Drosophila and human cellular models. Electronic microscopy revealed that human and Drosophila cells release similar EVs with diameters ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNAs, related pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these sizeable similarities suggest that EVs could potentially enable RNA-mediated intercellular communication in Drosophila.Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from  GFP mRNA leads to fluorescent protein expression in recipient endothelial cellsAlthough unprotected RNA molecules display short half-lives in biological fluids such as human serum2353291012131413Vibrio cholerae contain diverse RNAs enriched for intergenic sequencesTrypanosoma cruzi encapsulate large transcript populations dominated by short sequences derived from ribosomal RNAs (rRNA) and transfer RNAs (tRNA)Saccharomyces cerevisiae, Candida albicans, Paracoccidiodes brasiliensis and Cryptococcus neoformansDrosophila melanogaster stands as a key model organism that has enabled discoveries of paramount importance over the last century. In blastoderm Drosophila embryos, a high proportion of mRNAs adopts spatially resolved patterns, accumulating near subcellular structures such as plasma membrane domains20Drosophila larval development, where a pool of the Wnt ligand is released from imaginal discs in association with exosomal membranes22Drosophila cell culturesDrosophila and two human cell lines. Our morphologic and transcriptomic observations reveal considerable similarities across EVs from these distant metazoan systems: they contain comparable amounts of RNA largely consisting of short ribosomal sequences, retrotransposons, other non-coding RNAs and mRNA signatures enriched for translational functions.Recently, increasing characterization efforts have extended our appreciation of exRNA repertoires in multiple biological species. For instance, lipid vesicles released by the Gram-negative bacterium Drosophila cell lines, Dm-D17-c3 (D17), derived from third instar larvae haltere discs25Drosophila samples would provide instructive comparisons. We opted for the EGFR-driven, epidermoid carcinoma line A431We investigated EVs in two HepG2 = (9.01 ± 1.92) ×1010; μA431= (8.80 ± 0.76) ×1010; μD17 = (6.72 ± 2.2) ×1010; μS2R+ =(1.24 ± 0.35) ×1011]. The differences found when comparing HepG2 and A431 EV counts , D17 and S2R+ counts  and all four cell lines  were not clearly significant . Analysis of cell media supplemented with depleted FBS revealed the absence of contaminant particles. Having established that all four cell models release nanostructures, we performed a standard EV differential ultracentrifugation-based purification. Purified EVs from Drosophila D17 and human HepG2 cells were analyzed by transmission electron microscopy (TEM), confirming the prevalence of cup-shaped, exosome-like structures . Although the size distributions determined by NTA and TEM partially overlapped, we observed a slight discrepancy when comparing the two techniques, with NTA pointing at larger average size than TEM. Indeed, small EVs (<50nm) were abundant in micrographs of HepG2 and especially D17 EVs, but NTA profiles were largely exempt of bodies smaller than 75 nm. It should be noted that uranyl acetate staining can alter EV morphology, likely causing EVs to collapse and present smaller diameter than what would be observed in solutionAs a first approach to EV profiling, we performed nanoparticle tracking analyses (NTA) using a Nanosight device on cell culture supernatants cleared of floating cells (30 min at 2000 × g). We calculated the total number of particles found in each preparation based on NTA . Our calculations indicate that S2R + EVs contain significantly more protein  than D17 EVs. Individual RNA quantification revealed lower values in the range of attograms/ag (10−18 g) that displayed strong variability   and RNR2 (16S) rRNAs  rRNAs . Full-lela exRNA . The 28SAC079949.1 and AL161626.1, annotated as “novel miRNAs” in Ensembl and as “rRNA pseudogenes” in GATExplorer. These miRNA loci overlap with large ribosomal subunit repeatsAC079949.1 and AL161626.1 were first identified in human EV extracts and are mentioned in a patent request regarding the use of mesenchymal stem cell-derived EVs for wound therapyAC079949.1 in exRNA  that were highly abundant in human EVs and relatively rare in cells, in exRNA . Such seP = 3 × 10−3). The miscellaneous RNA category was strongly overrepresented in human exRNA, accounting for over one third of A431 and over half of HepG2 exRNA distributions, as opposed to 1% for HepG2 cell RNA. In Drosophila D17 EVs, snoRNAs represented nearly half of the distribution, whereas