Leishmania donovani and Leishmania braziliensis.Leishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes.We purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches.These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between The online version of this article (doi:10.1186/s12864-015-1260-7) contains supplementary material, which is available to authorized users. Leishmania are highly endemic to tropical and sub-tropical regions of the world. They are transmitted to humans and other mammals by sandfly vectors that inject the flagellated, promastigote life cycle stage of leishmania into the dermis of the host while taking a blood meal. After innoculation, promastigotes are engulfed by host mononuclear phagocytes either directly or indirectly as cargo of apoptotic neutrophils [Leishmania donovani and Leishmania braziliensis, cause visceral and mucocutaneous leishmaniasis, respectively. While the former is naturally the more serious threat as it can lead to death if left untreated, the latter can have an extremely high impact on the affected individual due to debilitating and disfiguring destruction of critical soft tissue structures.Protozoan parasites of the genus trophils . FollowiThe current paucity of effective and well tolerated drug treatments and even more so, the lack of highly efficacious, well standardized and widely available vaccination strategies can be attributed at least in part to the gap of knowledge about the intricate interplay between leishmania and host macrophages. Macrophages are universal key players in both innate and adaptive immune responses. Their primary function is to engulf and digest prey, whether pathogen or debris from cellular turnover, which makes their intracellular environment very nutrient-rich. Leishmania exploits these macrophage characteristics in a very sophisticated manner: it lets the phagocyte ingest it, and then uses the cell as its safe nursery, where it scavenges nutrients and replicates while remaining unrecognized by other immune cells. The mechanisms by which leishmania manages to survive within these potent immune cells are just starting to be elucidated. One key strategy employed by leishmania appears to be the prevention of macrophage activation, a step that is crucial to induce macrophage digestion and killing functions -4. At thIn principle, there are two categories of molecules –surface associated and secreted- made available by leishmania to communicate with the host and turn on and off macrophage cellular functions. Regarding secreted molecules, our group has recently discovered that leishmania use a non-classical secretion mechanism to export a majority of their secreted proteins, which involves the release of small vesicles called exosomes ,7.Exosomes are 50–100 nanometre-sized membrane vesicles secreted by a variety of single- as well as multi-cellular eukaryotic organisms. They are distinct from membrane microvesicles, which are produced by blebbing, since their release occurs through fusion of multivesicular bodies from the endocytic/exocytic pathway with the plasma membrane of the cell . ExtraceL. donovani exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages [L. donovani exosomes can modulate mononuclear cell phenotypes in vitro, rendering them anti-inflammatory by specifically inhibiting cytokine production. Studies with C57Bl/6 and Balb/c mice provided evidence that treatment with exosomes from L. donovani as well as Leishmania major prior to infection exacerbated disease in vivo [L. donovani exosomes cleaved the microRNA (miRNA) processing nuclease Dicer 1 in murine hepatocytes, resulting in downregulation of microRNA-122 expression, lowering of serum cholesterol and enhancement of murine liver infection [L. major exosomes reported that the vesicles globally affected macrophage gene expression, which was in part GP63-dependent [Importantly, in our studies, rophages . Moreove in vivo . These fnfection . In a seependent . In summToxoplasma gondii-infected fibroblasts released exosomes containing a set of host mRNAs and miRNAs that was distinct from that of uninfected, serum-starved cells [Trichomonas vaginalis exosomes were reported to contain RNA sequences, the biotype and function of which still remain to be determined [Trypanosoma cruzi was shown to release extracellular microvesicles containing a variety of non-coding RNAs including tRNA-derived small RNAs, which have a suspected regulatory nature [In addition to their protein cargo, exosomes and microvesicles were recently shown to be carriers of nucleic acids in the form of RNA. This observation was first made in mast cell exosomes, which were found to contain mRNA as well as miRNA . Surprised cells . Howevertermined , and Tryy nature ,23.in vitro. Using high throughput sequencing and bioinformatics analyses, we found that leishmania exosomes were enriched in small RNAs derived from largely non-coding RNAs. Notably, we discovered that these vesicles contained a relatively abundant and highly selective population of small RNAs derived from mature tRNAs. Furthermore, we found a number of novel transcripts, some of which were highly enriched in exosomes. Although exosomes released by both L. donovani and L. braziliensis had largely similar RNA content, L. braziliensis exosomes specifically contained transcripts derived from genes that also code for siRNAs.Based on the evidence that exosomes may serve as biologically important shuttle vectors for RNAs, in the present study, we sought to investigate the RNA content of leishmania exosomes. We indeed found that leishmania exosomes contained RNA cargo which they were capable of delivering to host cells Taken together, these findings show for the first time that leishmania exosomes are highly enriched in small non-coding RNAs, particularly tRNA-derived small RNAs with potential regulatory functions. This suggests that these RNAs may have functions in intercellular communication. These findings hint at a previously unrecognized potential mechanism of leishmania pathogenesis, mediated through the exosomal delivery of small, principally non-coding RNAs to mammalian host cells.L. donovani or L. braziliensis cultured in vitro under infection-like stressors . Notably, the length profile of exosomal RNA was distinct from that of L. donovani total RNA, with the bulk of exosomal sequences being short (25–250 nt). Furthermore, we did not detect full length ribosomal RNA (rRNA) peaks in exosome RNA profiles. In contrast, these full length rRNA peaks were prominent in the total RNA profiles. To confirm that the purified nucleic acid was in fact RNA, we incubated exosome RNA with DNase, RNase or KOH. As can be seen in Figure Exosomes were purified from supernatants of early axenic amastigotes and subjected to RNA extraction with phenol/chloroform. Results depicted in Figure To exclude the possibility that RNA was merely co-purified during exosome isolation but was not directly associated with or internal to the vesicles, we treated intact exosomes with RNase in the presence or absence of membrane-permeabilizing detergent. The results in Figure L. braziliensis early axenic amastigotes using the same procedures as described for L. donovani. This analysis showed that L. braziliensis exosomes also contain RNA, with similar characteristics to that of L. donovani exosome RNA  these two organisms cause distinct disease manifestations and hence can be expected to differ in their mechanisms of pathogenesis; b) they are spread through different vectors: L. donovani is transmitted by sandflies of the genus Phlebotomus in the Old World, whereas L. braziliensis is transmitted by Lutzomyia in the New World; and c) L. braziliensis was found to have a functional RNA interference pathway, which seems to be absent in L. donovani [L. donovani and ~1.1 million paired reads for L. braziliensis .In order to assess the global transcriptome present in leishmania exosomes, we constructed complementary DNA libraries for high-throughput sequencing. We chose to compare RNA purified from exosomes released by early axenic amastigotes of donovani . We hypoL. donovani and the L. braziliensis libraries with reference genomes, respectively LdBPK (Leishmania donovani strain BPK282A1) and LbrM (Leishmania braziliensis MHOM/BR/75/M2904) using Bowtie 2 and the very-sensitive-local option which sets the seed length to 20 nucleotides, allowing for only one mismatch within the seed alignment  than the strain used to generate the reference genome. This is supported by the fact that we were able to align significantly more reads (52.88%) from the L. braziliensis library with the L. major reference genome . The results of this analysis showed that 28.1% of reads from the L. donovani and 34.46% for L. braziliensis), which could potentially be novel transcripts. Interestingly, we only saw less than 4% of reads mapping to protein coding genes (CDS) or spliced leader (SL) RNA genes. These results indicated that the majority of sequences present in the leishmania exosome transcriptome are derived from non-coding RNAs and intergenic regions, whereas sequences derived from mRNAs are underrepresented.When categorizing reads into RNA biotypes based on reference genome annotations, we saw that for both libraries, the majority of reads were aligning with rRNA and tRNA genes, in the sense orientation Figure B. In addL. major is the species with the best assembled genome to date and presents the most complete annotation of non-coding RNAs, we also performed alignments of L. major annotated non-coding RNAs with the LdBPK and LbrM reference genomes, in order to identify non-annotated, non-coding RNA loci in our target genomes. The results of the screening using Artemis showed that the top 20 most abundant reads from both libraries mapped to three RNA classes in the sense orientation: rRNA, tRNA and snRNA  RNA library , we observed that the genomic loci giving rise to our identified novel transcripts had SL sites in the 5′ region upstream of them  within the sequences of the novel transcripts to see whether they have the potential to code for a protein or peptide and found potential ORFs for the majority of them  genomes to search for potential targets in these genomes, looking for complementarity. We obtained 60 hits for all of the 1288 reads representing novel transcripts in the L. donovani exosome library, one of which was positioned in between the genes 1170 and 1180 on chromosome 30 (LdBPK_301180_leftof) and the other in between the genes 1600 and 1610 on chromosome 29 (LdBPK_291610_leftof) for probe design. The regions that are complementary to the probes used are indicated in Figure L. donovani total and exosome RNA into a polyacrylamide gel, along with 21 nt and 150 nt size markers. The results from the Northern blots showed that both novel transcripts could be detected in total and exosome RNA  [L. braziliensis exosomal RNA cargo. The main classes of L. braziliensis siRNAs are derived from the spliced leader-associated conserved sequence (SLACS) retroposon [L. braziliensis-specific telomere-associated sequence (TAS) [L. braziliensis library, we found 4471 reads mapping to these elements  are believed to be 20–25 nt in length [L. donovani library, where we only found 353 reads mapping to the siRNA-coding genetic elements , possibly resulting in false positive alignments. Despite this, it is clear