion (13–33 g) was removed and washed with 15–30 mL deionized water , where 100 μl of leaf wash was diluted in sterile water as needed (1:10–1:1000) to obtain isolated colonies on a plate of growth medium. Thus, 100 μl of the dilution was applied to 4 Petri dishes containing acidified potato dextrose agar  and incubated at 25°C for 5 d. The colony forming units (CFU) for each fungal species on the plates were counted, and the mean CFUs per plate were used to calculate the original concentration (CFU/100μl) in each wash sample by multiplying the colony counts by the dilution factor.Aureobasidium sp., Cladosporium sp., and Rhodotorula sp. Single-spore isolates were grown on 2% potato dextrose agar (PDA) for 7–10 d at 25±3°C for DNA extraction. Pure culture mycelia were scraped directly from the medium using a sterile scalpel and the total genomic DNA was extracted using a FastDNA Kit . The internal transcribed spacer (ITS) regions, including ITS1, ITS2, and the 5.8S rRNA gene of the ribosomal DNA (rDNA), were amplified using primers ITS1 and ITS4 [Fusarium sp. from the citrus mealybug colony was handled similarly as above except the DNA was extracted using an AllPrep DNA/RNA/miRNA Universal Kit  and purified using Diffinity RapidTip . The resulting amplicons were sequenced at the University of California, Riverside, Genomics Core sequencing facility using the ITS1 primer. The sequence was input into the NCBI’s BLAST sequence database and identified to 99% identity. Identified fungi were isolated and grown on individual APDA plates and stored at 4°C up to 6 mo or placed in 25% glycerol at -80°C for longer term storage.Fungi were identified to genus or species when possible by morphology and color. Polymerase chain reaction (PCR) and sequencing was used to verify species identification of and ITS4 –27. An UAureobasidium pullulans or Cladosporium cladosporioides and grown at 25°C under otherwise sterile conditions. Cladosporium cladosporioides was incubated for 2 wks while stirring constantly to achieve some degree of homogeneity throughout the media. Aureobasidium pullulans was incubated for 4 wks and allowed to grow as a layer on the surface of the media. About 30 mL aliquots of the C. cladosporioides culture or equal sized sections of the A. pullulans surface layer were placed into 8 glass petri dishes (9 cm diameter) that had been washed with micro90 detergent, rinsed with tap water, deionized water and acetone, then baked at 140°C to remove any residual odors. Petri dishes containing either the fungal cultures or sterile PDB (uninoculated and held under sterile conditions for the same incubation time) were placed into treatment or control glass beakers, respectively, for the bioassay as described above. House fly attraction to C. cladosporioides was evaluated during one bioassay period while attraction to A. pullulans was evaluated over two bioassay periods to confirm the significant attraction noted in the first bioassay period.In preparation for use in bioassays, isolated fungi from the honeydew-contaminated plant washes were plated onto APDA and shipped overnight to UC Riverside, then stored at 4°C until use. Autoclaved potato dextrose broth (PDB) media (250 mL) was inoculated with either For bioassay comparison #1  the number of flies captured on each sticky trap (treatment and control) during each bioassay period was log transformed to normalize the data and then analyzed for differences among assay cage, assay period, and treatment (and for interactions among these factors) using multi-factorial ANOVA with treatment means separated using Fisher’s least significant difference (LSD) test. All remaining bioassay comparisons were analyzed by calculating the difference between the number of flies captured on the sticky cards above the treatment and the control in each cage (diff = treatment - control) and using a one sample t-test to compare the observed difference values to an expected value of zero, if the treatment and control were not different in their attraction to flies. The Shapiro-Wilk test was used to verify normality of all data sets prior to analysis. All analyses were performed using SAS statistical software v9.4 .House flies were significantly more attracted to mealybug-infested squash relative to the empty beaker control  . There wHouse flies were significantly attracted to mealybug-infested squash relative to uninfested squash , but not to uninfested squash relative to an empty beaker  or to needle-damaged, decaying squash relative to uninfested/undamaged squash . Flies were also attracted to aphid-infested faba bean plants relative to uninfested faba bean plants .House flies were attracted to combined samples of whitefly-infested orange and grapefruit leaves  and to citrus mealybug and cottony cushion scale-infested mandarin orange leaves  . In contAureobasidium pullulans and C. cladosporioides were identified in higher concentrations from most of the field collected honeydew samples relative to the corresponding uninfested (control) plant material. Neither fungus was detected from the laboratory cultures of citrus mealybug on squash, where Fusarium solani was predominant  but not to odors from C. cladosporioides in PDB  relative to sterile broth , [Aphidoletes aphidimyza (Rondani) [Episyrphus balteatus (De Geer) [Aedes albopictus (Skuse) mosquitoes when tested under field conditions [Honeydew is rich in simple and complex carbohydrates , and is Pallas),  and the Rondani) . HoneydeDe Geer) . Thus, fnditions  despite nditions .A. pullulans and C. cladosporioides) were repeatedly isolated from field-collected plant materials contaminated with honeydew that were attractive to house flies in laboratory bioassays. Volatile odors from one of these fungi (A. pullulans) grown on PDB media were attractive to house flies in the absence of any honeydew or insect odors, suggesting that house fly attraction to honeydew may be due, at least in part, to odors produced by this fungus. These odors are likely byproducts from the metabolic breakdown of the honeydew by the fungi [Two fungi Click here for additional data file.S2 FigHoneydew was produced by lerp psyllids infesting unidentified eucalyptus trees in Bakersfield, CA. These trees were in the proximity of an animal agricultural facility. Image was taken by ACG.(JPG)Click here for additional data file.


Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3′LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5′LTR promoter.Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the  Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic deltaretrovirus infecting cattle that shares common biological and structural features with the human T-cell leukemia virus I and II (HTLV-I and II)  and is regulated by cellular transcription factors for which several binding sites have been identified in the LTR9101112131415161718Recent publications from two independent laboratories have identified, by bioinformatics analysis and deep sequencing, a cluster of 10 micro-RNAs (miRNAs) encoded by the BLV genome26in vivo recruitment of the RNAPII and RNAPIII complexes to the BLV genome. We demonstrated the recruitment of a bona fide RNA polymerase III at the BLV miRNA cluster through a canonical type 2 RNAPIII promoter similar to the one responsible for transfer RNA (tRNA) transcription. We also established a direct functional link between BLV miRNAs expression and RNAPIII transcriptional activity. Next, we showed that both tumor- and quiescent-related isoforms of RNAPIII were recruited to the miRNA cluster and that the miRNA cluster exhibited a profile of positive epigenetic marks (histone acetylation and DNA hypomethylation). Interestingly, in addition to the RNAPIII recruitment, we demonstrated an RNAPII recruitment at the junction between the 3′LTR and the host genome which was also associated with positive epigenetic marks typical of transcriptionally active promoters. Therefore, we tested the potential promoter activity of the LTR and demonstrated that this region was able to drive transcription in the antisense orientation in the nucleosomal context of episomally replicating constructs through a new RNAPII-dependent promoter. Importantly, ChIP-seq experiments performed in a BLV-infected cell line confirmed the high RNAPIII recruitment to the BLV miRNA cluster and the RNAPII occupancy just downstream of this region, suggesting a collision phenomenon between these two polymerase machineries and stalling of RNAPII.In the present report, we investigated the et al. have identified by bioinformatics analyses RNAPIII initiation cis-elements in the BLV miRNA clustercis-regulatory elements are functional in the context of 293T cells transformed with plasmids coding for BLV miRNA2627in vivo recruitment to the BLV provirus of RNAPIII by performing