S2R + EVs contained a majority of mRNAs  and more closely resembled A431 exRNA levels (P < 10−4). In accordance with previous reports, three groups of “miscellaneous” Polymerase III products4445VTRNA1-1 shows full-length coverage in exRNA . Presumably, the few nucleotides that distinguish these paralogues lead to the establishment of differential interactions resulting in extensive EV targeting of RN7SL1/2 and cellular retention of RN7SL3.We then compared EV and cellular RNA libraries by correlating relative abundance values of individual transcripts  and 4. Ain exRNA , its eveG2 cells . MultiplDrosophila D17 exRNA levels presented lower correlations with corresponding cellular levels (P < 10−4) than with S2R + exRNA (P < 10−4). Multiple snoRNAs were overrepresented in D17 exRNA samples, especially H/ACA box species involved in site-specific pseudouridylation of 18S and 28S rRNA  antisense fragment of the retrotransposon TART, a telomere-specific LINE-like element involved in chromosome length maintenance61626465Drosophila retrotransposons, notably Copia and related sequences were also highly abundant. This 5 kb-long element displayed full-length coverage in S2R + and D17 exRNA  form a class of Piwi-interacting transcripts involved in transposable element silencing during Drosophila gonadogenesis676869Drosophila exRNA is unclear, but it is tempting to suggest that the short antisense sequences mapped to Copia and TART in exRNA may function as small interfering RNA precursor.Transposable sequences such as long interspersed elements (LINEs) and osophila . Interes17 exRNA . InteresHuman endogenous retrovirus long terminal repeats, notably members of the ERV1, ERVL and ERVK subfamilies were the most abundant repeats in HepG2 and A431 exRNA. A few full-length L1 retrotransposons, several Alu, 7SL and tRNA repeats were also prevalent in human exRNA . By contDrosophila D17 and human HepG2 samples. As expected, FPKM and cycle threshold values (CT) were significantly anti-correlated (−0.92 ≤ Pearson’s r ≤ −0.75) (P < 10−4). By contrast, human HepG2 mRNA levels in EVs were closer to corresponding cell levels than described above for global comparisons in human HepG2 (P < 10−4). Accordingly, several highly expressed cellular mRNAs were traced in EVs, notably mitochondrial mRNAs involved in the respiratory chain, such as cytochrome oxidase, NADH:ubiquinone reductase and ATPase subunit mRNAs , orthologous to a mammalian plasticity protein involved in synaptogenesisArc mRNA is targeted to rat neuronal dendrites via a cis-regulatory motif found in its 3′UTRDrosophila, it could promote Arc1 accumulation to plasma membrane domains, resulting in its preferential incorporation within EVs.Over 1,000 mRNA signatures were traced in all exRNA samples. We validated RNA-seq results by RT-qPCR analyses targeting 7 mRNAs in exRNA and cellular RNA of ≤ −0.75) . Importa≤ −0.75)  and mRNAit mRNAs . Numerouosophila . Gene onosophila . In addiDrosophila sequences should exhibit a common propensity to accumulate in EVs. To test this hypothesis, we took advantage of our vast EV repertoire of human and Drosophila mRNAs to investigate global abundance correlations across orthologs of the two species. Using an integrative ortholog prediction toolP < 10−4). These distributions were largely dominated by abundant mRNAs encoding ribosomal proteins. When comparing the relative abundance distributions of orthologs in EV, we documented a slightly weaker correlation (P < 10−4). Therefore, while the expression levels of orthologous mRNAs are strongly related, our comparative analysis does not suggest that gene-specific mRNA enrichment to EVs is a globally conserved feature.Whether a subset of cellular mRNAs undergoes selective targeting to EVs through a sequence-specific mechanism remains unclear. If specific RNA motifs are involved in sorting mRNAs to EVs and if such motifs are conserved, a fraction of orthologous human and Drosophila mRNAs encode neuronal membrane-associated proteins, notably Snap25 Muc26B, Mu4B)Drosophila embryogenesis21Arc1 mRNA. Among the 5 exRNA-exclusive human HepG2 mRNAs, ALB (Albumin) and APOB (Apolipoprotein B) encode secreted proteins involved in transmembrane transport85TSPN16 (Tetraspanin 16) belongs to a class of membrane-spanning factors described as EV protein markers85Drosophila, the exclusivity of these mRNAs within EVs suggests the prevalence of instructive targeting signals in their sequence that promote local translation of protein factors associated with membrane or extracellular localization.Previous comparative studies have identified so-called “exRNA-exclusive” transcripts, likely undetected in cognate