that there were >10 times more reads in the L. braziliensis library mapping to siRNA-coding genetic elements, indicating that our results are specific and providing evidence that L. braziliensis may export siRNAs or their precursors within exosomes. However, we cannot rule out the possibility that the sequences we found in exosomes may originate from regions of the SLACS/TATEs genes other than the ones giving rise to siRNAs.As discussed above, (siRNAs) . We weretroposon , the teltroposon , the L. ce (TAS)  and the ce (TAS) . We genen length . For comL. donovani and L. braziliensis exosome RNA libraries that mapped to tRNA genes. A few recent studies characterizing the RNA content of mammalian exosomes had reported the presence of tRNAs or their fragments in these vesicles. For example, reads mapping to tRNAs were found in sequencing libraries made with RNA from exosomes released from neuronal cells (13.5%) [L. donovani and L. braziliensis, respectively, mapped to tRNA genes when aligned to the Leishmania major MHOM/IL/81/Friedlin (LmjF) reference genome (which has the best curation on tRNA annotation amongst leishmania species). These frequencies exceeded by some measure those reported for mammalian exosomes in the studies cited above. Close inspection of the genome alignments revealed that a high percentage of these sequences were covering only parts of the respective tRNA genes  , indicating that the tsRNAs were unlikely to be random degradation products. Strikingly, we observed the same rank order frequency of tRNA isotypes as origins of tsRNAs in both libraries  , immune  (13.5%)  and plas (13.5%) . Strikin% , immunAsp and tsRNAGln were derived from the 5′end or mid-5′ end in both libraries (Table Glu and tsRNAGly) (Table Leu), and others to the length of tRNA-derived RNA fragments   Table . Furthere) Table .Arg) , the majority of which fell into non-annotated regions of the host genomes  and tsRNALeu (Leu1). The probes were designed to be complementary to the 5′ end of each tRNA and hence recognize both full length tRNA and 5′ tsRNA. We also included a probe that was designed against the middle region (anticodon loop) of tRNA-Leu for comparison (Leu2). When hybridizing blots of RNA isolated from L. donovani total cells and versus exosomes with these probes, we detected a common band corresponding to the full length tRNA in total and exosome RNA in case of both the probes Asp1 and Leu1 (72 nt and 82 nt respectively) . Thus, despite the abundance of longer sequences in total cell RNA, we were unable to detect them in exosomes. The enrichment of short RNA sequences in leishmania exosomes is concordant with the majority of reports on exosome RNA in other organisms published thus far . Alt. Altbonaar  subgenus have been shown to have a functional RNAi pathway and actively produce siRNAs [Leishmania (Leishmania) species, such as L. major and L. donovani [L. braziliensis and L. donovani in parallel in order to compare the exosome RNA trascriptome of an RNAi-competent organism with an RNAi-deficient one.Numerous studies on exosome RNA have reported the presence of small regulatory RNAs such as micro RNAs (miRNAs) in these vesicles. It was of interest, therefore, to examine the leishmania exosome RNA content in detail by high throughput sequencing. It is important to mention here that leishmania are a special case with regard to small regulatory RNA pathways: e siRNAs ,32. Convdonovani . With thL. braziliensis exosomes uniquely contained transcripts derived from siRNA-coding putative mobile elements and repeats, such as SLACS and TATEs [When sequencing exosomal RNAs from these two leishmania species, we found that they both contained a variety of small non-coding RNA species, the majority of which appeared to be cleavage products derived from longer known non-coding RNAs such as rRNA, tRNA, snoRNA and snRNA. We also saw a small number of reads mapping to protein coding genes. In addition, we discovered a number of novel transcripts that were conserved in both libraries, and nd TATEs . Other snd TATEs ,37,39,43bona fide exosomes from a protozoan parasite and provide a comprehensive analysis of high-throughput sequencing data of exosomal RNA cargo. By virtue of comparing two distinct (old and new world) species of leishmania, we have made the serendipitous discovery that the packaging of specific RNA sequences into exosomes appears to be a conserved phenomenon in leishmania. At the present time it remains unclear whether our findings are illustrative of what happens in other eukaryotic pathogens; however, there is some evidence indicating that the release of RNA within vesicles might occur in other parasitic organisms as well. In particular, there have been two articles published by independent groups that demonstrate the release of tRNA-derived small RNAs and other types of RNA within extracellular vesicles shed by the protozoan T. cruzi [bona fide exosomes. The distinction between exosomes and other extracellular vesicles is important, as their origin, mechanism of biogenesis and thus loading of cargo differs substantially [Trichomonas vaginalis) and the other two helminths (Heligmosomoides polygyrus and Dicrocoelium dendriticum). However, all of these studies have significant limitations in their experimental design and RNA analysis when compared with the present study. The study on T. vaginalis only shows a size profile of RNA purified from exosomes measured by Bioanalyzer, but no sequencing data on exosomal RNA [D. dendriticum describes the analysis of exosomes by high-throughput sequencing; however, data analysis was focused on looking at micro RNA and does not report on other types of RNA in the exosomes [H. polygyrus reports sequencing data of libraries that have been generated with RNA obtained from parasite secretions and a vesicular fraction collected by ultracentrifugation, but not RNA from bona fide exosomes that were purified with specific exosome purification protocols [Importantly, our study is the first to purify T. cruzi ,44; howeantially . Three oomal RNA . The artexosomes . Lastly,rotocols . The limL. donovani does not have the canonical proteins that are required for functional RNAi including AGO. However, an AGO/PIWI-like protein homolog, has been found in RNAi-deficient leishmania and other trypanosomes [cis-acting regulatory motifs within the 3′ UTRs of mRNAs and trans-acting RNA-binding proteins [T. cruzi. The authors identified a unique AGO/PIWI protein termed TcPIWI-tryp that is expressed in all life cycle stages of the parasite and localizes to the cytoplasm [Results from a number of studies -51 led tanosomes . The funanosomes . The preproteins -56. Otheytoplasm . Interesytoplasm . HoweverT. cruzi [A large portion of reads in both our libraries mapped to rRNA genes in the reference genomes and they appeared to be shorter fragments. The presence of rRNA-derived sequences in leishmania exosomes is in agreement with other recent reports on exosome RNA cargo ,58. SequT. cruzi ,44. At tT. cruzi . These sBotrytis cinera could silence Arabidopsis and tomato genes involved in plant immunity by binding host AGO [T. cruzi produces tRNA-derived small RNAs (tsRNAs) that may be delivered to susceptible mammalian cells [T. cruzi tsRNAs can modify the expression of specific genes as assessed by microarray [Our finding that leishmania exosomes are overall enriched in non-coding RNA fragments which are taken up by host mononuclear cells, raises the interesting possibility that these RNA fragments may interfere with gene expression in the host, possibly by binding to host AGO. This type of epigenetic regulation of gene expression across kingdoms has been proposed, but so far little consistent and conclusive evidence has been presented. One elegant study recently showed that small RNAs from the plant fungal pathogen host AGO . This waan cells . Moreovecroarray . It rematrans-splicing. The fact that the majority of novel transcripts contained one or more ORFs suggests that they may be translated into peptides or proteins. However, we were not able to find any homologous protein in any other species.When examining reads that were less abundant in the libraries, we discovered 12 distinct genomic loci Table  that appTwo of the twelve novel transcripts that were most abundant from the group were further examined by quantifying their expression in exosomes in comparison to total cells by Northern analysis Figure B. We fouOne question that remains to be answered is what type of RNA these novel transcripts represent  or whether they represent novel type(s) of RNA. Further studies will be needed to properly address the functions of these novel transcripts.L. braziliensis exosome RNA library that mapped to siRNA coding loci such as SLACS and TATEs in the L. braziliensis genome. Even though the functional significance of endogenous siRNAs in L. braziliensis is still unclear, they are thought to be a genome defence mechanism to control the spread of potentially harmful nucleic acids, such as mobile elements, repeats and viruses [L. braziliensis mature siRNAs (LbrAGO1-bound) [L. braziliensis packages these putative siRNA sequences as cargo of exosomes is of significant interest. It implies that these RNAs may function not only in the cell where they originated, but that they may also act in intercellular communication when taken up by other leishmania or by host cells or both. To our knowledge, no other parasite has been shown to release pathogen-derived siRNAs within vesicles directly. Further studies will be needed to confirm the identity of the sequences as siRNAs and delineate their function in parasite biology and in host-pathogen interaction.We detected a number of sequences in the  viruses . Althoug1-bound) . This miL. donovani and L. braziliensis exosome transcriptomes. Only recently tRNA fragmentation has been recognized as a specific process. tRNA fragments have been found in all domains of life and can be divided into several categories, depending on the cleavage site: cleavage within the anticodon loop gives rise to 5′ and 3′ tRNA halves (30–35 nt), and cleavage within the D-arm (5′) or T-arm (3′) gives rise to smaller tRNA-derived RNA-fragments (tRFs) (13–20 nt) [et al. showed that tRFs can associate with Argonaute proteins, however, they associated more effeciently with the non-silencing AGO3 and AGO4 [A striking finding of the present study was the abundance of tRNA fragments principally originating from a small subset of tRNA isoacceptors Figure B that we3–20 nt) . Each of3–20 nt) , whereas3–20 nt) ,63. Toge3–20 nt) . tRFs, o3–20 nt) . Further3–20 nt) . In addiand AGO4 . Furtherand AGO4 .Plasmodium berghei and Toxoplasma gondii, tRNA-halves were detected, and a relation between tRNA-half production and growth rate was observed [T. cruzi. Despite the fact that the lengths and origins of tsRNAs differed slightly from study to study, their production was convincingly demonstrated in both trypomastigotes and epimastigotes in a number of reports [T. cruzi tsRNAs were bound to TcPIWI-tryp, a distinct Argonaute protein that has been described in this RNAi-deficient organism [T. cruzi. The authors proposed that these vesicles may have a role in life cycle transition from epimastigote to trypomastigote as well as contribute to infection susceptibility of mammalian cells [T. cruzi may participate in non-canonical