chromatin immunoprecipitation (ChIP) assays. For this purpose, we prepared chromatin from the latently BLV-infected L267 cell line and immunoprecipitated the largest RNAPIII subunit (RPC1) using a specific antibody. As controls, we performed ChIP assays with a purified IgG to measure the aspecific background and with an antibody directed against the largest RNAPII subunit (RPB1). Next, purified DNA was amplified by real-time quantitative PCR with oligonucleotide primers hybridizing to thirteen specific regions along the BLV genome and its host environment  isolated from a BLV-infected sheep that developed leukemia. We demonstrated recruitment of RPC1 to the BLV miRNA cluster . Forty-eight hours post-transfection, cells were harvested and lysed. Total RNA and protein extracts were used in RT-qPCR and western blot experiments to measure BLV miRNAs expression levels and RPC6 protein levels, respectively.To avoid off-target effects of the shRNAs, we used two different shRNAs recognizing distinct target sites in the RPC6 mRNA. We first attempted to deplete RPC6 by transfecting or transducing latently BLV-infected L267 and YR2 cell lines. Unfortunately, due to the low transfection or transduction efficiencies in these cell lines, we were unable to show a decrease either in RPC6 protein level or in BLV miRNA transcripts level. Therefore, we performed transduction experiments in the 293T cell line, using lentiviral particles containing shRNAs against RPC6 or a non-targeting shRNA as control. Transduced cells were then transfected with a plasmid expressing the entire miRNA cluster (pmiR). As an internal control for transfection efficiency, cells were cotransfected with a plasmid expressing the Firefly luciferase mRNA level  have been found to encode two isoforms of RPC7  defining two different RNAPIII complexesFollowing a duplication event in an ancestor of vertebrates, 2 genes . As shown in As positive controls, we amplified immunopurified DNA by real-time quantitative PCR with oligonucleotide primers hybridizing in three known RNAPIII-dependent genes  to identify putative CpG islands in the region of the miRNA cluster. The three prediction tools identified a CpG island containing the BLV miRNA cluster and spanning a region from nucleotide (nt) + 6342 to nt + 6833 (nt + 1 defined as the first nucleotide of the 5′LTR after retrotranscription). Then, we evaluated the DNA methylation status of each CpG dinucleotide of the BLV miRNA cluster in the context of integrated proviruses present in L267 and YR2 cells by treating genomic DNA with sodium bisulfite. Our results showed that, except for the third and the fourth CpG located upstream of the first BLV pre-miR, the miRNA cluster was not methylated in both the L267  of the BLV LTR sequence in the antisense orientation to identify specific cis-regulatory elements present in RNAPII-dependent promotersTo further characterize this newly identified promoter, we performed cis-regulatory motifs, we introduced individually or in combination point mutations known to abolish the motif’s function52In order to functionally characterize these hbz geneTaken together, our results demonstrate the presence of a transcriptionally active RNAPII promoter in the BLV 3′LTR, associated with positive epigenetic marks and responsible for antisense transcription, a situation reminiscent of the previously reported HTLV-I 3′LTR antisense promoter activity responsible for transcription of the viral As presented in As shown in 44Altogether, our data are further confirming RNAPII recruitment to the transcriptional promoter located at the end of the 3′LTR  and indienv gene and the pX region. Some evidence support the notion that the miRNA cluster is transcribed by the RNA polymerase III, although a direct functional link between RNAPIII transcriptional activity and BLV miRNAs expression has not been reported to date. Indeed, the algorithm used by Kincaid and coworkers has been designed to find RNAPIII-specific initiation cis-regulatory elements that have been functionally characterizedcis-regulatory elements2627in vivo, and at epigenetically and functionally characterizing its promoter. To this end, we performed chromatin immunoprecipitation assays in two BLV latently-infected ovine cell lines and in BLV-infected primary B cells.One of the major features of BLV infection is the absence of viremia due to the RNA polymerase II 5′LTR-driven transcriptional repression. Nevertheless, recent reports using bioinformatics analysisin vivo (bona fide RNAPIII at this genomic region in the context of a complete BLV provirus and confirming previous evidence2627BLV (L267LTaxSN cell line) or by PMA/ionomycin treatment .L267 is a clonal lymphoma-derived B-cell line established from a BLV-infected sheep (S267) injected with naked proviral DNA of an infectious BLV variant23The eukaryotic expression vectors pTax is a gift from Drs. Luc Willems and Richard KettmannTo construct the plasmid expressing the BLV miRNAs, genomic DNA from the L267 cell line was firstly extracted using the DNeasy Blood and Tissue kit (Qiagen) according to the protocol of the manufacturer. The miRNA cluster was then amplified by PCR using specific primers  and 125 6 cells (1 106 from the epigenetic modifications) and 5 μg of antibodies (see e of the immunoprecipitated DNA and the PerfeCTa SYBRgreen SuperMix (Quanta BioSciences). Relative quantification using standard curve method was performed for each primer pair and 96-well Optical Reaction plates were read in a StepOnePlus PCR instrument (Applied Biosystem). Fold enrichments were calculated as percentages of input values or as fold inductions relative to the values measured with IgG. Primer sequences used for quantification (see ChIP assays were performed as previously describeddies see . Quantittion see  were desChIP assays were performed as described above. Recovered DNA (~100 ng) was then used for library preparation using the TruSeq ChIP Sample Preparation protocol following Manufacter’s instructions (Illumina Technologies). Paired-end sequencing was then performed with the Illumina HiSeq 2000 instrument. More than 20 millions of single reads were obtained for RPC1 and RPB1 libraries and 10 millions of single reads for IgG library. The single-matched reads were mapped to an hybrid ovine genome (OAR v3.1) containing the BLV provirus sequence (GenBank: KT122858.1) at the L267 integration site (chr10:86299813) using bowtie2 software (-N 1 and -k 5 parameters). Finally, our results were visualized using IGV program. Reads spanning LTR-host and LTR-BLV boundaries were extracted from the alignment file using an in-house R script and ggplot2 was used to plot the ChIP-seq coverage.Genomic DNA was isolated from L267 and YR2 cell lines using the DNeasy Blood and Tissue kit (Qiagen) according to the protocol of the manufacturer. One microgram of genomic DNA was treated with bisulfite with EpiTect Bisulfite kit (Qiagen). The miRNA cluster region of BLV provirus was amplified by two successive PCR reactions (primer sequences are in the 6 cells was extracted using TRIzol (Life Technologies) followed by treatment with turbo DNAse (Life Technologies) according to the manufacturer’s protocol. Retrotranscription reactions were performed with PrimeScript RT-PCR Kit (TaKaRa) either using 200 ng of RNA and random hexamer oligonucleotides (for luciferase and GAPDH genes) or specific stemloop oligonucleotides for miRNA quantificationTotal RNA from 3 106 /10 cm dish), with the different shRNAs-containing plasmids (9 μg), the pVSV-G (2.25 μg) and the psPAX2 (6.75 μg) packaging vectors by the calcium phosphate transfection method according to the manufacturer’s protocol (Promega). 72 hours post-transfection, viral stocks were collected.VSV-G pseudotyped lentiviral particles were produced by transfection of 293T cells (5 105) were transduced in 6-well plates by 1 mL of undiluted viral stock supplemented by Polybrene at the final concentration of 8 μg/mL. 6 hours post-transduction, the medium was removed and replaced by 2 mL of complete DMEM.293T cells (4.5 105) were transfected with 3 μg of DNA (pmiR and pTK-luc) using FuGENE 6 reagent (Promega) according to the manufacturer’s protocol in 6-well plates. Raji cells (3 106) were transfected with 750 ng of DNA  as internal control) using the DEAE-dextran procedure as described previously293T cells . Data sets from RNA quantifications were analyzed using a two-tailed unpaired T test comparisons between samples and the cells transfected with the non-targeting shRNA. The threshold of statistical significance α was set at 0.05. Analyses were performed using Prism version 6.0 (GraphPad software).How to cite this article: Van Driessche, B. et al. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome. Sci. Rep.6, 31125; doi: 10.1038/srep31125 (2016).