cells due to highly efficient subcellular transport processes, high cellular turnovers or a combination thereofDrosophila EVs. Our work revealed that several features of EVs are considerably conserved in these distant metazoan species, notably the abundance of ribosomal sequences and retrotransposons, including sense-antisense RNA pairs. Drosophila EVs released by S2R+ and D17 cells are enriched for diverse snoRNAs, while human EVs produced by HepG2 and A431 cells contain strong signatures of miscellaneous RNAs, such as vault and Y RNAs. While EM and NTA analyses have shown considerable heterogeneity in our preparations regarding EV size and morphology, transcriptomic analyses were performed on EV populations, and therefore can’t determine whether individual EVs display disparate RNA repertoires. Diverse methods such as immunoaffinity captures or density-based separation have been developed to isolate exosomes from preparations containing larger vesiclesDrosophila EVs. Previous studies have suggested that mammalian exosomes and microvesicles may contain contrasting molecular signatures, with exosomes showing higher amounts of RNA than microvesiclesDrosophila. Furthermore, the use of long-read RNA sequencing platformsDrosophila. In light of our findings, it seems likely that diverse Drosophila lineages, in line with their human counterparts, rely on exRNA to convey intercellular communication. This hypothesis could be explored via loss-of-function studies.To the best of our knowledge, we provide here the first morphological and transcriptomic comparative analysis of human and 2 in T-flasks of 175 cm2 and routinely detached using 0.25% trypsin upon reaching 80% confluence. Drosophila D17 and S2R+ cells were respectively maintained in Shield and Sang Insect Medium (M3) and in Schneider medium. Both media were supplemented with 1% of a 1:100 pen/strep solution and 10% depleted FBS. M3 medium additionally contained insulin (20 μg/mL). Drosophila cells were cultured at 25 °C. Cell death rate was routinely monitored by trypan blue staining (Sigma-Aldrich) and consistently remained below 5% for all cell types.Human HepG2 and A431 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% of a 1:100 solution of penicillin and streptomycin (pen/strep) and 10% fetal bovine serum (depleted FBS) (Wisent). Before use, FBS was depleted of bovine EVs by ultracentrifugation . Human cells were cultured at 37 °C, in an atmosphere of 5% CO7 cells. EVs were isolated according to an established differential ultracentrifugation protocolFor EV isolation, cells were kept at low passage (P < 10) and cultured for approximately 48 h starting from ~10Steps were performed as previously describedCell depleted supernatants (30 min at 2000 × g) were analyzed by nanoparticle tracking analysis using an LM-10 machine (Nanosight) according to the manufacturer’s instructions. Samples were submitted to 3 successive analyses of 30 s using the default settings of the instrument. 3 washes were performed with water between samples. DMEM, M3 and Schneider media containing depleted FBS were used as negative controls. Biological triplicates were analyzed.TM reagent (Ambion) and processed according to the manufacturer’s instructions RNA extracts were purified with the RNA Clean & ConcentratorTM-5 system (Zymo Research). In-column DNase I (New England BioLabs) treatment, RNA washes and elution steps were performed according to the manufacturer’s instructions. One additional centrifugation step was included  to ensure complete removal of the washing buffer. RNA samples were eluded in 12 μL of RNAse-free water (Wisent). Absorbance distributions were immediately quantified using a NanoDrop 2000c spectrophotometer. RNA samples were pure (A260/A280 ≥ 2.0)(2.00 ≤ A260/A230 ≤ 2.25). Aliquots of ~5 ng were submitted to capillary electrophoresis on a Bioanalyzer 2100 machine (Agilent). RNAse protection assay was performed as previously described−18 g × 6.02 × 1023 molecules × mol−1 × (50 nt × molecule−1 × 325 g × mol−1 × molecule)−1 = 37.04 molecules.EVs and corresponding cellular pellets were resuspended in 1 mL of TRIzolBiological duplicates of sequencing libraries were prepared from high quality RNA extracts (50 ng exRNA and 500 ng cellular RNA) using the Illumina TruSeq Stranded RNA Kit according to the manufacturer’s instructions. The TruSeq PE Clusterkit v3-cBot-HS was used on an Illumina HiSEq 2000 machine.Drosophila BDGP5.78/dm3 genomes, respectively. Alignment BAM files were used to generate bigWig files, which were submitted to the UCSC genome browser for read coverage visualization. Reads mapping to RepeatMasker v4.0.6 sequence coordinates were counted with