regulatory pathways and raise the question as to whether a similar phenomenon may be operative in leishmania.A small number of studies have looked at the presence of tsRNAs in protozoan parasites. In observed . However reports ,29,69. Iorganism ,57. The an cells . These fbona fide exosomes, competent for delivery to mammalian cells. The major fraction of tsRNAs found in both L. donovani and L. braziliensis exosomes were 5′ tRNA halves, however, we also found shorter tsRNAs derived from the D-arm or T-arm of the tRNA, corresponding to 5′tRFs and 3′tRFs. The production and presence of tRNA halves in exosomes from leishmania amastigotes might be a result of the elevated temperature and acidic pH the parasites were exposed to. We found that the vast majority of tsRNAs in leishmania exosomes were derived from tRNA-Asp, tRNA-Gln, tRNA-Leu, tRNA-Glu and tRNA-Gly  RNA pool.In the present study, we provide evidence that leishmania also produces specific tsRNAs, and that these potential regulatory RNAs are released by the intracellular amastigote stage within At this point, the mechanism of biogenesis and function of tsRNAs in leishmania is unknown. We are cannot, therefore, be 100% certain that the same classification of tRNA halves and tRFs as recently proposed by several groups ,63,70 apL. braziliensis appears to package putative siRNAs or their precursors into exosomes, whereas RNAi deficient L. donovani does not. Based on Northern analyses, our data indicate that specific RNA sequences are selectively, and in some cases quantitatively packaged into exosomes. This conclusion is supported further by the highly biased distribution of sequences detected in exosomes over only a subset of genes in the leishmania genome combined with a striking paucity of transcripts derived from protein coding genes which are otherwise abundant in total cellular RNA.In summary, this report provides evidence that leishmania exosomes are enriched in short sequences derived from non-coding RNAs such as rRNAs and tRNAs. Moreover, exosomes contain a number of novel transcripts, albeit in relatively low abundance. The RNAi-proficient L. donovani and L. braziliensis exosomes, 2) the vast majority of identified novel transcripts was present in exosomes from both species, and 3) the most abundant tsRNAs found in exosomes were derived from a highly biased subset of the same tRNA isoacceptors in both species. Taken together, the data argue strongly that leishmania exosomal RNA sequences are specifically produced and packaged into exosomes for release, likely with the purpose of modifying host cell phenotype to support chronic infection. The investigation of the precise functions of these small, non-coding, leishmania RNAs should contribute significantly to our understanding of parasite biology and mechanisms of pathogenesis.Importantly, our findings provide at least three lines of evidence arguing for the presence of an evolutionarily conserved mechanism for packaging small non-coding RNAs into exosomes in leishmania: 1) the high degree overlap between the top 20 most abundant sequences found in L. donovani Sudan strain S2 promastigotes were routinely cultured in M199 (Sigma-Aldrich) with 10% heat inactivated fetal bovine serum , 20 mM HEPES (Stemcell), 6 μg/mL hemin (Sigma-Aldrich), 10 μg/mL folic acid (Sigma-Aldrich), 2 mM L-glutamine (Stemcell), 100 U/mL penicillin/streptomycin (Stemcell) and 100 uM adenosine (Sigma-Aldrich) at 26°C. Every 3 days the organisms were subcultured 1:10 in fresh medium and were kept in culture for a maximum of 20–25 passages. Fresh parasites were obtained by purification of amastigotes from spleens of infected Syrian Golden hamsters followed by in vitro transformation into promastigotes by culturing for 7 days at 26°C in promastigote media.L. braziliensis  promastigotes were routinely cultured in the same media as above except for supplementation with 20% FBS. L. braziliensis promastigotes were subcultured 1:5 every 3 days in fresh media and kept at 26°C.L. donovani and L. braziliensis axenic amastigote culture supernatant as described previously [L. braziliensis and 37°C for L. donovani. After 24 hours of incubation, exosomes were purified from the 400–800 mL culture supernatant under endotoxin-free conditions by a series of centrifugation and filtration steps, followed by flotation on a sucrose cushion, as described in [Exosomes were purified from eviously ,18. BrieThe size and concentration of the isolated exosomes were analysed using the NanoSight™ LM10-HS10 system . For analysis, a monochromatic laser beam (405 nm) was applied to the diluted exosome solution (1:100 in 0.02 μm filtered PBS) that was injected into a LM12 viewing unit using a computer controlled syringe pump. NanoSight™ tracking analysis (NTA) software version 2.3 was used to produce the mean and median vesicle size together with an estimate of particle concentration. Samples were measured 3 times to confirm reproducibility.2EDTA, 0.01 M Tris-Cl pH = 7.4, 0.2% SDS, all Sigma-Aldrich) was added to 50 μL of exosomes resuspended in PBS followed by addition of 200 μL Ultra-Pure buffer-saturated phenol pH = 7.4 (Life Technologies). The mixture was vortexed vigorously and centrifuged for 2 minutes at 13,000×g in a microcentrifuge at room temperature. The upper aqueous phase was collected and the phenol extraction was repeated once more followed by two extractions over 200 μL chloroform each (Fisher Scientific). RNA was precipitated by addition of 0.3 M NaCl, 2 μg/mL glycogen (Ambion) and 75% EtOH, and incubation at −20°C over night. RNA was pelleted by centrifugation at 13,000×g for 30 minutes at 4°C. RNA pellets were washed with ice-cold 75% ethanol and resuspended in 10–20 μL ddH2O. RNA concentration was determined by measuring the OD260 with the nanodrop (Thermo).RNA was purified from leishmania exosomes by phenol/chloroform extraction using all RNA-grade reagents. For this purpose, 150 uL of LETS buffer . Alternatively, DNase-treated RNA was run on a 15% polyacrylamide gel, stained with SYBR green (Life Technologies) and imaged with UV-imaging.To look at length profiles of exosome-derived RNA, 2 μg of purified leishmania total RNA and 1 μg of exosome RNA were first treated with 5 units DNase I (Thermo) to remove potential DNA contamination. After incubation for 30 minutes at 37°C, DNase was inactivated by addition of 2.5 mM EDTA and incubation at 65°C for 10 minutes followed by phenol-chloroform extraction and ethanol precipitation as above. RNA was resuspended in 4 μL ddH32PdATP (Life Technologies) and run on 15% polyacrylamide gels followed by imaging with a Typhoon phosphor-imager .To confirm identity of nucleic acid purified from exosomes as RNA, 1–2 μg of phenol/chloroform extracted RNA was treated with DNase (as above), followed by treatment with either 0.4 mg/mL RNase A (Thermo) for 15 mins at 37°C or hydrolysis with 50 mM KOH (Sigma-Aldrich) for 15 min at 95°C. Samples were then 5′ end labeled according to the manufacturer’s instructions using polynucleotide kinase (PNK)  and γ2O and run on the Agilent Bioanalyzer to determine whether or not RNA had undergone degradation.To assess whether the exosomal membrane was protecting the vesicular RNA content from degradation by exogenous RNases, intact exosomes resuspended in PBS  were treated with 0.4 mg/mL RNAse A for 15 mins at 37°C in the presence or absence of 0.1% Triton X-100 (Sigma-Aldrich). As a control for RNase activity, 1 μL of prepared RNA pico ladder (Agilent) was treated with RNase A under the same conditions. After incubation, samples were extracted with phenol/chloroform 2× each and RNA was precipitated with ethanol as above. Samples were then treated with DNase, again phenol/chloroform extracted and ethanol precipitated, resuspended in 4 μL ddHL. donovani axenic amastigotes as described above. Pelleted exosomes were resuspended in 100 μL PBS. Protein concentration in the exosome preparation was determined using the Micro BCA Protein Assay kit (Pierce). Exosomes were then stained with the membrane-permeant, RNA-specific dye SYTO RNASelect (Life Technologies) according to the manufacturer’s recommendations. For this purpose, the SYTO dye was diluted in DMSO and added to the resuspended exosomes at a final concentration of 10 μM, followed by 20 minutes incubation at 37°C. Excess unbound dye was removed by washing twice with 1 mL PBS, pelleting the exosomes at 100,000×g for 1 hour at 4°C. Exosomes were then resuspended in the original volume of PBS (100 μL). Labelling efficiency was assessed by fluorescence microscopy using an Axioplan II epifluorescence microscope equipped with 63×/1.4 Plan-Apochromat objective (Carl Zeiss Inc). Images were recorded using an AxioCam MRm Camera coupled to the AxioVision software Version 4.8.2 (Carl Zeiss Inc.).Exosomes were purified from 400 mL culture supernatant of To investigate the exosome-mediated delivery of RNA to host macrophages, THP-1 cells were differentiated over night with 10 ng/mL phorbol-12-myristate 13-acetate (PMA), followed by washing and resting cells for 24 hours. Differentiated cells were then treated with labeled exosomes for 2 hours at 37°C. As a negative control, cells were treated with labelled exosomes and incubated at 4°C for 2 hours, preventing phagocytosis. For quantification of exosome RNA uptake, exosome-treated THP-1 cells were washed 3× with PBS to remove non-internalized exosomes. Cells were then fixed with 2% paraformaldehyde (Sigma) in PBS for 15 minutes at room temperature. After fixation, cells were again washed with PBS and then analyzed by flow cytometry . To verify that exosomes were in fact internalized and not just bound to the cell membrane, the same experiment was performed with THP-1 cells grown on coverslips to be analysed by confocal microscopy. After incubation, cells on coverslips were washed and fixed as above, permabilized with 0.1% Triton X-100 in PBS for 5 minutes, and stained with Alexa Fluor 594 phalloidin (Life Technologies) for 1 hour at room temperature in the dark. After 3× washing with PBS, coverslips were mounted with Prolong Gold antifade mounting media containing DAPI (Life Technologies) to detect macrophage nuclei. Confocal microscopy was done with a Leica DMIRE2 inverted microscope equipped with a SP2 AOBS laser scanning head. This is a filter-free spectral confocal and multiphoton microscope, and all imaging operations which include selections of laser, detection channels and other functions are fully automated and computer controlled. Pictures were taken with a 63× magnification oil immersion objective.L. donovani and L. braziliensis exosomes  as starting material. RNA was first treated with DNase I (as described above) to remove potentially contaminating DNA. To remove 5′ phosphates on the RNA, we first performed a calf intestinal alkaline phosphatase (CIP) treatment using 1 unit of CIP (Roche) per 10 μL reaction and incubation for one hour at 37°C. Once incubation was completed, samples were phenol-chloroform extracted twice and ethanol precipitated as described above. In order to monitor the efficiency of the CIP treatment, a parallel reaction was spiked with a 24 nt long radiolabelled RNA, and pre and post incubation with CIP were loaded in a 20% denaturing polyacrylamide gel to follow the disappearance of counts. Next, the CIP treated RNA sample (resuspended in 10 μL ddH2O) was treated