Injury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neuro-degeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves. Axonal injury to the peripheral nervous system (PNS) triggers active translation of the localized transcripts that have been transported into axons from the cell body –4. IntraMicroRNAs (miRNAs) are a class of small regulatory non-coding RNAs (~22 nt long). They have been proposed to negatively regulate translation in the nervous system at the posttranscriptional level in a sequence-specific manner –20. ReceAlthough a recent study reported altered levels of miRNA in the proximal stumps of the sciatic nerve to the injury site at five post-injury time points , a big qWe find that axoplasmic miRNA levels in rat sciatic nerve are altered, upon injury. We identify a total of 141 rat miRNAs from the proximal stump of the sciatic nerve to the injury site. We further focus on bioinformatic analyses of seven most up-regulated and eight most down-regulated miRNAs with at least a mean fold change of 2 in regenerating nerve, compared to those in naïve nerve. By integrating predicted potential target mRNAs and 2,924 transcripts known to be localized to distal axons of DRG neurons , we suggAnimal procedures were approved (Approved protocol number NBR-2014-001) by the Institutional Animal Care and Use Committees (IACUC), and the experiments were conducted under the IACUC at Alfred I. DuPont Hospital for Children. For injury, 150–225 g male Sprague Dawley rats were deeply anesthetized using intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). Once completely anesthetized, a small incision (approximately 0.5 cm) was made over the sciatic nerve at the mid-thigh level. The sciatic nerves were surgically exposed by gentle separation of the quadriceps muscle and manually crushed using fine jewelers’ forceps twice for 15 seconds each. After closing the incisions with 4·0 Ethilon sutures, animals were returned to cages and observed for the degree of nerve crush injury as evidenced by contracted hind paw. From closely observing animals with peripheral nerve injury, they experienced little pain or discomfort after the nerve crush procedure. To minimize possible pain and distress, animals were visually checked daily for any signs of systemic or localized infection  as well as any evidence of pain or distress until sacrifice for the study. Any complications including gnawing at the hindlimb or incision site or open incision, excessive indurations of the incision, weight loss or lack of grooming were recognized as the evidence of pain or distress. Although we had only rarely seen any complications, animals that exhibited these features were euthanized and carcasses disposed by the regulations.in vivo, animals were euthanized at 1, 4, 7, and 14 days post-injury (PDI) by asphyxiation with CO2 using compressed sources of gas, and sciatic nerves ipsilateral (injured) and contralateral (uninjured) to crush injury were collected and processed as previously described , and non-neuronal cell [glial fibrillary acidic protein (GFAP) and Receptor tyrosine-protein kinase ErbB family-3 (ErbB-3)] mRNAs were used to assess purity of RNA isolation. Purified RNA was further fractionated to <200 nucleotides in length by using an RNeasy Mini spin column followed by the RNeasy MinElute Cleanup Kit (Qiagen).To isolate total RNA from whole sciatic nerves, we utilized the mirVana™ miRNA Isolation Kit (Invitrogen) following the manufacturer’s instructions. For comparison purposes, a mechanical squeezing method was used to isolate total RNA from nerves as previously described with a few modifications . After dFor small RNA library construction and deep sequencing, a total of 15 samples of sciatic nerves collected at different time points  including uninjured nerves (n = 3/each time points) were subjected to Illumina HiSeq 2500 sequencing by the University of Delaware DNA Sequencing and Genotyping Center (UDSGC) located in the Delaware Biotechnology Institute (DBI) in Newark, DE. In brief, adapters were ligated to the 5’ and 3’ termini of the purified small RNAs followed by cDNA synthesis . The resultant cDNA was PCR amplified using a common primer and Illumina index primer for the purpose of multiplexing in a single flow cell on Illumina HiSeq 2500. Resulting sequence data were analyzed by the CLC Genomics Workbench (Qiagen) software package with support from the University of Delaware Center for Bioinformatics and Computational Biology (CBCB) Bioinformatics Core. After trimming off adapters and counting reads, length distribution was calculated and filtered to partition miRNA from other small non-coding RNAs. Trimmed sequences ranging from 16–25 nt were annotated to rat miRBase 21 and sequences ranging from 23–36 nt were annotated to piRNABank rat database. Only sequences containing 50 reads or greater were use for differential expression analysis.Cluster 3.0 open source clustering software. The similarity metrics across different time points after injury including uninjured control were calculated using average linkage and Euclidean distances metric.To assess the relationship between significantly altered miRNAs in response to injury, we used hierarchical clustering method using NCode™VILO™miRNA cDNA Synthesis Kit (Invitrogen) to optimize reverse transcription efficiency from the small RNAs. The resulting cDNA was then used as a template for qPCR analyses using NCode™EXPRESS®SYBR GreenER™miRNA qRT-PCR Kit (Invitrogen) on the ABI Prism 7900HT. We initially evaluated whether the most common reference RNAs used in qPCR experiments, such as 5S and 12S rRNAs, tRNA, and U6 snRNA, showed uniform level across the samples by plotting the raw CT values for each samples. Our variance analysis in expression levels of each of the reference RNAs showed that U6 snRNA was the most consistently expressed RNA, followed by tRNA, 12S, and 5S rRNAs. Thus, the relative level of miRNAs was normalized to that of U6. The final results of qPCR in relative level were expressed as the ratio of miRNA in the injured nerve to that in the uninjured nerve using the 2−ΔΔCT method. To verify miRNA-specific primer specificity, we confirmed a single peak in the melting-curve analysis, as well as run the reaction products on a 5% agarose gel  for all genes analyzed.To validate Illumina deep sequencing data, we determined the profiles of miRNAs by quantitative PCR (qPCR) analysis using miRNA-specific LNA primers (Exiqon). In brief, the purified small RNA was converted to cDNA using A combinatorial strategy was used to predict target mRNAs for the differentially expressed miRNAs using two algorithms, TargetScan and miRanda. The target mRNAs commonly predicted by these algorithms were then overlapped with a dataset that previously identified axonally localized transcripts  to identGene Ontology (GO) enrichment analysis was used to identify biological functions that were most significant to the predicted axonal target of each significantly altered miRNA following injury.p value that determine the probability of the association between the predicted molecules and the pathway.Ingenuity Pathway Analysis (IPA) (Ingenuity System Inc.) was used to identify the significant pathways in the nervous system for the predicted intra-axonal target mRNAs according to the KEGG database. The significance was measure by the number of predicted molecules that map to the pathways and a p values; the smaller FDR indicates the smaller error in judging the p value. p values <0.05 were considered statistically significant. The edgeR program was used for all statistical analyses.Differences in altered miRNA levels across different time points after injury was assessed using the Chi-squared test, and the false discovery rate (FDR) was calculated to correct the A similar study recently identified 225 known miRNAs from rat sciatic nerve and 201 miRNAs from DRG neurons showing significant changes in levels at five time points after nerve injury . HoweverWe first assessed the purity of axoplasmic RNAs extracted from the proximal stumps of the sciatic nerve. The conventional method for extracting axoplasmic RNA in the previous study was based on whole nerve axoplasm of dissected sciatic nerve segments . To direAfter trimming off adapter sequences from the raw sequencing data of Illumina HiSeq 2500, a dataset of 10.9 ± 1.5 (uninjured), 12.4 ± 1.0 (1 DPI), 11.0 ± 0.8 (4 DPI), 11.4 ± 1.1 (7 DPI), and 10.1 ± 1.5 (14 DPI) million reads, ranging from 17 to 38 nt, was obtained . FurtheredgeR program. Of 141 annotated axoplasmic miRNAs, the statistical analysis revealed 63 rat miRNAs that were differentially regulated with different false detection rate (FDR) between the contralateral uninjured nerve and the ipsilateral injured nerve at any time points after injury (p<0.05 and FDR<0.05)  . ClusterTo cluster axoplasmic miRNAs by their temporal changes with similar responses to nerve injury, the datasets from each time point were directly compared with the uninjured control and the mean fold-change values were calculated for hierarchical cluster. As shown in To verify Illumina RNA sequencing data, we examined the axoplasmic level of a subset of miRNAs including four up-regulated  and four down-regulated miRNAs  by qPCR using miRNA-specific primers . These sp<0.05)  , showingOnly a few other studies assessed altered miRNA levels in nervous system relative to uninjured neuron. Yu et al.  performeS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