BEDTools. Ribosomal RNA sequences were filtered out by first mapping the reads to FASTA files of genes annotated as rRNA in Ensembl. Remaining reads were then re-aligned to reference genomes with Tophat. BAM files were used for expression analyses with Cuffdiff v2.2.1 without effective length correction. Transcripts per million (TPMi = [FPKMi/ΣjFPKMj] ×106) were used as relative abundance units. A 5 FPKM threshold was applied. For biotype and correlation analyses, the 1,000 most abundant transcripts were considered, which accounted for over 90% of identified transcripts.Read quality was confirmed using FastQC v0.10.1 and trimming was performed with Trimmomatic when deemed necessary. Read alignment was performed using Tophat v2.0.10 on the human GRCh37/hg19 and the The M-MLV reverse transcriptase (Invitrogen) was used according to the manufacturer’s instructions to synthesize cDNA in triplicate starting from 100 ng of exRNA and cell RNA. Priming was performed with random hexamers (Promega). Real-time PCR was performed on the ABI ViiA7 instrument (Life) using SYBR Green reagent (Applied Biosystems). Cycling conditions were as follows : 50 °C for two minutes and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 59 °C for 60 s. Melting curve analysis was performed. Validated primer sequences targeting exon junctions were retrieved from the GETprime database−3 were considered.The 1,000 most abundant mRNAs in each EV type were used for functional analysis. Process, function and component GO terms were retrieved with DAVID. Terms associated with FDR < 10Statistic tests were performed with GraphPad Prism v6.GSE76173.Datasets are available on GEO under the accession number How to cite this article: Lefebvre, F. A. et al. Comparative transcriptomic analysis of human and Drosophila extracellular vesicles. Sci. Rep.6, 27680; doi: 10.1038/srep27680 (2016).


C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons.Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the DOI:http://dx.doi.org/10.7554/eLife.14599.001 The brain contains billions of nerve cells, or neurons, that communicate with one another through connections called synapses. As the brain develops, these circuits are extensively modified as new synapses are created and others are removed. Neurological disorders may emerge if these processes are not regulated correctly. Identifying the biological pathways that control the addition and removal of synapses could therefore provide new insights into how to treat human brain diseases.To communicate across a synapse, the signaling neuron releases chemicals called neurotransmitters that alter the activity of the receiving neuron. Some neurotransmitters, such as GABA, inhibit the activity of the receiving neuron. The activity of a neuron – and hence how often it releases neurotransmitters – depends on different ions moving into and out of the neuron through proteins called ion channels that are embedded in the cell membrane. For example, the movement of calcium ions into the neuron can trigger the release of neurotransmitters.Caenorhabditis elegans is often used as a model organism to study how the brain develops. During development, the worm nervous system eliminates synapses that release GABA and reassembles them at new locations. However, the nervous system does not eliminate these synapses at random. Miller-Fleming, Petersen et al. now show that a C. elegans protein called UNC-8 is responsible for this effect. UNC-8 forms part of an ion channel that allows sodium ions to enter the neuron and is selectively produced in GABA neurons that are destined for remodeling.The roundworm Miller-Fleming, Petersen et al. found that inside GABA-releasing neurons, calcium ions stimulate an enzyme called calcineurin that may in turn activate UNC-8. Sodium ions then enter the neuron through UNC-8 channels. This boosts the activity of the calcium ion channels, which further increases how many calcium ions enter the cell. Ultimately, the amount of calcium inside the neuron becomes high enough to activate an additional pathway that eliminates the synapse. This downstream pathway involves components of a cell-killing (or “apoptotic”) mechanism that is repurposed in this case to remove the GABA release apparatus at the synapse.Other proteins are likely to help UNC-8 sense the activity of neurons and destroy synapses in response. Further work is required to investigate these additional components and to determine how they work with UNC-8 to remove synapses in the nervous system during development.DOI:http://dx.doi.org/10.7554/eLife.14599.002 Neural circuits are reorganized during development to produce a mature, functional