with tobacco acid phosphatase (TAP) to remove 5′ caps. Half of the CIP treated RNA sample was combined with 2.5 units of TAP (Epicentre), 1X TAP buffer, brought to a final volume of 10 μL with ddH2O and incubated for one hour at 37°C. In order to control for the efficiency of 5′ cap removal, a parallel reaction was spiked with γ32PdATP and pre-and post ligation samples were loaded in a 20% plyacrylamide gel to monitor the disappearance of counts. RNA was ethanol-precipitated and resuspended in 10 μL ddH2O. The CIP and TAP treated RNA were then labeled with polynucleotide kinase (PNK) to have the same 5′ phosphate in all RNA molecules about to be ligated. Ten units of PNK (NEB) were used along with 1X PNK buffer (NEB), and γ32PdATP in a 10 μL reaction and incubation for one hour at 37°C. Next, ~ 10% of the sample was loaded onto a denaturing 15% polyacrylamide gel. The remainder of the PNK reaction was taken to a 30 μL volume with ddH2O and run through a dye terminator removal (DTR) cartridge (EdgeBio) following manufacture’s indications in order to remove ions and unincorporated γ32PdATP. The sample was ethanol precipitated and resuspended in 5 μL of ddH2O. The next step was to ligate a custom adenylated AppDNA adaptor (5′ App-GAAGAGCCTACGACGA) to the 3′ end of RNA molecules. This adaptor was slightly modified so that the 3′ end was blocked in order to prevent self ligation. Half of the pre-treated exosomal RNA sample was combined with T4 RNA ligase buffer  was added. The ligation reaction was gel purified from a denaturing 10% polyacrylamide gel and the recovered material was used as a template in a reverse transcription (RT) reaction. For this purpose, half of the recovered sample was combined with 100 μM RT primer (5′ TCGTCGTAGGCTCTTC), ddH2O and incubated at 80°C for two minutes. After cooling samples down slowly, 1X First Strand Buffer (Life Technologies), 0.8 μM dNTP and 200 U of Superscript II Reverse Transcriptase (Life Technologies) were added. Controls with no enzyme and no template in the reaction were prepared in parallel. Reactions were incubated for 1 hour at 48°C. The RNA template was hydrolyzed by heating in the presence of 100 mM KOH followed by neutralization with 1 M Tris–HCl pH = 5 , and the resulting cDNA was isolated on a 10% denaturing polyacrylamide gel. 20 cycles of PCR amplification were performed in the presence of 5 mM MgCl2, 100 μM dNTPs, 1 μM each forward primer (5′ ATCGTAGGCACCTGAAA) and reverse primer (same as RT primer), 1X Taq buffer and 2.5 Units Taq polymerase (UBI). PCR products were then gel purified and quantified by Qubit (Life Technologies) and used as input material for ligation of TruSeq adapters (Illumina) according to the manufacturer’s recommendations. 150 base pair, paired-end sequencing was performed using an Illumina MiSeq instrument (Illumina) at the Epigenomics core of Albert Einstein College of Medicine, NY.We used 1–2 μg of RNA extracted by phenol/chloroform extraction from lycerol; , 2.5 U ohttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/), both L. donovani and L. braziliensis exosomal RNA reads had their adapters trimmed by cutadapt version 1.0 . For each library, the output files from the trimming were separated into RNA adapter-trimmed reads and DNA adapter-trimmed reads, and the former was used to guide the assignment of correct orientation for all reads sequenced. We ran FLASh (settings: −M 100 –x 0.2) [http://hannonlab.cshl.edu/fastx_toolkit) to respectively combine the mates, for the cases where DNA inserts were shorter than twice the length of reads, and then collapsed identical sequences into single ones to facilitate handling the data in subsequent specific analyses. Bowtie2 version 2.1.0  [Leishmania donovani strain BPK282A1) and LbrM (Leishmania braziliensis MHOM/BR/75/M2904) from TriTrypDB version 6.0), as well as against the species with the best assembled and annotated genome . The very-sensitive-local setting of Bowtie 2 uses a seed length of 20 nt for the alignment, and the –N1 command allows for only one mismatch on that seed alignment. The alignments with LdBPK and LbrM were used to categorize the exosomal RNAs for the respective species, relying on htseq-count script [After completion of Illumina paired-end sequencing and read quality control checking by FastQC ( –x 0.2)  and FASTcal –N1)  was usedt script  and the t script  for the t script  was usedL. major tRNAs for a better categorization of potential tRNA-derived small RNAs present within the exosomes. An ad-hoc PERL script was written to calculate the cReads position within each tRNA feature they mapped onto: 5′end , mid-5′ (cReads starting on the first 1/3 and ending before the last 1/3 of the tRNA gene length), 3′end , mid-3′end (cReads starting after the first 1/3 and ending within the last 1/3 of tRNA gene length), mid . The same method was used to calculate the cReads position within each rRNA feature for rRNA fragments found in exosomes.As mentioned above, we used reads mapping to The discovered 12 novel transcribed loci had their nucleotide sequences translated by the getorf program from the EMBOSS package  with theL. braziliensis-specific SLACS/TATEs elements (extracted from TriTrypDB-6.0_LbraziliensisMHOMBR75M2904_ AnnotatedTranscripts.fasta downloadable file at tritrypdb.org). The following thresholds were applied during this screen: e-Value < = 1, identity > = 80% and query (cRead) coverage > = 70%.To determine whether there were transposable elements-derived RNA fragments within leishmania exosomes and also discard any possibility of cross-contamination between the libraries, we performed a BlastN search  for L. bLutzomyia longipalpis and Phlebotomus papatasi,https://www.vectorbase.org/) reference genomes. Bowtie2 version 2.1.0  [http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html)  were used for this purpose.To search for any sequences homologous to mammalian miRNAs within the leishmania exosomal RNA libraries, we ran blastn from the BLAST Plus package  version cal –N1)  and htseL. donovani axenic amastigote total RNA from a single culture, or exosome RNA pooled from 4 separate exosome preparations) were loaded onto 8% denaturing polyacrylamide gels. Gels were stained with SYBR Green and visualized, then the samples were blotted onto Hybond N+ nylon membrane . The membranes were UV cross-linked using a Stratalinker (1200 μJ for 30 seconds), blocked, probed and washed according to [in vitro transcribed RNAs were used as size markers. For hybridization, 5′end labelled DNA probes were used . For 5′erm DNA; ) in a HyR environment version 3.0 was used to generate read length distribution histograms for each library , as well as to perform the Pearson’s Correlation analysis regarding the tRNA-derived small RNA reads abundance and the amino acid usage frequency of the respective predicted proteomes. Other graphs were generated with GraphPad Prism 4.0 and EXCEL.


The aim of the present study was to examine the biological differences between seminomas with occult and clinically apparent metastases at the time of diagnosis of the primary tumor to gain insight into the biology of these tumors and facilitate the identification of novel predictors of seminoma metastasis.Total RNA including small RNAs was isolated from testicular tumors of patients with pure seminoma presenting with lymphogenic metastasis  and occult metastasis . The regulation of biological processes was examined (1) throughout the mRNA transcriptome  and (2) the miRNA transcriptome employing small RNA next generation sequencing . Protein coding genes (mRNAs) and small RNAs showing a significant (≥2-fold) difference between the groups were identified. Finally (3), we examined 95 candidate miRNAs in 36 apparent metastasized and another 5 occult metastasized seminoma using logistic regression analysis.6/13.94×106 small RNA reads were identified for apparent/occult metastasized seminoma. These reads on average convert into 9,901/9,675 small RNAs including 422/404 mature microRNAs. None of these mRNAs/small RNAs met our selection criteria for candidate genes. From 95 candidate miRNAs 44 appeared expressed, with 3 of them showing weak but significant (p = 0.05) differences among both groups.Among 19,596 genes, on average 12,894 mRNAs appeared expressed  and 16.99×10Occult and apparent metastasized seminomas are biologically almost indistinguishable and probably represent no separate tumor entities. These findings may simplify future research on seminoma metastasis. Testicular tumor is the most common tumor among young men and is associated with a 5-year survival rate of approximately 100% in the early stages. Pure seminoma is currently the most frequent histological subtype (55%) and in up to 70% of cases, it presents without visible metastasis at primary staging Recent studies propose the existence of certain risk factors associated with both, apparent and occult seminoma metastasis. For instance, multivariate analyses showed that large tumor size (>6 cm) and infiltration of the rete testis are risk factors associated with clinically apparent metastasis Considering these similarities, we hypothesized that primary tumors with clinically apparent metastases and those with occult metastases might share a considerable number of biological characteristics (namely the process of metastasis). If seminomas with apparent and occult metastasis do not represent different metastatic subtypes, this would simplify future studies considerably, because we would only have to discriminate metastasized seminoma from non-metastasized seminoma without considering subtypes of metastasis. Investigation at the molecular level appear promising: a study from our group showed that clinical risk factors discriminated metastasized from non-metastasized seminomas in approximately 65% of cases In the present study, we investigated differences in the regulation of biological processes at the mRNA and miRNA transcriptional level between seminomas with occult and those with apparent metastases. As a first approach we performed a whole genome microarray analysis to screen for genome-wide mRNA transcriptional gene expression changes. As a second approach whole genome changes of all small RNAs were assessed by next generation sequencing (NGS). In previous analysis we identified miRNAs which completely discriminated non-metastasized from metastasized seminoma (accepted for publication). As a third approach we used 95 from these miRNA species and examined their potential to discriminate apparent metastasized seminoma (n = 36) from occult metastasized seminoma (n = 5) using qRT-PCR.Patients in both groups comprising occult metastasis seminoma (n = 2×5) presented without visible metastasis at primary staging, received no adjuvant treatment, and developed retroperitoneal tumors during the 2-year follow-up. Patients in both groups comprising apparent metastasis at primary staging (n = 5 and n = 36) were limited to those with clinical stage IIb and IIc to include lymphogenic metastatic spread only and to provide a high level of diagnostic accuracy (avoiding questionable lymph nodes).Samples from testicular tumor biopsies were incubated in RNA-later solution  immediately after collection and later stored at −20°C. All tissue samples were examined by an experienced pathologist for histological and TNM classification . The locTissues frozen in