MYH7)-related myopathies are emerging as an important group of muscle diseases of childhood and adulthood, with variable clinical and histopathological expression depending on the type and location of the mutation. Mutations in the head and neck domains are a well-established cause of hypertrophic cardiomyopathy whereas mutation in the distal regions have been associated with a range of skeletal myopathies with or without cardiac involvement, including Laing distal myopathy and Myosin storage myopathy. Recently the spectrum of clinical phenotypes associated with mutations in MYH7 has increased, blurring this scheme and adding further phenotypes to the list. A broader disease spectrum could lead to misdiagnosis of different congenital myopathies, neurogenic atrophy and other neuromuscular conditions.Myosin heavy chain 7  contains supplementary material, which is available to authorized users. MYH7 gene on chromosome 14 encodes slow/b-cardiac myosin heavy chain (MyHCI), a class II myosin expressed in slow, type 1 muscle fibers as well as in the heart ventricles. MyHCI is the molecular motor of muscle and forms the backbone of the sarcomere thick filaments [The MYH7. MYH7- related cardiac diseases are more frequent and include familial hypertrophic/dilated cardiomyopathy (MIM 192600), and left ventricular non-compaction (LVNC) cardiomyopathy (MIM 613426) with more than 200 mutations described so far [As expected both cardiac and skeletal muscle disorders can arise from a defect of hy MIM 19600, and MYH7 mutations were initially classified in two main subgroups according to clinical and pathological findings: myosin storage myopathy  and Laing distal myopathy . Additional phenotypes have been reported over the years, including a scapulo-peroneal or limb-girdle muscle form with or without morphological features of myosin storage [de novo mutations or inherited in a dominant fashion although cases with autosomal recessive inheritance have seldom been reported [Skeletal myopathies due to  storage , 5 and fMSM, MIM 08358 andreported .MYH7 [Slow/b-cardiac myosin in vivo exists as a dimer of two globular heads attached to a long, α-helical coiled-coil region. The N-terminus heads bind actin and ATP, which is required for motor activities. The C-terminus rod assembles myosin into thick filaments of the sarcomere through charge-based interactions between adjacent rods . MutatioMYH7 . HoweverMYH7 to better characterize the emerging genotype–phenotype correlation in these myopathies.Here we report clinical, morphological, and myoimaging data from 21 patients with confirmed mutations in Patients were recruited from a multicenter study on congenital myopathies which gathered 13 Italian tertiary care centers for pediatric and adult neuromuscular disorders. Written informed consent was obtained from all subjects or their legal guardians when primary diagnostic procedures were performed, with explicit consent for future use for research purposes and publication of images. All patients underwent a systematic clinical characterization by at least two experts in neuromuscular disorders, including neurological, cardiac (ECG and echocardiogram), and respiratory (spirometer) assessments. CK level was recorder in all patients. Follow up ranged from 5 to 30 years. Neurophysiological studies (EMG and nerve conduction velocity) were performed in 12 patients.MYH7. Twelve cases were diagnosed as unspecific myopathies and, in absence of disease markers, underwent Motorplex analysis, a targeted molecular tool in next-generation sequencing (NGS) designed to analyze 93 genes known to be responsible of different inherited myopathies [In 9 cases the presence of suggestive clinical and histopathological signs directly targeted the Sanger sequencing of the coding regions of opathies .http://www.fruitfly.org/seq_tools/splice.html.The experimental strategies, variants annotation and prioritization in Motorplex were as described elsewhere . In partMYH7 were confirmed by Sanger sequencing. Segregation analysis was performed using DNA from all available family members.All the mutations identified in RNA from the skeletal muscle biopsies in patients 6 and 19 was extracted using TRIzol reagent according to the manufacturer’s instructions . Messenger RNA was retrotranscribed using the SuperScript III kit (Invitrogen). For the cDNA analysis, the following primers were used: 36_37F: 5'-AAGGCCATCACGGATGCC; 3'UTR_R: 5'-CCTCAAGGGCGGCAAGAA.Having reached a confirmatory molecular diagnosis, we reviewed the muscle histology features in 16 patients adopting standard histological and histochemical stains. In four patients, we also performed immunohistochemical analyses to study the expression of slow MyHC .Muscle MRI was performed in 14 patients according to conventional protocol examining axial planes of the pelvis and lower limbs with T1-weighted spin echo sequences. All muscle MRI imaging data were reviewed and scored by two experts  in the network.In silico prediction of the effect of novel mutations falling in the LMM region was performed using the COILS program [ program , a web-bTwenty-one patients are described. Twelve are women and nine men. Age varied between 7 and 70 years. Age at onset was between 0 and 49 years. Age at diagnosis was between 6 and 68 years. Nine cases were sporadic, nine displayed positive family history Fig.  whereas Detailed clinical data of patients are summarized in Table Heart was affected in seven cases , and ranged from bundle branch block to overt cardiomyopathy with features of non-compaction of left ventricle. Two patients required a pace-maker. Another case (case 5) suffered from episodes of pericarditis. Serum CK levels were normal or mildly elevated (below 1500 U/l) in all patients. EMG was usually myopathic, however three patients  presented also neurogenic features such as polyphasic and increased amplitude motor potentials and two (cases 8 and 14) had been diagnosed with a motor neuropathy for several years before genetic studies allowed the correct diagnosis.Case 4 is an 8-year-old boy who came to medical attention at the age of 4 for dropped head due to isolated weakness of neck extensors Fig. . Muscle MYH7, one inherited from his 70-year-old mother who is clinically normal  and one 64-year-old woman (case 2) and the 35-year-old daughter of case 1. All family members share a similar phenotype characterised by late onset distal myopathy mainly affecting lower limbs with subsequent involvement of proximal limbs in the two eldest patients. In no case there was respiratory involvement; however case 1 also displays hypertrophic cardiomyopathy. Muscle biopsy displayed hypotrophy of type 1 fibres and presence of cores like area.Family 2 consists of a 48-year-old woman (case 5) and her 16-year-old daughter (case 6). An elder sister of case 5 and her 93-year-old mother were reportedly affected by a severe scoliosis and tetraparesis with loss of ambulation; they were not available for our clinical examinations. Case 5 has seven unaffected siblings and shows an early onset distal myopathy with typical bilateral hand involvement. Her daughter, case 6, has mainly axial and distal myopathy. Interestingly, face weakness and scoliosis were also prominent in this family, whereas there were no pulmonary or heart involvements. Muscle biopsy performed only in the mother showed marked uniformity of fibre type and presence of diffuse mini-cores.MYH7 testing and heart ultrasound revealed the presence of non compacted left ventricle. Neurological investigation is currently normal at the age of 7 years.Family 3 has previously been described elsewhere by same authors . BrieflyIn Family 4, case 8 harbors the c.4855G > A/p.Glu1619Lys on the maternal allele in compound heterozygosity with c.4850_4852del p.Lys1617del. Parents are reportedly healthy, though the father had died before our studies because of a cancer. Although the missense variant affecting residue 1619 has already been associated with familial dilated cardiomyopathy  and it aMuscle sections were available for revision in 16 cases  to test for myofibrillar alteration and demonstrated a normal expression of slow myosin in myofilaments.1w images were reassessed in 13 of 20 patients in whom myoimaging had been performed . Two missense mutations are located in exons 14 and 16 thus affecting the NH domains. Additional file We identified 14 heterozygous mutations in Four novel mutations, including two missense, a change affecting the splice site junction and a variant affecting the stop codon (stop loss) were identified.in silico tools used for its interpretation  predicted a putative damaging effect. Even if a segregation analysis is hampered by the lack of further relatives, we considered the variant probably causative of the phenotype observed in patient 16.In particular, a novel change in exon 31  was detected in Family 1. Patient 6 showed a novel c.5655 + 1G > A variant leading to an in-frame skipping of exon 38  and in patients 13 and 15, we identified already described changes to Proline , 17. A dTwo missense mutations (c.5779A > T/p.Ile1927Phe and c.1322C > T/p.Thr441Met) were identified in patients 17 and 21. These variants have previously been associated with cardiomyopathy –20 a clide novo synonymous mutation c.5655G > A/p.Ala1885Ala was identified in patient 18. The variant, very recently described by Pajusalu [The recurrent p.Lys1617del [Pajusalu , causes We performed functional prediction studies using the COILS program of the following mutations: p.Ser1435Pro, p.Ala1439Pro, p.Ala1603Pro and p.Glu1619Lys.All mutations affect the ability of the protein to form stable and functional thick filament, changing the shape of the heptad repeat motif in the LMM region.  