nervous system. Circuit refinement involves both the elimination of specific synapses and the strengthening of others. These developmental changes in brain architecture are temporally coordinated with critical periods in which neuronal wiring can be shaped by activity. The establishment of binocular vision in the mammalian brain, for example, depends on visual input during a distinct developmental period in which the circuit is uniquely sensitive to neural activity. GABA signaling regulates the onset and duration of this critical period of plasticity in the visual circuit, but the molecular mechanisms that drive synaptic remodeling in this pathway are poorly defined .C. elegans, the cell death pathway component CED-3/caspase-3 and its upstream regulator CED-4/Apaf1 promote synaptic disassembly by activating the F-actin severing protein gelsolin; the calcium sensitivity of this pathway points to a potential role for neuronal activity in synaptic remodeling  triggers neurotransmitter secretion in active neurons . Elevatemodeling .Degenerin/Epithelial Sodium Channels (DEG/ENaCs) are voltage-insensitive, trimeric cation channels. The term degenerin arises from the finding that constitutively active forms of DEG/ENaC channels induce neurodegeneration . ENaCs aC. elegans, a simple, well-defined GABAergic circuit is remodeled during development in a mechanism that is accelerated by neural activity  Ven program ; (3) VD  mutants ; (4) Themodeling . We explsynapses . Here wesynapses . The necsynapses . Thus, wunc-8 expression is elevated in GABA neurons in unc-55 mutants  that includes a 1.6 kb genomic region upstream of the unc-8 coding sequence  that deletes 197 nucleotides of the first transmembrane domain and shifts the reading frame to introduce a premature stop codon (unc-8(tm5052) should result in an unstable unc-8 mRNA and likely null allele.Our RNAi results suggested that UNC-8 promotes synapse removal in remodeling GABA neurons  Peterse. To confop codon . In thisflp-13) to drive expression of the presynaptic vesicle-associated proteins, GFP::RAB-3 and SNB-1::GFP/synaptobrevin. DD remodeling is initiated toward the end of the first larval stage (L1) and involves the relocation of ventral presynaptic components to the dorsal side  to confirm this function in VD neurons that remodel in unc-55 mutants. This test is important because previous evidence suggests that VD remodeling in unc-55 mutants is driven by the ectopic activation of the native DD remodeling program  from VD neurons  transgene show significant retention of GABAergic synapses in the ventral nerve cord    . Althoug ± 0.04) .10.7554/unc-55; unc-8. At the EM level, both GABAergic and cholinergic synapses show electron-dense presynaptic terminals and abundant synaptic vesicles, but can be distinguished by morphological criteria . We identified ventral GABAergic synapses in wild-type, unc-8 and in unc-55; unc-8 animals ; whereas ventral cholinergic motor neuron synapses were preserved (data not shown). The finding that GABAergic synapses were detectable in unc-55;unc-8 double mutants is consistent with the partial restoration of fluorescently-labeled presynaptic markers to ventral GABA neuron processes in unc-55; unc-8 animals (unc-8 gene promotes disassembly of multiple components of the presynaptic apparatus.We used electron microscopy (EM) to test this idea by examining the structure of the GABAergic synapses in  animals  and therunc-55; unc-8 animals contain GABAergic presynaptic domains we next used electrophysiological recordings to ask if these residual synapses are functional. Spontaneous inhibitory postsynaptic events (iPSCs) arising from GABA release were recorded from ventral body muscles . Robust, iPSCs were detected for wild-type and unc-8 animals  in unc-55; unc-8 animals in comparison to wild type; UNC-49::GFP is closely co-localized with presynaptic mCherry::RAB-3 at ventral unc-8 and unc-55; unc-8 GABAergic synapses both before remodeling and in adults . Ventral SNB-1::GFP puncta were scored in adult animals. As expected, unc-55 animals from control plates show few ventral SNB-1::GFP puncta in GABA neurons  suggesting that calcium ions enhance Benzamil binding . This result favors a model in which unc-2 and unc-8 act together in a linear pathway to remove ventral synapses in remodeling GABA neurons  priming . We confsynapses   expression in DD neurons  are required for this effect; DD neurons that express ChR2 (GABA::ChR2) remodel earlier with significantly more dorsal puncta after 13 hr of light exposure in comparison to controls  should induce precocious synapse disassembly. To test this idea, we used the presynaptic marker GFP::RAB-3 