RNA-later solution  were carefully thawed, homogenized  and digested using proteinase K. Total RNA including small RNAs was isolated  and the remaining DNA was digested. RNA was stored at −80°C until use. The quality and quantity of isolated total RNA were measured spectrophotometrically , and RNA integrity was assessed by the 2100 Agilent Bioanalyser . Only RNA specimens with a A260/A280 ratio ≥2.0 (Nanodrop) and RNA integrity number (RIN)≥8.0 were used for whole genome microarray or small RNA Next Generation Sequencing  or qRT-PCR.http://www.pantherdb.org/), which groups genes with similar biological functions based on their annotation. All data are stored and accessible under the Gene Expression Omnibus platform of NCBI (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55198).Genome-wide expression profiling was performed using the Agilent oligo microarray GE 8×60 K  combined with a one-color based hybridization protocol for 4 samples per group. In brief, total RNA was reverse transcribed into cDNA, converted to cyanine-3-labeled-cRNA , purified, fragmented and hybridized on the microarray. Signals on the microarray were detected using the Agilent DNA Microarray Scanner. GeneSpring GX12 software was used to quantile normalize the raw data. Gene expression  was analyzed as an outcome and compared between the two groups using the non-parametric Welch’s approximate t-test. Genes had to be expressed in >50% of samples per group. Only significant and ≥2-fold differences in gene expression between the two groups were considered for comparisons, and unadjusted p-values and those adjusted for multiple comparisons  were calculated. All gene candidates underwent gene set enrichment analyses using PANTHER pathway software (http://www.mirbase.org/) and known non-coding RNAs . Small RNAs showing a significant Genome-wide small RNA sequencing was performed using the SOLiD5500xl Next Generation Sequencing Technology . In brief, total RNA (5 samples per group) was purified (PureLink miRNA isolation Kit), enriched small RNAs were ligated to SOLiD adaptors and reverse transcribed (SOLiD RT primer and ArrayScript RT). The cDNA was purified , a cut-off size of 60–80 nt was selected , the cDNA was in-gel amplified, and samples were barcoded using a SOLiD 3′Barcode primer at the same time. Amplified cDNA was purified  and used in emulsion PCR (SOLiD EZ Bead). Thereafter, the emulsion was broken to recover enriched beads and the so-called di-base probes were used by the SOLiD system in the sequencing-by-ligation procedure. In addition to the SOLiD5500xl inherent software (LifeScope) used for image and signal processing, the software CLC Genomics Workbench 5.1 (CLC bio) was used for clustering, counting, and annotation of all generated 75 bp reads. After discarding reads without an adaptor sequence and those shorter than 15 bp (trimming), reads were assigned to known miRNAs  was used to simultaneously detect 95 miRNAs (TaqMan primer probe assays) on a 384-well platform (LDA). A 20 ng RNA aliquot of each RNA sample was reverse transcribed using the TaqMan microRNA Reverse Transcription Kit. 100 µl cDNA  was mixed with 100 µl 2× RT-PCR master mix and pipetted into 2 fill ports of the LDA. Four different samples could be processed per LDA. Cards were centrifuged twice , sealed, and transferred into the 7900 qRT-PCR instrument. The qRT-PCR was run for two hours following the qRT-PCR protocol for 384-well LDA format. Ahead of our experiment we established the upper limit of the linear-dynamic range of our qRT-PCR using replicate measurements on one of our samples. The upper limit occurred at CT≤29. CT values were normalized relative to the median gene expression of the examined microRNAs. Normalized CT values of both groups were examined on their discriminative capability employing logistic regression analysis.All chemicals for qRT-PCR using TaqMan chemistry were provided by Life Technologies, Darmstadt, Germany. All technical procedures for qRT-PCR were performed in accordance with standard operating procedures implemented in our laboratory in 2008 when the Bundeswehr Institute of Radiobiology became accredited according to DIN EN ISO 9001/2008.For whole genome microarray analysis and NGS methodology we isolated on average 38.9 µg of total RNA  per 10 mg of biopsy samples. The average RNA integrity number (RIN) was 8.8 .For qRT-PCR analysis we isolated on average 30.6 µg of total RNA  per 10 mg of biopsy samples. The average RNA integrity number (RIN) was 8.3 . One outlier with RIN 4.6 in additional quality control experiments did show no signs of degradation including the qRT-PCR controls. No DNA contamination could be detected in our samples.Of 19,596 gene mRNAs  spotted on the whole genome microarray, on average 65.8%  were distinguishable from the background (expressed). The biological replicate samples showed high correlation coefficients (Pearson’s correlation) within and between experimental groups:R-squared values from 0.912–0.975 within apparent metastasisR-squared values from 0.938–0.967 within occult metastasisR-squared values from 0.872–0.978 between the two groups.No differentially expressed RNAs were identified by applying the stringent approach . Therefore, the non-stringent filtering approach  was applied. No up-regulated and only 30 down-regulated genes were identified. Median p-values of 0.03 (range: 0.001−0.05) and median fold-changes of 2.7 (SD+/−2.5) with higher fold-changes associated with higher p-values (due to higher variance) suggested that most of these 30 genes met the selection criteria at the border of becoming significant. Because downstream enrichment analysis requires lists of at least 100 differentially expressed genes, to run this analysis we changed our inclusion criteria and generated lists with fold-change cut-offs ≥1.5 and uncorrected p-values ≤0.05 knowing that this would increase the number of false positive candidate genes. The resulting list comprised 149 up-regulated and 132 down-regulated genes. Using this list for downstream enrichment analysis of biological processes, molecular functions, cellular components, protein classes and pathways did not generate any significant enrichment for up- or down-regulated genes.6/13.94×106 (SD+/−2.35×106/1.99×106) and on average 30.4%/30.1% reads (SD+/−15.3%/14.6%) remained for further analysis after trimming of the reads. Among these reads, 53.3–69.6%/53.2–66.1% appeared as annotated reads with 7.1–11.9%/7.4–13.6% representing annotated small RNAs. These small RNA reads convert to an average number of 9,901/9,675  small RNA species eligible for statistical analysis in apparent/occult metastasized seminoma, respectively. Among these, we counted an average of 422/404 mature microRNAs . None of these small RNAs met our selection criteria for candidate genes.The average total number of reads for apparent/occult metastasized seminoma was 16.99×10From 95 miRNAs  altogether 61 miRNAs appeared expressed . Of them 44 miRNAs were expressed in at least 50% of the samples and eligible for logistic regression analysis . Only 3 In the present study, we examined biological differences between seminomas with clinically apparent metastasis at the primary staging and those without (occult). Whole genome screening for protein coding genes detected approximately 66% of all known genes. Further analysis of these 12,894 genes using our stringent selection criteria showed no differentially expressed genes, and only 30 down-regulated genes were identified using uncorrected p-values. Those genes appeared as weak candidates based on their fold-change differences and the p-values close to 0.05, in addition to the bias associated with false positive results due to the p-values not corrected for multiple comparisons. Therefore, they were considered questionable candidate genes. To detect changes in gene expression associated with the regulation of different biological processes, we used a less stringent filtering process to identify candidate genes for PANTHER analysis, keeping in mind that the candidate RNAs identified by this filtering approach must be interpreted with caution. Despite this modification, no biological processes, molecular functions, cellular components, protein classes or pathways appeared significantly over or underrepresented in the PANTHER analysis.We also examined approximately 9,800 small RNAs, including 413 mature microRNAs, and no significant changes in gene expression were observed between the two metastasis subtypes.With a third approach we increased the sample size (n = 41) and focused on those 95 miRNAs which according to recent work significantly discriminate metastasized from non-metastasized seminoma (accepted for publication). However, when using these genes to discriminate apparent from occult metastasized seminoma we only found weak associations with p-values close to 0.05 for three miRNAs. These p-values were partly calculated based on a small sample size (n = 3) in the group of occult metastasized seminoma and, therefore, should be judged cautiously.These similarities in the regulation of biological processes at the mRNA as well as the miRNA transcriptome suggest that the two metastasis subtypes do have more features in common (e.g. process of metastasis and corresponding gene expression regulation) than differing from each other.Our findings are also in agreement with prior clinical and epidemiological findings showing that metastasis incidence is positively associated with an increased size of the primary tumor Another important factor is the identification of parameters capable of discriminating metastasized from non-metastasized seminoma. In this regard, modifications at the mRNA and miRNA transcriptome are superior to already identified clinical or epidemiological risk factors The present study has several limitations. The number of biopsies examined for whole genome transcriptome changes was low. However, with the screening methodologies used, false positive and false negative results are a bigger concern than low power: from a methodological and statistical point of view, there is a greater likelihood of “seeing more” than of overlooking associations, in particular in the absence of adjustment for multiple comparisons as in the present study. In our experience, the use of whole genome microarrays is associated with a rate of false positive/negative results of approximately 20% In order to increase the tumor sample size we performed a third examination comprising 95 genes (miRNAs) and 41 samples with 5 samples originating from patients who decided in favor of surveillance and developed a later metastasis. Still, the additional number of occult metastasis employed was only 5, but these patients are rare when bearing in mind that it represents the output of collected samples from 3 testis cancer centers over the last 7 years.In conclusion, seminomas with occult metastasis and those with apparent metastasis at the time of the diagnosis of the primary tumor do have more features in common than differing from each other, thus making them almost indistinguishable from the biological point of view. Hence, both metastasis subtypes may not represent separate tumor entities. This will simplify future molecular biological examinations using mRNAs or small RNAs as potential risk factors to discriminate metastasized from non-metastasized seminoma.