and it has becoming clear that alterations in MYH7 are no longer an uncommon cause of skeletal myopathy. The forms of MSM and LDM are fronting a growing number of isolated or familial cases, which do not exhibit distal muscle weakness or do not have hyaline bodies nor other myofibrillar alteration in muscle biopsy [To date 327 mutations in e biopsy , 5. In te biopsy . Our datWhilst clinical presentation of MYH7 skeletal myopathy is starting to be better clarified, more complexity arises with the concomitant presence of cardiomyopathy. It was initially postulated that mutations in the NH globular head mainly led to a cardiomyopathy phenotype, whereas mutations in the COOH tail domain, in particular the LMM domain, were responsible for the skeletal myopathies variably associated with cardiomyopathy. This distinction is no longer pertinent as several cases with skeletal myopathies and mutations in the globular head have been reported , usuallyMYH7 related myopathies [Several previous reports describe familial variability in MYH7-related myopathy , 26, 27.opathies , 9. In bIn our cohort, the most severely affected patients (case 15 and 18) have early onset with diffuse muscle weakness and wasting, progressive scoliosis and respiratory involvement very much resembling congenital myopathies without cardiac involvement. At the opposite spectrum, three patients  have mainly distal weakness with onset in later adulthood similar to most common neuropathies. Two of these patients presented cores in muscle biopsy, whereas the third has features of FTD and severe cardiac involvement (case 9). A milder and mainly proximal weakness is also described in a 60-year-old patient (case 14) with unspecific changes at muscle biopsy.All patients presented in this study have a progressive course of the disease sometimes with proximal involvement in later stages as suggested also by Lamont . CertainMYH7 mutations from early onset forms with features resembling a congenital myopathy to adult forms with also proximal involvement and heart disease.All these considerations propose a continuum in the disease spectrum related to Disease progression can be also depicted by evolution of the muscle histopathology as shown in case 4. In this patient clear cores and mini-cores lesions were evident in the first biopsy  affecting intron 38 in case 7. In both cases, as expected, we confirmed an in-frame skipping of the exon 38 upon mRNA analyses in skeletal muscle Additional file Our study also added a second case with the c.5655G > A variation in exon 38 (case 18) after the case described by Pajusalu et al  and a fuIn seven patients (of which three in family 1 and two in family 2) we identified different missense variants in the tail LMM domain changing highly conserved amino acids to proline and likely influencing protein domain structure, including the novel c.4303 T > C p.Ser1435Pro (Family 1). Previous works reported that mutations in the LMM region might affect the ability of the protein to form stable and functional thick filament, changing the shape of the heptad repeat motif.Considering this broad and overlapping spectrum of clinical presentations, genetic analyses of MYH7-related phenotypes can be difficult to target and it is not surprising to have a delay in the conclusive diagnosis . From ouMYH7 demonstrated a typical pattern consisting in early and more severe involvement of the tibialis anterior muscle, followed by extensor hallucis longus and extensor digitorum longus muscles, whereas the lateral gastrocnemius muscle was always spared [Muscle MRI of lower limbs in previously reported patients harboring mutations in s spared , 34. In More relevant MRI finding in the 13 patients from our study corroborated the notion that early involvement of the tibialis anterior muscle can be considered the “red flag” of a myosinopathy, also in patients with atypical features, with a pattern of involvement that reflects the clinical progression from distal to proximal muscle weakness.MYH7-myopathyes, a distinction from other congenital myopathies with distal affection is essential. Particularly a predominant anterior lower leg involvement is always presents in NEB-myopathies and in ACTA1-myopathies with tibialis anterior involved first [ACTA1 and NEB mutations, comparing with the prevalent involvement of vasti muscles in myosinopathies could direct genetic tests. On the other and, in cases of MYH-7 mutations and predominant proximal or axial weakness the striking affection of tibialis anterior compared to thigh involvement is strongly suggestive. Finally, few patients in our cohort were first diagnosed as neuronopathies; also in these cases muscle MRI can play an important role in differential diagnosis considering the striking differences of the pattern of muscles involvement. In patients with distal spinal muscular atrophy (or SMALED), in fact, there is the prevalent involvement of thigh muscles with sparing or hypertrophy of adductor longus and tibialis anterior (i.e. DYNC1H1 mutated patients) or of biceps femorii and medial gastrocnemius (i.e. TRPV4 mutated patients) [MYH-7 related myopathies.However, considering the wide phenotypic spectrum related to ed first , but theatients)  differenMYH7 and displaying a broad spectrum of clinical presentations, such as different disease onset, muscle weakness distribution, and the co-occurrence of heart involvement. Under this blurred clinical-genetic scenario, we foresee three forms of MYH7-myopathies: 1) an early onset distal myopathy with cores, 2) a late onset form of distal myopathy without histological evidence of cores and with variable association of cardiomyopathy and/or FTD and 3) the least frequent form of limb girdle involvement with myofibrillar damage resembling the myosin storage myopathy. We confirm that sites of mutations do not correlate with severity and cannot predict phenotype. This is particularly true for cardiomyopathies which appear to be most common in men irrespective of genotype and unrelated to mutations falling in specific domains. Functional studies and more extensive molecular analyses are warrant to explore these aspects.To sum up, this study describes a cohort of 21 Italian patients carrying a total of 14 different mutations in MYH7-related myopathies, and a striking difficulty in targeting the underlying molecular etiology. Thus, NGS tools such as MotorPlex can be dually useful to pinpoint the MYH7 mutations in unspecific cases and to identify associated alterations in additional muscular genes that could act as modifiers and provide an explanation for different phenotype.Unlikely the other, more frequent forms of congenital myopathy, our findings also propose a progressive nature of CMP, cardiomyopathy; DM, distal myopathy; FTD, fibre type disproportion; LDM, laing distal myopathy; LMM, light meromyosin domain; LVNC, left ventricule non compacted; MRI, magnetic resonance; MSM, myosin storage myopathy; NGS, next generation sequencing


Genomic studies of glioma sub-types have amassed new disease specific mutations, yet these only partially explain how mutations are linked to predisposition or progression. We hypothesized that microsatellite variation could expand the understanding of glioma etiology. Furthermore, germline markers for gliomas are typically undetectable; therefore we also hypothesize that the predictability of cancer-associated microsatellite loci in germline DNA may support the current hypothesis of a glioma cell of origin.−3). The preservation of these glioma-specific loci could therefore serve as valuable diagnostic and therapeutic markers; especially since the heterogeneity of tumor cell populations can obscure the identification of mutations preceding a metastatic phenotype.In this study, “normal” germline exome sequenced DNA from the 1000 Genomes Project (n=390) were compared with exome sequences from germlines of subjects with WHO grade II and III lower-grade glioma  and WHO grade IV glioblastoma  from The Cancer Genome Atlas to identify microsatellite loci non-randomly associated with glioma. From germline data, we identified 48 GBM-specific loci, 42 Lower-grade glioma specific loci and 29 loci that distinguish GBM from LGG (p≤ 0.01). We then attempted to distinguish WHO grade II glioma (n=67) from GBM resulting in 8 informative loci. Significantly, in all glioma grades, comparisons between tumor and matched germline sequences demonstrated no significant differences in these variants (p≥ 0.01). Therefore, these microsatellite loci are considered to be components of grade-specific signatures for glioma which distinguish germline sequences of individuals with cancer from those of individuals that are “normal”. In order to better understand the significance of these loci, we identified biological processes enriched in genes with these variants. Most strikingly, six helicase genes were enriched in the GBM cohort (p≤ 1.0 ×10 Every year in the U.S. approximately 10,000 new adult malignant brain tumors are diagnosed. TreatmeHigh-throughput sequencing studies of tumor genomes have produced new molecular markers that have enhanced classification of GBM and highlighted molecular pathways that propagate pathogenesis and progression -9. In thTherefore, we hypothesize that gliomagenesis is correlated with microsatellite variability at specific loci in germline DNA. Additionally, we demonstrate that germline (non-tumor) DNA is representative of the tumor and these glioma-specific microsatellite loci discriminate glioma grades . With validation, such markers could be developed to aid clinical predictions. We further hypothesize that these loci could influence tumor biology, including a potential contribution to early glioma tumorigenesis.Classifications were provided by The Cancer Genome Atlas (TCGA) and we have chosen to discuss our results using the same assemblage for scientific comparisons . Microsatellite loci were identified from exome sequencing data for a cancer germline and a “normal” population. Those sequences that varied in length significantly between the two populations were compiled to create a ‘signature’ list . Our next goal was to determine, if specific microsatellite variants could be further used to distinguish Grade II from GBM. Most Grade II samples (75%) had between from 0% to 80% CAMLs, while 90% of GBM samples exhibited 100% CAMLs  associated with signature loci were analyzed using DAVID annotation tools. From thWe discovered CpG islands (≤10 kB from MST loci) and transcription factor binding sites  near CAMLs. Ten MST loci from GBM and 9 LGG loci (with 3 loci shared between both glioma cohorts) were linked to CpG islands. Nine MST loci from GBM and 20 MST loci in LGG (with 2 loci shared between both populations) were located within or near TFBS see . IllustrLNX2 and FGD6 