to determine that DD ventral synapses are removed prematurely in tax-6(d) mutants; dorsal synaptic assembly is also precocious and occurs significantly earlier than in wild-type controls. Additionally, we determined that treatment of tax-6(d) animals with the DEG/ENaC inhibitor Benzamil significantly delays the precocious remodeling phenotype  of remodeling GABA neurons  and consequent neurotransmitter release  class of the DEG/ENaC family proteins are activated by low pH and have been implicated in learning and memory . In one paradigm . Our finparadigm . This obparadigm . A recen release . This moeostasis . Our resXenopus oocytes indicate that UNC-8 preferentially transports sodium and is impermeant to calcium , unless otherwise stated (unc-55(e1170) and unc-8(tm5052) alleles were used for these studies. For a complete list of the strains used in this study, see Strains were maintained at 20°C on NGM plates seeded with OP50 (e stated . The wilunc-8(tm5052) allele was obtained from a UV/TMP mutagenized library, as described previously  with the insertion of CT resulting in a premature stop codon prior to the first transmembrane domain. We used the following primers to detect the tm5052 mutation: Forward 5’-TGGGGCCCTAATAATTTCGA-’3 and Reverse 5’- AGTGACAGTATGAAGCCAGG-’3.The eviously  and was Construction of GABA csRNAi transgenic lines unc-8 target the first 7 exons of the unc-8 coding region. To clone the unc-8 sense construct pSA76, a 2.3 kb region of unc-8 cDNA was amplified with the following primers containing 5’AscI/3’SacII adaptors: Forward 5’- GGCGCGCCATGTCACCTTTGCTGACGT-3’ and Reverse 5’-GCCAGGAGGTGATATTCTAGCCGCGG-3’. This fragment was cloned into pCR2.1 via TOPO-TA reaction  to yield pSA75. The 2.3 kb unc-8 cDNA fragment was then subcloned into the existing GABAergic cell-specific RNAi (csRNAi) plasmid pSA47 via AscI/SacII to yield pSA76  to yield pSA73. The unc-8 fragment from pSA73 was then inserted into the GABA neuron-specific RNAi (csRNAi) plasmid pSA47 via AscI/SacII to yield pSA77. The unc-8-containing region of pSA77 between ScaI and SacII was then inserted into plasmid pSA49 to yield pSA78. The pttr-39 promoter in pSA78 drives expression of the 2.3 kb unc-8 antisense fragment and mCherry. pSA76 (unc-8 sense) and pSA78 (unc-8 antisense) were linearized and ligated, then transformed into unc-119 worms via microparticle bombardment to yield a spontaneous integrant (strain NC2601) as indicated by 100% transmission of rescued (unc-119+) movement (indicating unc-8 sense) and mCherry expression in all GABAergic motor neurons (indicating unc-8 antisense)  . A contrunc-8 genetic locus was obtained from GeneService  and purified. Fosmid DNA was transformed into electrocompetent SW105 cells and was verified by PCR. A GFP-galK recombineering cassette was amplified with 50 kb homology arms from pBALU1 and gel-purified. The GFP-galK PCR product was transformed into electrocompetent, λRed recombinase-activated, fosmid-containing SW105 cells. The cells containing the fosmid and GFP-galK were grown for more than 60 hr at 32°C and streaked on MacConkey and galactose plates with chloramphenicol to ensure the insertion of recombineering cassette. To excise galK from the GFP intron, colonies were incubated with 0.1% arabinose to create an unc-8::GFP expression fosmid. This unc-8::GFP fosmid was then purified and confirmed by sequencing. The fosmid was injected into unc-8(tm5052) animals at 25 ng/μl with co-injection marker pceh-22::GFP at 15 ng/μl.The UNC-8::GFP fosmid was recombineered as previously described . Brieflypunc-25::coq1cDNA::GFP::unc-54) in place of the coq-1 fragment. The resultant plasmid, pTWM62 (punc-25::UNC-8::GFP) was injected (10 ng/μl) with co-selectable marker pmyo-2::mCherry::unc-54 (2.5 ng/μl) into unc-8 (tm5052) animals.UNC-8 cDNA was PCR-amplified from pSGEM/pTWM60  with pripttr39::arx-5::GFP::unc-54) in place of the ARX-5::GFP fragment. The resultant plasmid, pTWM92 (pttr-39::UNC-8cDNA) was injected (25 ng/μl) with co-selectable markers pmyo-2::mCherry::unc-54 (2 ng/μl) and punc-25::mCherry::RAB-3 (5 ng/μl) into unc-55; unc-8 (tm5052) juIs1 or juIs1 animals.UNC-8 cDNA was PCR-amplified from pSGEM/pTWM60  with pri0. All adults were removed from the plates after 1 hr. Plates were maintained at 23°C until assayed. Puncta arising from localization of fluorescent presynaptic markers were counted with a Zeiss Axiovert microscope (63X oil objective) in immobilized animals. For timecourse experiments puncta were counted between DD1 and DD6 (unc-8(csRNAi)   or unc-8nc-8cDNA , puncta pttr-39::mCherry. The cells were traced in ImageJ and the background was subtracted. The unc-8::GFP fluorescence intensity was then normalized to the pttr-39::mCherry fluorescence intensity for each cell. Fluorescence intensity plots in 2r) was calculated in ImageJ using the Manders coefficients macro, from at least 10 animals for each genotype. 