How did life emerge on Earth? The aim of the Network of Researchers on Horizontal Gene Transfer and the Last Universal Cellular Ancestor (NoR HGT & LUCA) is to understand how the genetics of LUCAs were reorganised prior to the advent of the three domains of life. This paper reports the research of eminent scientists who have come together within the network and are making significant contributions to the wider knowledge base surrounding this, one of science’s remaining mysteries. I also report on their relevance in relation to LUCAs and life’s origins, as well as ask a question: what next? It is perplexing, the origin of life. This is because understanding the processes of life’s origin is mind-bogglingly complex. It has been over 60 years since Stanley Miller carried out his world famous “electric discharge” experiment, and the newspapers subsequently heralded “life in a test tube” after he had succeeded in making amino acids from “electrified” primordial gases . To dateThe more widely-recognised modes of HGT include transduction and transformation, and these are thought to be the result of Darwinian evolution. If this is the case, then how did HGT occur during the genetic evolution of pre-LUCAs in the absence of Darwinian evolution  are enzymes that bring about replication of RNA from an RNA template, and these enzymes are part and parcel of the genomes of all RNA-containing viruses, with no DNA involvement at any stage. More importantly, they are prone to introducing mutations, especially during viral genome replication, thus causing the virus to evolve quickly. To study the rare biological events, a “single molecule technique” is an amazing toolbox that could be deployed; a particularly relevant process is the mechanism of error incorporation by RNA polymerase involved in genome replication, which is the basis of evolution. Approaches, such as stop flow assays, have been developed and have shed light on many sub-steps in nucleotide incorporation. However, they struggle to distinguish low probability events from the bulk, as error incorporation. On the other hand, single molecule techniques were not parallelised enough to reach the necessary statistic to characterise events happening only one in a thousand to ten thousand. Therefore, Dulin et al.  have devet al. . This meet al. . Such teet al. . The preet al. [versus 5'–3' phosphodiester bonds during preRNA chemistry and, in the absence of sophisticated enzymes in general, with the eventual formation of 5'–3' phosphodiester bonds, results in an important step forward for the canonical position of RNA as a self-replicator [The emergence of life depended on the chemical evolution of RNA and its subsequent ability to carry chemical information, to perform catalytic activity, to self-replicate and to be subjected to mutation. The latter event allowed the molecule to evolve according to the changing environmental conditions in the form of selection pressures. Krishnamurthy  asserts et al. ,25, demoplicator . In ordeDuring the LUCA epoch in particular, ribozymes were very important catalysts, which modified RNAs by splicing them ,28. Twiset al. [What is the origin of such ribozymes? In a new hypothesis entitled: “tRNA core hypothesis: a new model for the origin of the biological system” put forward by Farias et al. , it is eThe lipid-world hypothesis is a direct alternative to that of an RNA world, claiming that compositional assemblies, such as vesicles with multiple amphiphile types, can replicate and undergo evolution . A compui.e., the “LUCA world” [A further step would have been the formation of evolvable vesicles made from non-living molecules, which contained strands of RNA. These initial RNAs replicated through the translation of encoded RNA replicase for 600 generations, and during such replications, mutations in the initial RNA genomes were introduced spontaneously. This soon led to the formation of highly “replicable parasitic (selfish) mutants”; with continued replication, the initial RNA genome gradually reinforced its interaction with the translated replicase, thereby gaining a competitive edge against the mutant RNAs. This showed the presence of Darwinian evolution that probably began during the “RNA world”, A world” . et al. [et al. [2COH) and then on to a whole host of other “living” compounds. This included the formation of carboxylic and amino acids (metabolism first hypothesis), as well as the synthesis of 3',5'-cyclic nucleotides , which formed RNA polymers, such as ribozyme-like catalysts (genetics first hypothesis). The eventual result was that there was no bifurcation of metabolism and genetics, but that both were inter-mingling to form the biology as we know it today; such conclusions were reached via additional experimental, theoretical and computer simulation studies [The presence of these replicators is speculated, and it is also asserted that, according to the literature, nucleotides could spontaneously form and even join RNA oligomers in high concentrations of formamide ; such anet al.  and Pino [et al. . Their i studies ,39.Other biologically-necessary compounds are cofactors, of which one of the most important is nicotinamide adenine dinucleotide (NAD) . NAD is etc. Allen [Paulinella chromatophora has been investigated with the objective being to gain insight into how the genetic repertoire of P. chromatophora evolves from the merger of two physiologically- and genetically-different cells that allowed the host cell (an ancient heterotrophic protist) to control and manipulate the chromatophore (a cyanobacterium) according to its needs [The alkaline hydrothermal vent hypothesis predicts that life arose in the watery environment on the seabed, and that life’s reactions were taking place in tiny clay bubbles, formed during the escape of gas from the mantle, with energy being derived from geothermal sources. In order for life to break out of these bubbles and become an independent, free floating entity, it became necessary for the electro-chemical potential and ionic concentration gradient across cellular boundaries to be regulated and maintained (homeostasis). Such an arrangement would generate the proton motive forces needed for metabolism and active transport, c. Allen  addressets needs ,43,44. Eetc., can create a good foundation upon which to build. Other significant questions remain: what environmental conditions were needed? In what location did life first form? Is RNA chemistry a red herring? We have no definitive answers; however, as can be seen from the scope and quality of the research by such a high calibre group of scientists, there is a willingness to develop further insights into this exceptionally stimulating scientific area and then disseminate the knowledge amongst the widest audience possible. To fully understand the evolution of life from simple non-living chemicals and chemical reactions to the formation of three-dimensional life forms is a challenging matter, encompassing multi-faceted research and investigation into diverse areas of both chemistry and physics. The research pinpoints crucial areas in life’s early development, but intriguing gaps still remain. Key indicators in the report, such as the importance of lightning during HGT, the role played by eutectics in icy water that led to the emergence of RNA and the chemical evolution of viruses from early prehistoric chemistry,


We show that human RIG-I maintains an auto-inhibited resting state owing to the intra-molecular HEL2i-CARD2 interactions while apo MDA5 lacks the analogous intra-molecular interactions and therefore adopts an extended conformation. Our work demonstrates that RIG-I binds and responds differently to short triphosphorylated RNA and long duplex RNA and that sequential addition of RNA and ATP triggers specific allosteric effects leading to RIG-I CARDs activation. We also present a high-resolution protein surface mapping technique that refines the cooperative oligomerization model of neighboring MDA5 molecules on long duplex RNA. Taken together, our data provide a high-resolution view of RLR activation in solution and offer new evidence for the molecular mechanism of RLR activation.RIG-I and MDA5 are the major intracellular immune receptors that recognize viral RNA species and undergo a series of conformational transitions leading to the activation of the interferon-mediated antiviral response. However, to date, full-length RLRs have resisted crystallographic efforts and a molecular description of their activation pathways remains hypothetical. Here we employ hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to probe the  In the vertebrate immune system, RIG-I (Retinoic acid inducible gene 1) like receptors (RLRs) are the major cytoplasmic surveillance proteins that defend the host against viral infection by sensing viral RNA . There aThe RNA sensing modules of RIG-I and MDA5 are homologous in sequence and structure. The HEL domain comprises a conserved Rec-A-like-helicase core (HEL1 and HEL2) and a novel insertion domain HEL2i. Because of this unique domain architecture RLRs are classified as double-stranded RNA stimulated ATPases (DRAs), which also include Dicer and Dicer-related helicases . DRAs prapo duck RIG-I suggests that the CARDs are trapped in an inactive conformation through intra-molecular interactions between CARD2 and HEL2i  has emerged as a sensitive way to study protein folding, protein-ligand interaction and protein intra- and inter-molecular interactions in solution, providing dynamic structural data –46. BackStudies have pointed out that multiple states and sequential conformational changes are essential for the activation of mammalian RIG-I and MDA5 ,41–42,49Escherichia coli cells (Novagen). As described previously, the proteins were expressed in Luria Broth (LB) media at an OD600 of 0.8 upon the addition of 0.5-mM isopropyl-β-D-thiogalactopyranoside (IPTG) and grown at 18°C overnight for 20 h , 115 971 M−1 cm−1 for the mouse MDA5, 82 992 M−1 cm−1 for the MDA5(ΔCARDs:1-306) proteins. The storage buffer contained 25-mM Hepes, pH 7.4, 150-mM NaCl, 5% glycerol, 5-mM β-ME. The protein preparations were flash frozen with liquid nitrogen and stored at −80°C. The purity for each protein was >95% as verified on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).The constructs were cloned into the pET-SUMO vector (Invitrogen) and transformed into Rosetta II (DE3) for 20 h . The celUUCGGCCGCGCC, 3p10L: 5′pppGGACGUACGUUUCGACGUACGUCC) was transcribed with T7 polymerase and purified on 20% denaturing polyacrylamide gels. Low molecular weight (LMW) polyIC (Invivogen) was dissolved in buffer containing 25-mM Hepes (pH 7.4), 150-mM NaCl, 5% glycerol, 5-mM β-ME to a final concentration of 10-mg/ml. Concentrations were determined spectrophotometrically.As described previously , 5′ trip2O was replaced with D2O (99.99%) and glycerol was excluded. Specifically, we added 1-mM ATP into D2O buffer (ATP&D2O buffer) for ATP hydrolysis studies associated with all proteins. All samples with and without RNA ligands were incubated on ice for 1 h prior to HDX. Four microliters of 18-μM RIG-I or RIG-IΔCARDs in the presence or absence of RNA ligand (100-μM 3p10L or 5-mg/ml polyIC) were mixed with 16 μl D2O buffer  or H2O buffer for 0-s samples, and