and up-regulation of CRISP1 in LGG. In GBM, a significant down-regulation of SEMA3E and up-regulation of SLC44A4 were measured  were compared with normal adult brain RNASeq data, previously described by Ameur, A. et. al, Table  16]. In. In16]. ured see .We analyzed TCGA gene mutation data  to identify connections between driver mutations loci ) closely identified with gliomas and the signature. Our data show that GBM samples contain 80% of the GBM CAML genotypes regardless of the number of driver mutations found within those samples and that the majority of LGG samples contain 60% of LGG CAML genotypes. However, an increase in CAMLs was identified in most samples with an average of 5 driver mutations, in both GBM and LGG. Interestingly, LGG samples with only 1 driver gene mutation  were the most likely population to demonstrate CAMLs, and on average 81% of these LGG samples had 43% of the LGG CAMLs Fig. . In geneThese data show microsatellite genotypes can differentiate cancer from non-cancer populations and also lower grade from GBM. Since these variant loci are identifiable in somatic DNA and are conserved in tumors lends support to the hypothesis that glioma initiating cell populations exist and are inherent to the individual and their disease.Interestingly, we observe that a small number ≤3% of normal subjects have 100% of the GBM, CAML genotypes. This may be due to sample population age biases such that some individuals are as yet undiagnosed, or that our measure over-predicts given that the rate of gliomas in the general population is less than 3%. Therefore risk screening may be valuable only in a subset of the population presenting with an abnormal MRI. Additionally, when we inspected samples with driver mutations, observed in gliomas, the relative percentage of CAMLs is similar in individuals with 1-4 mutations. However, for individuals with 5 mutations there appears to be an increase in identifiable CAMLs which may correspond to overall instability in the genome's of these individuals. The mix of glioma types in the LGG cohort may lend to smaller populations of LGG DNA sequences with identifiable CAMLs compared with GBM  and amplification of FGFR2; thus intronic CAMLs harbored by these genes could be biological indicators for tumorigenic activity. Additionally, loss of 10p is found in 47% of GBMs while 10q loss is found in 70% of primary and 63% of secondary GBMs[FGFR2, BRWD2 (WDR11), GLUD1, and NRP1; none were identified in the LGG signature.Since some microsatellites are associated with DNA ‘fragile sites’, locations within chromatin susceptible to constrictions or break-points that are linked to cancers and neuro-developmental disorders, we analyzed our MST loci to determine which are located in these regions, as a possible mechanism for tumor potentiation , 19. BRWal tumors, 21. Givdary GBMs. In our Rb, rendering individuals more susceptible to the disease later in life. Our data suggests the ‘first hit’ may also be somatic microsatellite instability which then increases the sensitivity to, and probability of, greater genomic aberrance theoretically by stress factors such as environment, inflammation or age leading to the ‘second hit’[SRC, NPAT and CBL); thus, these MST variants may be additive to gene mutations  that contribute to cancer.In the original account of ‘the two-hit hypotheses’ for somatic retinoblastoma, the first hit was an inherited mutation in cond hit’-24. ThesRYR1 [RYR1 expression is notably down-regulated in GBM; our GBM data demonstrates CAML genotypes unique to RYR1 with transcripts and isoforms also unique to GBM. Thus, CAML loci at genes such as RYR1 may render them to be more susceptible to mutation (for example from stresses associated with treatment) and may be valuable indicators for potential treatment outcomes. Additionally, this further supports the possibility of tumor initiating cells with irregular genetic variations that generate disease relative to an underlying combination and abundance of affected microsatellite loci.More recently, mutable genes commonly identified in GBM patients post chemotherapy and radiation treatment were discovered, and included RYR1 . RYR1 exDICER1 and POLQ, both genes associated with RNA and DNA modification and metabolism.The aberrant alteration of six helicase genes in GBM suggests that genes important to microsatellite identification and correction, along with transcription and RNA synthesis are themselves modified with MST variants. Previous studies have demonstrated that in addition to mis-match repair genes, helicases (notably RecQ family DNA helicases ) are impDDX60 and TRIM25; SPOPL and BRMS1L; FGFR2, BRWD2 and CBL; SEMA3E and NRP1. Other functional associations important to changes in RNA transcription included, SLC44A4 transcript isoforms uniquely identified in GBM . Similar results were observed for LNX2 in LGG supporting our hypothesis that CAMLs may modify RNA transcription and expression.With the abundance of helicase and UPS associated genes in our glioma signatures, another cancer promoting scenario may be introduced through changes to gene-products that compose spliceosome complexes ; through these genetic modifications, alternatively spliced RNAs may support spliceosome-associated proteins differently, which may further modify mature RNAs. A third mechanism may be modifications to ubiquitin proteasome system proteins (ligases and ubiquitin complex proteins) which could alter protein degradation or signal transduction. Supporting these two hypotheses we identified functionally associated genes with CAMLS, including: SEMA3E (a GBM signature locus) compared with normal samples; as previously noted SEMA3 competes with VEGF-165 for NRP1 (a GBM signature locus) and suppresses angiogenesis [EVC transcript were identified in GBM and LGG but not in normal brain tissue, and in GBM the novel isoform was the common transcript; expression data suggests that EVC is expressed more in GBM and LGG tumors when compared with normal germline expression, although these data did not pass our stringent 2-fold difference in expression .Additional important biological changes observed and linked to CAMLs included significantly decreased expression of ogenesis . Therefoessential for cancer cell survival (and conserved across a cancer type) (2) contribute intermittently – to cancer cell phenotypes like metastasis, heterogeneity, or aggressiveness, and (3) are tissue-specific, associated with only one type of tumor or tissue origin. If so, we can predict that specific microsatellite instability contributes to cancer-specific genomics and may occur during embryogenesis, which has also been predicted in other MST associated diseases, including Huntington's disease and Fragile X syndrome[Since these loci can be identified from germline (non-tumor) DNA, it further suggests that there is a tumor initiating cell population in which developmental cell signaling and patterning pathways may result in aberrant differentiation and proliferation due to an underlying transcriptional landscape determined by cancer-associated MST variants. Therefore, we further hypothesize that DNA microsatellite variability underlies an adaptability that is conserved in all cancers and can be investigated by determining the frequency of microsatellite variability in those genes (1) that are  syndrome. Thus, tExome sequencing data, from Illumina HiSeq sequencing machines were obtained from The Cancer Genome Atlas (TCGA) and the 1000 Genomes Project (1kGP). These sequences were aligned to the human reference genome using BWA, by their respective projects, and re-alignment followed with microsatellite loci and variant identification by methods and software established in our laboratory -31. OnlyWe produced a set of over 850,000 microsatellite loci which have flanking sequences unique in the human genome. Initially a set of over a million microsatellites was found in the human genome (NCBI36/hg18) using Tandem Repeats Finder (TRF) , with prReads are mapped to the reference genome using BWA or BWA-sw  . A custohttp://discovery.vbi.vt.edu/MicrosatDB/) [Accuracy estimations of our microsatellite-based genotyping method: Using Sanger sequencing and data from HapMap, we were able to validate 96.5% of a subset of 85 non-synonymous variations composed of repeat length variations and SNPs contained in microsatellites. The novel variants we validated using Sanger sequencing were submitted under the lab handle SGARNER and are available on-line in the latest release of dbSNP. All microsatellite-based genotyping calls from these studies were made publically available at MicrosatDB (osatDB/) .To increase uniformity of coverage and genotyping rates across samples sequenced at different times with different methods by different studies, we required at least 15,000 microsatellite loci to be called per sample for inclusion in this study. This filtered out one 1kGP-F sample and 235 1kGP-M samples. From the TCGA samples, this filtered out 23 LGG germline samples, 57 LGG tumor samples, 238 GBM germline samples, and 254 GBM tumor samples. The large number of GBM samples filtered is expected as these samples were sequenced much earlier by TCGA than the LGG samples. So we would expect lower coverage and shorter reads. Only those loci with at least 15x coverage are considered “callable” in a given sample . A locus had to be called in a minimum of 10 exomes to be included in the genotype distribution comparison analysis and to remove loci which may be called at insufficient frequency in one of the two data sets.We processed 339 GBM tumors, 268 LGG tumors, and 57 LGG germline RNAseq Illumina data sets with Cufflinks  by usingLocations of CpG islands and transcription factor binding sites associated with the human reference genome were downloaded from the UCSC Genome Browser . A Perl Variant calls for the GBM and LGG samples were downloaded from TCGA. The driver mutations and MMR genes of interest  were analyzed with respect to the CAML status of the samples.