2r values were averaged and presented as mean ± SEM. An 2rvalue of 0 represents no co-localization, whereas 2r= 1 represents complete co-localization.Animals were immobilized with 15 mM levamisole/0.05% tricaine and mounted on a 2% agarose pad in M9 buffer as previously described . Z-stackunc-55; juIs1 animals were placed on either Amiloride, Benzamil, or control plates at room temperature and progeny examined at the young adult stage (tax-6(d); wyIs202 animals were grown on Benzamil or control plates and their larval progeny were collected on control or Benzamil plates for timecourse assays  stock solution was prepared in sterile water (50 mg/ml) and stored at -20°C. A final concentration of 3 mM Amiloride diluted in OP50 bacteria was seeded on NGM plates. Control NGM plates contained the same volume of sterile water added to OP50. Benzamil hydrochloride hydrate  stock solution was prepared in sterile water (1 mg/ml) and stored at 4°C. A final concentration of 3 mM Benzamil diluted in OP50 bacteria was seeded on NGM plates. Control NGM plates contained the same volume of sterile water added to OP50. Plates were stored at 4°C for up to one week. Five adult lt stage . Adult te assays . The numtrans retinal  was dissolved in ethanol to prepare a 100 mM stock and stored at -20°C. 300 μM of all-trans retinal stock solution (ATR plates) or ethanol (control plates) was added to OP50 bacteria and seeded onto NGM plates. Plates were protected from light and were stored at 4°C for up to one week. 100 adult hermaphrodites were placed on either ATR or control plates, allowed to lay eggs for 1 hr and then removed from the plate. The midpoint of this hour is considered T0. Plates were exposed to blue light pulses with a 470-nm LED light  for 13 hr . Light stimulation was controlled using NI Max software through TTL signals generated by a digital function generator . 13 hr after egg laying, animals were assayed for DD remodeling by counting the number of dorsal SNB-1::GFP puncta. The examiner was blinded to the treatment. Data were collected from three independent time course experiments with at least six animals per treatment.All-C. elegans dissection and electrophysiological methods were as previously described . The low Cl intracellular patch pipette solution used to isolate outward GABA minis at a 0 mV holding potential was composed of 115 mM KGluconate, 25 mM KCl, 0.1 mM CaCl2, 1 mM BAPTA and 50 mM HEPES. Data were acquired using Pulse software  run on a Dell computer. Subsequent analysis and graphing was performed using Pulsefit (HEKA), Mini analysis  and Igor Pro .The escribed . AnimalsXenopus Laevis were purchased from Ecocyte Bioscience US LLC . Oocytes were injected with 10 ng/oocyte of cRNA and incubated in OR2 solution  and 2 mM Na-pyruvate) plus 500 µM amiloride (to prevent channel hyperactivation-dependent cell death) at 20°C for 2–3 d before recordings. Currents were measured using a two-electrode voltage-clamp amplifier  at room temperature. Electrodes (0.2–0.5 MΩ) were filled with 3 M KCl, and oocytes were perfused with a physiological NaCl solution  and divalent cation free plus EGTA NaCl solution . pH was adjusted at the indicated values using NaOH. The oocyte membrane was clamped at −30 mV and stepped from −160 to +100 mV. Benzamil was added to the solutions from a stock of 10 mM. A saturating concentration of benzamil (1mM) was added at the end of each experiment to confirm that endogenous/leak currents were similar in amplitude to non-injected oocytes within each oocyte batch. Oocytes that had larger endogenous/leak currents were not further analyzed. We used the pCLAMP suite of programs (Axon Instruments) for data acquisition and analysis. Currents were filtered at 200 Hz and sampled at 1 kHz. We used OriginPro 8  to generate graphs, Ki, and for statistical analysis.UNC-8(G387E) cRNA was synthesized using T7 mMESSAGE mMACHINE kit . cRNA was purified and examined on a denaturating agarose gel to confirm correct size and integrity. cRNA quantification was performed spectroscopically. Stage VI defolliculated oocytes from E. coli. The specimens were frozen rapidly in a high-pressure freezer  at −180°C and high pressure. Freeze substitution was performed on frozen samples in a Reichert AFS machine  with 0.1% tannic acid and 2% OsO4 in anhydrous acetone. The temperature was kept at −90°C for 107 hr, increased at 5°C/hr to −20°C, and kept at -20°C for 14 hr. The temperature