incubated at 4°C at various time intervals for on-exchange, e.g. 10, 30, 60, 300, 900 and 3600 s. To study ATP hydrolysis, 4 μl 18-μM of RIG-I or RIG-IΔCARDs with respective RNA ligands (100-μM 3p10L and 5-mg/ml polyIC) were incubated on ice for 1 h before mixing the RNA–protein complex with 16-μl ATP&D2O or ATP&H2O buffer for 0-s samples, and incubated at 4°C at the same time intervals. Similarly, 4 μl of 18-μM MDA5 or MDA5ΔCARDs (with or without 5-mg/ml polyIC) were mixed with 16-μl D2O buffer  or H2O buffer for 0-s samples, and incubated at 4°C at various time intervals, e.g. 10, 30, 60, 300, 900 and 3600 s. To study ATP hydrolysis, 4 μl of 18-μM MDA5 or MDA5ΔCARDs with 5-mg/ml polyIC were mixed with 16-μl ATP&D2O or ATP&H2O buffer for 0-s samples, and incubated at 4°C at the same time intervals. Ice-cold 20 μl quench solution consisting of 1-M guanidine hydrochloride and 1.5% (v/v) formic acid were added to each on-exchanged sample after specific time intervals prior to rapid freezing by liquid Nitrogen until HDX-MS analysis. For fully deuterated control samples, 4 μl of 18-μM RIG-I (±200-μM 3p10L) or MDA5 (±5-mg/ml polyIC) were incubated with 16-μl D2O solvent at 37°C for 20 h. The following steps were performed in the same manner as other HDX time point samples. Three replicates were performed for each HDX time point.The buffer for HDX on-exchange was the same composition except that H2O buffer for 1 h, quenched and analyzed by liquid chromatography (LC)-MS. Ice-cold 20 μl quench solution consisting of 1-M guanidine hydrochloride and 1.5% (v/v) formic acid were added to each on-exchanged sample after specific time intervals prior to rapid freezing by liquid Nitrogen until HDX-MS analysis. For ATP analog studies, 4 μl of 18-μM RIG-I were incubated with 100-μM 3p10L on ice for 1 h. Stabilized RIG-I&3p10L complex was then incubated with or without ATP/ADP/ADP-AlFx  for another hour to reach an equilibrium conformation prior to the HDX experiment. Four microliters of RIG-I&3p10L&ATP/ADP/ADP-AlFx  were then incubated with 16 μl D2O buffer for 5 min, quenched and analyzed by LC-MS. Ice-cold 20 μl quench solution consisting of 1-M guanidine hydrochloride and 1.5% (v/v) formic acid were added to each on-exchanged sample after specific time intervals prior to rapid freezing by liquid Nitrogen until HDX-MS analysis. The following steps were performed in the same manner as other HDX time point samples. Three replicates were performed for each HDX time point.For RNA ligand-RIG-I activation studies, 4 μl of 18-μM RIG-I were incubated with 100-μM 3p8L or 100-μM 3p10L or 5-mg/ml polyIC on ice for 1 h prior to HDX experiment. Four microliters of each sample were then mixed with 16 μl of D2O containing 0.3% (v/v) formic acid, and buffer B was acetonitrile containing 0.3% (v/v) formic acid. Protein samples were then digested online by passing through an immobilized pepsin-coupled column (2.1 mm i.d. × 30 mm) (Invitrogen) and were de-salted for 3 min on a house-packed C4 trap . The mobile phase for online pepsin digestion was buffer A and the flow rate was 150 μl min−1 driven by the LC loading pump (Dionex 3000 RSLC). A 20-min gradient on a house-packed C4 column  was used to separate and elute peptic peptides prior to MS analysis. All parts are connected by 1/16″ OD × 50-μM ID PEEK tubing and the flow rate was 15 μl min−1 driven by LC NC pumps (nano/capillary pumps) (Dionex 3000 RSLC). The gradient started from 5% buffer B and increased to 35% buffer B within 20 min, followed by washing with 90% buffer B for 3 min and equilibration with 1% buffer B for 5 min. MS raw files were acquired in the range of m/z 300–2000 (with mass resolution set to 100 000 at m/z 400) for 30 min in positive mode on an Linear Trap Quadropole (LTQ)-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with an Electrospray ionisation (ESI) source (capillary temperature 275°C and spray voltage of 5 kV). All the HDX systems were strictly performed at 0°C (fully buried on ice and water) and online pepsin digestion was carried out at 16°C. Blank injections were made between every two samples to remove carryover peptides. Data for each time point were repeated three times. All HDX data were normalized to 100% D2O content, corrected for an estimated average deuterium recovery of 70% and analyzed by the software HDX Workbench (http://integratedproteomics.com/products/ip2/) for high-confident peptide identification .For capillary-flow LC, buffer A was Hapo human RIG-I structure was modeled based on the apo duck RIG-I structure (PDB code: 4A2W) and the human RIG-I CTD structure (PDB code: 3LRR). The full-length apo mouse MDA5 structure was modeled based on the RIG-I CARDs (PDB code: 4NQK) and the human MDA5 HEL-CTD (PDB code: 4GL2). The homology modeling program is HHPred (http://toolkit.tuebingen.mpg.de/hhpred) (The full-length /hhpred) .in vitro and in cell-based IFN production assays  as a control, which lacks the N-terminal CARDs and the linker region to the helicase domain Figure . We usedn assays . As illuapo state, the latch peptide Y103-114 on RIG-I CARD2 exhibited the greatest magnitude of protection to HDX with less than 30% deuterium uptake after a 1-h incubation in deuterated solvent. In contrast, in the presence of 3p10L, this protection was greatly compromised with deuterium incorporation of up to 70% during the same time interval  compared to that of RIG-I (5%)  for the gate motif Y566-574 was observed upon 3p10L RNA binding. In contrast, the same surface patch in the RIG-IΔCARDs showed statistically insignificant deuterium incorporations relative to the RNA bound state. Thus, RNA binding allosterically destabilizes the CARD2-HEL2i hydrophobic interface by releasing the RIG-I tandem CARDs. We also observed an intriguing HDX pattern in a second human HEL2i gate motif, V522-539. In X Figure . In cont%) Table .Among the CARD2 peptides that exhibited an RNA-dependent increase in uptake of deuterium Table , peptideIn the absence of the RNA ligand, amide hydrogen within the RNA binding motifs of the HEL and CTD domains consistently exhibited greater exchange with the deuterium solvent Table ,58–59.In order to confirm the opening of the CARD2-HEL2i interface as the first step of RIG-I activation and to validate the HDX-MS method to monitor the activation process, three RNA species—3p8L, 3p10L and polyIC—were used in parallel to stimulate RIG-I in our HDX-MS assay. 3p8L and 3p10L are short RNA hairpins with 5′ triphosphates but of different length (8 and 10 base pairs). 3p8L was shown to bind RIG-I but not elicit RIG-I-mediated interferon production in a cell-based reporter assay, whereas 3p10L both bound and effectively stimulated an RIG-I-mediated interferon response ,20. In cHDX profiles of polyIC bound RIG-I displayed significantly different characteristic patterns of RNA binding compared to that of 3p10L Table . In contapo RIG-I and 3p8L bound RIG-I, indicating that 3p8L is unable to allosterically disrupt the CARD2-HEL2i interface, thus explaining why 3p8L cannot elicit an RIG-I-mediated interferon response. This observation provides direct structure-based evidence in solution that the minimum length requirement of RNA PAMP is at least 10 base pairs.3p8L binds to the RIG-I HEL and CTD domains, as indicated by the HDX protection profiles of the HEL1 motif Ia F293-310 and CTD RNA binding peptide F882-891 Figure . Howeverapo and RNA bound states . This ATP-dependent HDX enhancement also applied to the CTD domain, as shown by the kinetic profiles of the RNA capping loop (V843-856) and RNA binding residues (Y905-913) . These results suggest that the RIG-I:RNA complex becomes less stable upon ATP binding and hydrolysis, which may drive the helicase and CTD to partially disengage from contacts with the RNA ligand and reach a conformational equilibrium in which thermo fluctuations occur more freely.We next examined the role of ATP in driving the sequential conformational rearrangements of RIG-I subsequent to RNA recognition. HDX perturbations of 3p10L bound RIG-I were compared in the presence and absence of ATP  on RNA binding and CARDs opening mechanics of RIG-I , compared to 3p10L alone, 3p10L+ADP and 3p10L+ATP (∼21%) groups  in parallel Figure . To asses Figure . Its adjs Figure . Therefoapo MDA5 and apo RIG-I, we observed that the peptide F630-642 on MDA5 HEL2i surface, corresponding to the RIG-I HEL2i gate motif, showed considerably higher deuterium uptake  in the absence of polyIC . Indeed, we achieved high sequence coverage across seven HDX time points for RIG-I, MDA5 and the corresponding CARDs-deleted constructs. Direct comparisons of the time-resolved HDX-MS snapshots across these apo, RNA bound and ATP-stimulated enzymes enabled us to dissect the early activation steps of the RIG-I and MDA5 signaling cascades. Furthermore, our work also provided direct evidence for distinct RNA recognition and activation mechanics by using three RNA species of different lengths and secondary structure.Understanding the early activation process of the RLR signaling cascade promises to provide important insight into combating viral infection and consequently will be of great biomedical interest. However, the complete delineations of the RLR activation steps require high-resolution structures of full-length RIG-I and MDA5 captured in many different states. This is challenging if not impossible for the conventional methods such as crystallography and electron microscopy. To study the relatively large RLR apo state, the HEL and CTD domains are flexible and fully solvent exposed. This model based on our HDX data agrees with the crystallographic studies on duck apo RIG-I, so it is likely that all vertebrate RIG-I proteins share a common auto-inhibited conformation in the resting state  compared to that of RIG-I (∼60 disordered residues). These intrinsic properties of RIG-I and MDA5 in their resting states ensure complete surveillance against virus infections by sensing all types of foreign RNAs which are heterogeneous both structurally and chemically (Figure Our HDX data clearly show that human RIG-I adopts an auto-inhibited conformation in solution. This conformation is maintained by the intra-molecular interactions between the CARD2 latch peptide Y103-114 and the HEL2i gate motifs Y566-574 and V522-539. In this Our HDX data indicate that for RIG-I, RNA loosens the CARD2-HEL2i intra-molecular interactions and then ATP binding further liberates the CARDs Table . We demoet al. demonstrating that RIG-I can recognize the diphosphates at the 5′ ends of polyIC and trigger interferon production (Despite exhibiting no cooperativity upon binding to polyIC, the CARD2-HEL2i interface in RIG-I is clearly disrupted Table . As suggoduction . Becauseoduction . Based ooduction , althougoduction . Althougoduction ,20,23.In contrast, we found that the MDA5 tandem CARDs are not locked into an auto-inhibitory state and show no remarkable changes in conformation in the presence or absence of polyIC or ATP Table . From ouIn summary, our data illustrate the early events in the RLR activation pathways, which occur in a carefully choreographed order. RIG-I and MDA5 undergo completely different molecular pathways of activation Figure , despiteSupplementary Data are available at NAR Online.