Anelloviruses are nonenveloped single-stranded DNA viruses infecting a wide range of mammals. We report three complete genomes of novel anelloviruses detected in laboratory rats. Phylogenetic analysis demonstrates that these viruses are related to but distinct from recently described rodent Torque teno viruses (RoTTVs) found in wild rodent species. Apodemus sylvaticus), field voles (Microtus agrestis), and bank voles (Myodes glareolus) (Mus musculus) in both wild and laboratory settings.The prototype anellovirus, Torque teno virus (TTV), was described in 1997 in a Japanese patient with posttransfusion hepatitis of unknown etiology , 3, thei4–areolus) . These vRattus norvegicus) from 2 separate groups, group A (n = 4) and group B (n = 6). All 10 rats were found to be anellovirus positive. Sequencing of the cloned PCR amplicons revealed three distinct sequences. The first was present in three members of group A, the second in a single individual from group A, and the third in all members of group B. No evidence of mixed infections was seen. Complete viral DNA was amplified using the previously described technique of rolling circle amplification followed by PCR with inverted primers based on the screening amplicon sequence  in length, respectively, and sequences share greater than 90% nt identity across the whole genome. All three sequences show a genomic organization consistent with other anelloviruses possessing unidirectional overlapping open reading frames (ORFs). Two of these ORFs were identifiable as ORF1 and ORF2  based on sequence homology with other anelloviruses. A third predicted ORF (nt 1615–1911) was present in all genomes but showed no homology to any known viral proteins by NCBI BLAST analysis. Pylogenetic analysis of the partial ORF1 sequences (equivalent to the region described in reference This is the first description of prevalent anellovirus infection in commonly used laboratory rodents. The effects of these infections on other experimental procedures, including coinfections, remains to be determined.KM668486, KM668485, and KM609325, respectively.The complete genome sequences of RN2_Se15, RN_5_SE5 and RN_8_SE11 were deposited in GenBank under the accession numbers


Arabidopsis thaliana, the most common type of alternative splicing is intron retention. Many studies in the past focus on positional distribution of retained introns (RIs) among different genic regions and their expression regulations, while little systematic classification of RIs from constitutively spliced introns (CSIs) has been conducted using machine learning approaches. We used random forest and support vector machine (SVM) with radial basis kernel function (RBF) to differentiate these two types of introns in Arabidopsis. By comparing coordinates of introns of all annotated mRNAs from TAIR10, we obtained our high-quality experimental data. To distinguish RIs from CSIs, We investigated the unique characteristics of RIs in comparison with CSIs and finally extracted 37 quantitative features: local and global nucleotide sequence features of introns, frequent motifs, the signal strength of splice sites, and the similarity between sequences of introns and their flanking regions. We demonstrated that our proposed feature extraction approach was more accurate in effectively classifying RIs from CSIs in comparison with other four approaches. The optimal penalty parameter C and the RBF kernel parameter One of the important modes of pre-mRNA post-transcriptional modification is alternative splicing. Alternative splicing allows creation of many distinct mature mRNA transcripts from a single gene by utilizing different splice sites. In plants like  Arabidopsis reports that ∼42% of the multi-exon genes undergo AS with ∼40% of those genes having retained introns (RIs) but only 3% having spliced exons Syed et al.Arabidopsis. Accumulating evidence indicates alternative splicing in invertebrates and plants might have different mechanisms in comparison with vertebrates and especially mammals, and the extent and complexity of intron retention in plants still need to be specifically characterized.As an essential post-transcriptional process, alternative splicing (AS) can increase transcriptome plasticity and protein diversity Mastrangelo et al.Palusa et al.Arabidopsis. On the other hand, there are many studies indicating that intron retention is a major AS phenomenon in plants Transcript samples with RIs that are examined by RT-PCR are shown to co-purify with polyribosomes, suggesting that these intron retention events are not the result from incomplete splicing but are found in their nuclear exports Boulesteix et al.Machine learning approaches have been widely applied to knowledge extraction from biological experimental data Using random forest and in-house implemented PSOSVM that utilizes PSO to optimize parameters C and RIs are defined if the introns are spliced out in at least one isoform (mRNA) but entirely retained in at least one other isoform for the same genes. In addition, for multiple RIs founded in different isoforms of the same genes, if the differences in the 5′ splice sites (or the 3′ splice sites) of these RIs are less than 6 bp, we define these RIs as redundant ones. Hence the longest one is selected among them for downstream data analysis. CSIs are defined as ones that are always spliced out in all isoforms of individual genes.ftp://ftp.arabidopsis.org/home/tair/Genes/TAIR10_genome_release/) by a Perl script. Then using GMAP Arabidopsis from the genome sequence files (ftp://ftp.arabidopsis.org/home/tair/Sequences/whole_chromosomes/). R quantile function was employed to generate intron length quantiles for analyzing the intron length distribution in Arabidopsis.Based on TAIR10 gene annotation, coordinates of introns in genome sequences were determined using TAIR10_GFF3_genes.gff  and 23 bases for acceptor sites (−21∼+2) from introns and their flanking exons  sites were separated into two datasets from splice sites (see details in Let Here For the two Here http://cran.r-project.org/web/packages/seqinr/index.html).By the above-mentioned feature extraction approach, the sequence information in our dataset was changed into feature vector using R codes that utilize “seqinr” package  and LibSVM (http://www.csie.ntu.edu.tw/~cjlin/libsvm). The program of our PSOSVM was written in java.In this study, parameters of our proposed PSOSVM were set as shown in In order to select optimal parameters C and ss of   was set Several assessment measures were used to evaluate the classification performance using random forest and PSOSVM in this study. All of them were deduced from the numbers of true positives  and mitochondrion (ChrM) while only 42 CSIs cases were found in these organelle genomes. For the 8 different RNA types, both RIs  and CSIs  were detected overwhelmingly in mRNAs whereas they  were rarely discovered among other 7 RNA types. Therefore, all the RIs  and CSIs  detected in mRNAs within chromosomes Chr1–Chr5 constituted our data set for downstream analysis.In TAIR10 gene annotation for NA types . It is cArabidopsis has much shorter introns. As shown in quantile in terms of the given probabilities , 96% RIs and CSIs were found within the range from 44 to 501 bp and from 70 to 631 bp respectively. This suggested that extremely large introns  and extremely small introns  became outliers, which would cause a negative effect on classification. Consequently, we obtained the high-quality dataset including 2,520 RIs and 110,254 CSIs after removing these outliers  and 229 redundant RIs  Supervised machine learning approaches for the identification of RIs and CSIs require a set of labeled samples A+B+C features) for each intron in the experimental dataset. A denotes basic features, including both global features  and local features  have negative values of ta, at, atgt, taat, tatat, atatt, aaata, ttata, attat) possess positive values of C denotes the signal strength features of the splice sites  and the similarity level features  of two datasets, which include sequences from −20 to −1 and from +1 to +20 sites for 5′ and 3′ splice sites (As shown in ce sites .52) and optimized features (27) to classify RIs and CSIs , we firstly employed numFeatures, and the other parameter (numTrees) of random forest was set from 30 to 50 with a step size of 2 to find the optimal value using grid search algorithm.By using PSO, the optimal parameters C and A feature set, (2) A+C feature set, (3) our combined A+B+C feature set, (4) complete 52 feature set and (5) optimized 27 feature set  and PSOSVM . On the other hand, based on these three assessment measures, the random forest classifier always achieved better classification performance than PSOSVM. The differential performance between these two classifier reached 0.056 obtained by AUC assessment measure using our combined feature set   set see . For eacture set .A, in which the performance of F-Measure increases by 0.001 than that of Accuracy for random forest while the opposite case happens for PSOSVM. Interestingly, these five different feature sets display the same change trend of classification performance in terms of these three assessment measures for both random forest and PSOSVM. We focus on F-Measure (In -Measure  to illusA or A+C), complete 52 feature set, or optimized 27 feature set. This result suggests, not only for PSOSVM but also for random forest, that our hybrid feature extraction approach selected useful features for better classification between RIs and CSIs.As shown in C feature set made the greatest contribution to improve the classification performance: for example, 3% F-Measure increase using PSOSVM, and 3% AUC increase using random Forest for A+C feature set in comparison with A feature set  have higher frequencies in RIs than CSIs whereas others  occur higher frequencies in CSIs than RIs.Meanwhile, we also found that some short motifs were relatively frequent but quite different between the RIs and CSIs. So we extracted ArabidopsisArabidopsis the unusual GC-AG introns appear to be more frequent in RIs than CSIs while the canonical GT-AG introns are richer in CSIs than RIs. Another interesting phenomenon in our data analysis is that more than half of RIs (58.4%) occurs in CDS, CDS+3′UTR or CDS+5′UTR regions. Such positional distribution characteristic of RIs indicates the potential that these introns are partly or entirely translated to proteins. Previous studies demonstrate that growing examples of cellular mRNAs with RIs express functional proteins by avoiding degradation through the nonsense-mediated decay (NMD) Different from previous bioinformatics analyses of AS in Pertea et al.Arabidopsis by a computational approach. Although intronic splicing enhancers and silences (ISEs and ISSs) are less understood than ESEs and ESSs, a previous study also suggests B feature set, we analyzed all ggg-containing motifs with length from 3 to 5 bp included g triples ) may be ISEs in Arabidopsis. We checked all at/ta-rich motifs with length 4 and 5 bp and obtained the mean value of taat”, “tatat”, “atatt”, “ttata” and “attat” have been proved to help recognizing the CSIs in our data analysis. Overall, ggg-containing and ggag-containing motifs seem to be ISSs because of their obvious abundance in RIs than in CSIs. On the other hand, at/ta-rich motifs appear to be ISEs because of their significant abundance in CSIs than RIs in Arabidopsis, which would potentially promote the identification of intronic splicing regulatory elements in plants.It is well known that cis-acting sequences or motifs numFeatures  = numTrees is obtained by grid search), and the optimized parameters were beneficial to obtain stable classifier performance. In contrast, different values of  for alternate isoforms derived from the same genes. It is likely that highly expressed retained introns have different signal strength than retained introns with low expression levels. Therefore, utilizing RNA-Seq data to extract and incorporate expression information in intron level will definitely facilitate the development of more accurate and robust classifier by machine learning strategies. In fact, a recent RNA-Seq data analysis already shows evidence for novel transcripts and alternative splicing events in Arabidopsis that are not annotated in TAIR10 Arabidopsis, which can help us enhance the classification performance by providing more members within the RIs class. Moreover, we can do further classification of RIs that might be related to different environmental and/or developmental cues. Obviously, more RIs with different meta-data can be further analyzed to extract stress-, tissue-, or growth stage-specific features so that we can better understand how RIs are affected by both external and internal conditions in plants. On the other hand, RNA secondary structures have been demonstrated to affect alternative splicing in vivo genome-wide RNA structure map in ArabidopsisIn this study, we utilized current TAIR10 mRNA (transcript or isoform) annotation in A+B+C feature set could obtain the optimal classification performance by applying random forest and PSOSVM classifiers after tuning parameters. Follow-up analysis of these features has revealed interesting information about RIs in comparison with CSIs:The primary contribution of this work is our novel hybrid feature extraction approach that reveals overall features of introns, splice sites and flanking exons. These features can be utilized to effectively categorize and differentiate between RIs and CSIs. The experiments on five different feature sets verified that our combined In average RIs have shorter length (145 bp versus 160 bp), higher GC content (35.76% versus 32.43%) and lower AT content (64.24% versus 67.57%) than CSIs.RIs show different features of segmental nucleotides composition, such as higher RIs possess lower signal strength of 5′ and 3′ splice sites , and terminal dinucleotide GC-AG appears a higher frequency in RIs than CSIs.The RIs show higher similarity with their flanking exons than CSIs.ggg-containing and ggag-containing motifs as ISSs as they are enriched in RIs. Accordingly, at/ta-rich motifs seem to be ISEs because of abundant in CSIs.We here propose Arabidopsis.These features information about RIs can effectively facilitate an understanding of recognition mechanism of RIs in File S1Detailed introduction for how to extract data  using ou(DOCX)Click here for additional data file.File S2All extracted data used in the article.(ZIP)Click here for additional data file.File S3All source codes used in the article.(ZIP)Click here for additional data file.


To test the hypothesis whether enriched air nitrox (EAN) breathing during simulated diving reduces decompression stress when compared to compressed air breathing as assessed by intravascular bubble formation after decompression.2) in a hyperbaric chamber, with identical diving profiles (28 msw for 55 minutes). Intravascular bubble formation was assessed after decompression using pulmonary artery pulsed Doppler.Human volunteers underwent a first simulated dive breathing compressed air to include subjects prone to post-decompression venous gas bubbling. Twelve subjects prone to bubbling underwent a double-blind, randomized, cross-over trial including one simulated dive breathing compressed air, and one dive breathing EAN . Three decompression incidents, all presenting as cutaneous itching, occurred in the air versus zero in the EAN group (P = 0.217). Weak correlations were observed between bubble scores and age or body mass index, respectively.EAN breathing markedly reduces venous gas bubble emboli after decompression in volunteers selected for susceptibility for intravascular bubble formation. When using similar diving profiles and avoiding oxygen toxicity limits, EAN increases safety of diving as compared to compressed air breathing.ISRCTN 31681480 Decompression sickness (DCS) may occur after compressed air breathing during open water Self Contained Underwater Breathing Apparatus (SCUBA) diving, even if empiric safety procedures are respected. During and/or after decompression, dissolved inert gas may convert to gas bubbles. Presence of gas bubbles precedes clinical symptoms . It is g2) may reduce decompression stress by augmenting the so-called “oxygen window”, i.e. the diffusion gradient between the partial gas pressures into bubbles  and surrounding blood and/or tissues . Theoretically, higher blood and tissue oxygen partial pressures should lead to increased nitrogen dissolution and subsequent clearance from the body. Indeed, increasing the FiO2 from 0.4 to 1.0 during the ascent from dives, in which a rebreather circuit (closed circuit) was used, led to reduced intravascular bubble scores, demonstrating the relevance of the oxygen window during decompression .,11.10,11Body height and weight were measured in all subjects. Body mass index (BMI) and body surface area (BSA) were determined using standard calculations. Additionally, body fat percentage by skinfold measurements according to Durnin and Womersley and waist-hip ratio were obtained .Retrospective registration was necessary since trial registration was uncommon at the time of design of the trial. The original study protocol and an English summary of the study protocol are presented in Data were analyzed using R software , version 3.2.4 Each parameter was tested for normality with Kolmogorov-Smirnov tests and handled accordingly. All values are expressed as mean ± standard deviation or median with interquartile range [IQR] as appropriate. All included volunteers were considered as their own control subject since they each underwent one EAN and on Air dive. Since the bubble scores were calculated on an ordinal scale and given the size of analyzed sample, we used nonparametric tests for comparisons. The Higgins & Tashtoush formula for repeated measures  was used for the interaction alignments with the aligned Koch rank score , and the resulting p-value was reported ,14 BetweOut of the 47 included volunteers, 19 subjects were found to produce intravascular bubbles (Bubble score ≥2) upon decompression. The inclusion flow diagram is shown in All intravascular bubble scores were determined independently by two investigators blinded for group assignment. Inter-rater agreement was excellent with a Kappa level of 0.915 (P < 0.001).Three minor DCS incidents occurred during the cross-over study protocol. None of the twelve EAN dives was associated with any incident, all three incidents took in the air dives . Another eleven DCS incidents were observed in the 47 initial air dives. All DCS incidents presented as cutaneous itching during the ascent (3 events) or within one hour post decompression (11 events) without concomitant objective clinical signs. Dives leading to DCS incidents were not associated with an increase in the sum of the bubble scores , two incidents even occurred in subjects with a total bubble score of 0.2 vs. 1.8 ± 0.2 m2; P = 0.075).There was a weak relationship between the sum of the intravascular bubble score and age , but not with BMI or body fat percentage. Subjects with decompression incidents had lower waist/hip ratios , higher body fat percentages , and a tendency towards lower a BSA  breathing during wet dives at 22.5 msw did not affect the maximum bubble score when compared to compressed air breathing [2 partial pressures. Hence, any oxygen window effect of EAN versus compressed air breathing was less pronounced  than in our study . Thus, our findings are in good agreement with important role of the O2 suggested by Blatteau et al: increasing the FiO2 from 0.4 to 1.0 at 10 msw during the final part of the decompression from a simulated dry chamber dive markedly reduces venous bubble formation [et al investigation had different endpoints than the current investigation, did not have a double-blind randomized design as in our study, and was designed to match inspiratory N2 partial pressures between groups [et al [At first glance, our data of an EAN-induced reduction of venous bubble formation are in contrast with Thom reathing . Howeverormation . No difformation . It is uormation . Howevern groups . These ds [et al .Several recent studies have shown that the pathophysiology of DCS is more complex than decavitation of inert gases, and additionally includes microparticle formation, neutrophil activation and endothelial dysfunction ,17,18. Aet al in wet dives [2 dissolution and elimination. Regardless of the explanation, our data demonstrate the possible presence of a time-dependency of the bubble formation, resulting in poor intra-individual reproducibility of the diving effects. Therefore, the randomized cross-over protocol proved to be mandatory to discriminate differences of the two gas mixtures on venous bubble formation independent of within subject variability.The cross-over study protocol allowed for the subjects to serve as their own control and thus minimized bias due to between-subject variability. Bubble formation was lower after the second than the first simulated air dive, in accordance with a previous observation of Zanchi et dives . We can 2 bubble formation and possibly DCS symptoms. In fact, both the number of DCS incidents (20% of simulated dives) and the number of subjects with high bubble scores (40% of study subjects) were high. Nevertheless, all decompression incidents were minor, and consisted of cutaneous itching (no cutis marmorata or any other objective erythema). Even with a maximal O2 partial pressure of 1.37 atm, our diving schedule was still under below the accepted threshold of O2 toxicity threshold and we did not observe and sign of O2 toxicity [Our diving schedule was chosen to provoke Net al did not control for physical fitness between leaner and fatter subjects, and thus any reduced bubble formation in the leaner divers was most likely due to higher fitness in these subjects [There was a weak correlation between the total bubble score and age, whereas bubble production was not directly related to body fat percentage or BMI. Previous reports also demonstrated increased bubble formation associated with older divers (above 30–40 years old) ,21. Equi2 supersaturates in particular in fat tissue, due to its higher solubility in lipid than in aqueous solutions. It remains unknown, if number of venous emboli is a valid outcome parameter for N2 saturation of fat tissue. This question is underscored in our study by the discrepancy between the relation of demographic parameters and either bubble scoring or DCS symptoms: while there was no relation between body fat and intravascular bubbles, the presence of DCS symptoms was associated with higher body fat percentages. In fact, 81% of fatal diving accidents were reported to occur in divers with BMI ≥ 25 [The commonly accepted hypothesis on bubble generation is that NBMI ≥ 25 .A limitation of this study includes the use of a HC instead of non-simulated dives. Conversely, circumstances could be optimally controlled, which is important for the current proof-of-concept and results should be reproduced for wet dives .In conclusion, our results provide evidence for increased safety of EAN breathing during SCUBA diving as compared to compressed air breathing, when using identical diving profiles and not exceeding oxygen toxicity limits. EAN gas is widely available and these results could therefore confirm immediate utility to reduce DCS risks.S1 File(DOC)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(DOC)Click here for additional data file.