TM Viral RNA Isolation Kit, sufficiently separating nucleic acid analytes from PCR-inhibiting contaminants within the lysate while also critically maintaining high integrity of the viral genome. We find that this design has great potential for rapid, efficient and sensitive nucleic acid separation from patient sample.The effective and robust separation of biomolecules of interest from patient samples is an essential step in diagnostic applications. We present a platform for the fast extraction of nucleic acids from clinical specimens utilizing paramagnetic PMPs, an oil-water interface, a small permanent magnet and a microfluidic channel to separate and purify captured nucleic acids from lysate in less than one minute, circumventing the need for multiple washing steps and greatly simplifying and expediting the purification procedure. Our device was able to isolate influenza RNA from clinical nasopharyngeal swab samples with high efficiency when compared to the Ambion® MagMAX The extraction of DNA, RNA and protein from biological specimens is the first and most essential step for numerous biomedical applications includinMany commercial kits  have been developed for the solid phase extraction (SPE) of nucleic acids by utilizing paramagnetic particles (PMPs) or columns that selectively bind DNA and/or RNA–9. ThougRecent years have thus seen the emergence of microfluidic isolation platforms utilizing SPE, which can be implemented more easily than its liquid-liquid counterpart. These technologies typically involve the flow of lysate through micropillars, silica With a simple design, facile operation and no need for peripheral equipment, immiscible phase filtration has potential to spawn technologies for low resource, POC use as well as for high throughput sample preparation. In the area of NA extraction, Bordelon et al. have constructed a promising design using air surface tension valves to isolate respiratory syncytial virus RNA. RecoverIn this report we describe a stationary microfluidic method for extracting and recovering RNA from patient samples using oil-water interfaces. Our oil chip device differentiates itself by being a closed microfluidic system, which in the POC setting is a necessity to prevent contamination. This device was then tested by extracting influenza RNA from nasopharyngeal swab specimens, representing the first instance of a technique of this kind being used on clinical samples. After mixing the samples with silica PMPs and lysis/binding solution, the PMPs were pulled through an oil-filled microchannel and into a well containing elution buffer. Sample extractions were performed in tandem with the commercial Ambion kit, with the aim of achieving equivalent sensitivity. Eluted total RNA was then quantified with a UV-vis spectrometer, vRNA integrity was confirmed by RNA gel electrophoresis and extraction efficiency was assessed by RT-qPCR.The device consists of three wells joined by two channels intersecting to form a T-junction . The lonThe device is composed of a microscope slide bonded to solid polydimethysiloxane (PDMS) chip, fabricated via soft lithography techniques. PDMS precursor and curing agent  were mixed in a 10:1 mass ratio, degassed for 1 hr, poured over a solid SU-8 structure and allowed to cure for 1 hr at 70°C. The solidified polymer was then excised, hole-punched and finally bonded to a microscope slide via oxygen plasma treatment with a high frequency generator . The chip was then incubated at 70°C for an additional 30 min to enhance bonding.Upon complete bonding, the chip was rinsed with RNAse decontamination solution , followed by several subsequent washes with nuclease-free water. The channels were then cleared of liquid with high-pressure nitrogen gas. Once decontaminated, the channels were filled with olive oil by pipetting approximately 10 μL into the OR and allowing the oil to spread under capillary action. The particular olive oil used in this device had a viscosity of 93 cP at 22°C, as measured by a rheometer ; as well as an interfacial tension of 6.45 mN/m with Ambion lysis-binding solution, as measured by a goniometer . 10 μL of priming solution was then pipetted into each aqueous-phase well (LW and EW). The priming solution consisted of 1 mg/mL bovine serum albumin  and 0.01% Tween 20  in nuclease-free water, with the intention of blocking non-specific binding sites on the channel surfaces, as well as decreasing PMP adsorption to the glass bottom. Once primed, the chip was incubated overnight at 4°C.TM Viral RNA Isolation Kit , eschewing the wash steps and scaling down reagent volumes. The protocol begins with single-step cell lysis and RNA-PMP binding, which involves the mixture of a lysis/binding solution , biological sample and PMP mix. After 4 min of gentle mixing, the PMPs with adsorbed RNA are drawn to the bottom of the tube with a small, cylindrical , grade N52 neodymium magnet  and pipetted out along with 10 μL of lysate, which is then added to the LW after 10 μL of priming solution has been aspirated. This step serves to preconcentrate the PMPs before loading, though a modified device with a larger LW could be used to the same effect. Similarly, the priming solution in the EW is replaced with an equal volume of elution buffer .For RNA separation, we adopted the procedure for the Ambion® MagMAXOnce in the LW, the PMPs are coerced by the magnet to form a compact aggregate, which can then penetrate the oil-water interface and enter the oil-filled channel. Upon interface traversal, the PMPs will remain in a tight clump and glide easily along the channel, following the movement of the magnet. Optimal PMP transfer typically occurs when the speed of the magnet below 1 mm/s; greater speeds may lead to break-up of the PMP aggregate. This process of PMP aggregation, interface penetration and immiscible phase traversal can be completed in 0.5–2 minutes. Once pulled into the EW, the PMPs along with 10 μL of elution buffer are drawn out and vortexed vigorously to elute the RNA from the silica surfaces. Serial clinical samples were obtained from patients presenting with influenza-like symptoms to Memorial Hospital of RI during the 2013–2014 winter influenza season. These nasopharyngeal swab samples, which were suspended in viral transport medium and heat-inactivated, were assigned a randomized code number and then patient de-identified as per Institutional Review Board protocol, which allowed for maintenance of patient confidentiality and for a waiver of informed consent. RNA extraction was performed using both the commercially available MagMAX kit and our oil chip. Eluted samples were then analyzed with a Nanodrop spectrophotometer  for analyte concentration and purity. Viral RNA integrity was assessed with the Bioanalyzer RNA 6000 Pico assay .RT-qPCR was performed on a PikoReal 24 Real-Time PCR System  following the CDC protocol for swine influenza A (H1N1) using the SuperScript III One-Step RT-PCR kit  with all-type influenza A forward (5’-GACCRATCCTGTCACCTCTGAC-3’) and reverse (5’-AGGGCATTYTGGACAAAKCGTCTA-3’) primers and TaqMan probe (5’-TGCAGTCCTCGCTCACTGGGCACG-3’) . PurifieViral RNA extracted from clinical nasal swab samples was examined via gel electrophoresis to initially confirm the successful isolation of influenza RNA. Samples isolated by both kit and chip methodologies were run on an RNA 6000 Pico gel chip  and prodtot as the ratio of OD260 measurements yielded by the oil chip and commercial kit , the oil chip on average performs at Etot = 89.1%  over 28 distinct patient samples. Defining the spectrophotometrically determined percent efficiency E = 89.1% . This siM1/M2 gene segment. Standard and sample curves for patient samples 177, 78 and 193  is likely due to the small amount of inhibitors that are carried over with the PMPs during extraction. Evir was also measured for samples spiked with known amounts of input template. Amplicon was spiked into phosphate buffered saline at various concentrations (ranging from 108 to 106 copies/mL) and processed via kit and oil chip, giving average template recoveries of 36.2 ± 4.4% and 21.9 ± 2.4%, respectively, to give Evir = 60.5 ± 9.8%.The ratio of chip extracted-to-kit extraction vRNA yield, or E samples . Viral ltot and Evir values suggests that there is some inhibition of PCR occurring in the chip-extracted samples. One simple method for determining RNA purity is to quantify the 260/230 optical density ratio, which can signal the presence of lysing agents present in the eluted RNA. Chip-extracted RNA typically gave 260/230 ratios between 0.03 and 0.07, indicating that some amount of lysate was carried over with the PMPs. This may explain why the qPCR-determined viral RNA yield is lower than the OD-determined total RNA yield. It is likely that the oil chip is able to extract vRNA with the same efficiency as with total RNA, but the presence of isopropanol and GuSCN in the eluted sample may inhibit amplification and lead to lower Evir. Nevertheless, if used within a point of care diagnostic device that is mainly concerned with obtaining a positive/negative result, the oil chip technique will still extract sufficient RNA in roughly one third of the time, compared to the commercial kit.Overall, the successful operation of our oil chip device came about without the need for exhaustive optimization, though a comparison of the average Etot and Evir may be explained by the nature of the clinical samples. Different amounts of inflammatory cells and varying viscosities between the patient samples may have differentially affected the operation of the oil chip. Large Evir values, however, typically corresponded with large Etot values, as seen in four patient samples with among the highest Evir values . Additionally, reduction in the extent of RNA shearing may have contributed to variation. Occasionally but not consistently, peak shifting in RNA electropherograms were observed for chip-extracted samples, such that the 900–2500 nt band became skewed towards the right . Because the oil chip procedure requires much less vigorous vortexing, a possible advantageous outcome might be gentler treatment of the RNA, which is prone to shearing.The large variances of EWe found the oil chip to be flexible in its design and operation parameters; different magnet strengths, oil types  and channel dimensions were implemented to roughly the same effect. Ultimately, device efficacy depends on the movement of PMPs as one aggregate across the oil-water interface. Once this step is accomplished, the captured analyte can be separated with (as in the case of influenza RNA from patient sample) negligible contaminant carryover. PMP mobility and aggregation is thus critical to successful separation. For a given channel geometry there exists an optimum PMP aggregate size that ensures successful penetration; too few PMPs and there is an insufficient magnetic dipole force to overcome interfacial capillary forces; too many, and the PMP aggregate will fill the width of the channel and simply pull the interface with it as the magnet is moved, preventing the oil phase from pinching off the aqueous tail. The magnet should therefore be held for a certain amount of time (roughly 30 seconds for this device) in order to coalesce the PMPs. The time scale for this event depends on the priming solution used, magnet strength and magnetic susceptibility of the PMPs.LO), lysate-substrate (γLS) and oil-substrate (γOS) interfaces determine whether complete separation of the PMPs and lysate occurs. To prevent the formation of an aqueous bridge, high γLO and γLS must be utilized to favor the pinching off of the PMP aggregate.As the PMP aggregate moves through the oil it travels as a closed aqueous microenvironment. Because it is surrounded by an immiscible phase, it is energetically unfavorable for the aggregate to partition or break up, thus facilitating the transport of PMPs through the oil. This scheme could potentially be used for other separation applications that involve the capture of analyte by PMPs , as the local chemical environment of the PMPs remains constant even as they enter the oil, thus preventing unwanted or premature elution. Furthermore, more stringent purification or step-wise processing with various labels (such as in an ELISA) may be achieved by multiple oil-water barriers in series. Insufficient interfacial tension between the lysate and oil can however lead to excessive stretching of the oil-water interface and the formation of an aqueous phase bridge connecting the LW and EW. It is therefore necessary to fine-tune the surfactant concentration to ensure a balance between PMP mobility and high surface tension, thus dictating the balance between PMP transfer efficiency and carry-over minimization. As discussed by Berry et al. , the intWe have demonstrated with a simple design the efficient separation of RNA from patient sample without the need for washing and centrifugation. Our device, when compared to the Ambion MagMAX kit, can be run in a third of the time with nearly equivalent efficacy, using only a pipette and permanent magnet. Furthermore it requires less volume of reagents and sample, and can be applied to not just solid phase extraction but likely any separation technique involving analyte capture on PMPs. With the oil chip, we were able to isolate influenza RNA from clinical samples at high concentration while also maintaining vRNA integrity. The carryover of lysate had a small inhibitory effect on amplification, as shown by the slight drop in vRNA extraction efficiency compared to total RNA extraction efficiency; however, the overall performance was promising given the rapidity and ease of operating the oil chip. We conclude that this technique has great potential for both point-of-care as well as high-throughput applications.S1 Fig9 to 105 copies/mL. Amplification efficiency was calculated to be 99.8%. b) RT-qPCR amplification plot for samples 177, 78 and 193.a) The standard curve was formed from serial dilutions ranging from 10(TIF)Click here for additional data file.S2 FigElectropherogram of Bioanalyzer DNA 1000 gel assaying RT-PCR products of processed patient samples 590, 177 and 474, the last of which tested negative for influenza A. cDNA concentrations (ng/μL) are given next to respective peaks.(TIF)Click here for additional data file.S3 FigRNA Electropherogram for patient sample 193 processed both by kit and oil chip method. The peak shift towards the right for the oil chip sample was observed.(TIF)Click here for additional data file.S1 Tabletot for Column 4 is calculated as the ratio of OD260 measurements of chip-extracted RNA to kit-extracted RNA.E(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.