was then increased by 10°C/h to 20°C. Fixed specimens were embedded in Epon resin after infiltration in 50% Epon/acetone for 4 hr, 90% Epon/acetone for 18 hr, and 100% Epon for 5 hr. Embedded samples were incubated for 48 hr at 65°C. All specimens were prepared in the same fixation procedure and labeled with anonymous tags so that the examiner was blinded for genotype. Ultra thin (40 nm) serial sections were cut using an Ultracut 6 (Leica) and collected on formvar- covered, carbon-coated copper grids . Grids were counterstained in 2% aqueous uranyl acetate for 4 min, followed by Reynolds lead citrate for 2 min. Images were obtained on a Jeol JEM-1220  transmission electron microscope operating at 80 kV. Micrographs were collected using a Gatan digital camera  at a magnification of 100k. Images were quantified using NIH ImageJ software. Dorsal and ventral cords were distinguished by size and morphology. GABAergic synapses were identified by previously established criteria, including position in the cord as well as the morphology of the synapse  fixation as described . 10–15 a synapse . GABAerg In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Kang Shen as the Reviewing Editor and Gary Westbrook as the Senior Editor.Thank you for submitting your article "The DEG/ENaC Cation Channel Protein UNC-8 Drives Activity-Dependent Synapse Removal in Remodeling GABAergic Neurons" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.General assessment:This manuscript addresses the molecular mechanisms that govern a poorly understood, but critical biological process, synapse remodeling. UNC-55 is a transcription factor that prevents the VD class GABAergic motor neurons to adopt the fate of another subclass of GABAergic motor neuron (DD), which undergoes post-embryonic synapse remodeling. Previously, the corresponding author's group performed micro-array experiments to compare the transcriptional profiles of GABAergic motor neurons in the presence and absence of UNC-55, identified a dozen of UNC-55-regulated transcripts . This manuscript explores the functional involvement of and potential mechanisms through one of these targets, the UNC-8 ENaC sodium channel affect remodeling.unc-55 mutants, and to access the effect of removing UNC-8/ENaC, and its putative pathway components . They confirmed that removing these components, either individually, or in combination with removing UNC-8, led to a similar degree and partial restoration of the ventral presynaptic structures in unc-55 background. They performed EM analyses to confirm the anatomic presence of these structures, and, by electrophysiology analyses, that these structures  appeared to lack the ability to release synaptic vesicles. Because a previous study from the group  revealed that IRX-1, a TF, is an UNC-55-target that functions similarly to UNC-8 to promote the remodeling of GABAergic neurons, author probed the genetic relationship between unc-8 and irx-1. They found that removing both in unc-55 background led to an increased effect on promoting the remodeling. Hence, placing UNC-8 and IRX-1 in two parallel pathways negatively regulated by UNC-55.In this paper, the authors used several presynaptic fluorescent markers to quantify the remodeling phenotype of GABAergic motor neurons in wild-type and Both reviewers found the manuscript contains a lot of data, and two semi-related studies that address two UNC-55-dependent mechanisms that regulate synaptic remodeling. Both reviewers are impressed with the comprehensive EM and electrophysiological analyses. They found that the manuscript is appropriate for publication after the authors address the following questions. As you will see, many of the comments are about writing and the strength of the conclusions. Some comments need to be addressed experimentally.Essential revisions:unc-8 mutants. One reviewer wrote: "In several places, the manuscript made strong statements along the line that 'UNC-8 drives presynaptic structure removal'. I feel that authors may want to soften these statements and may need additional data to enforce such statement, for following reasons: a) the rescuing effect of removing UNC-8, UNC-2, TAX-6 and CED-4 is not only partial, and appears to reflect morphological, but not functional rescue of unc-55's GABAergic neuron defects. Compared to the effect of removing IRX-1 , the role UNC-8 pathway is less prominent. B) if UNC-2 is to be stated as 'required for remodeling', it would be useful to analyze the remodeling phenotype in unc-2 single mutants (as shown for unc-8 in 1) Most importantly, both reviewers raised concerns about the interpretation of "synapse elimination" phenotypes in the unc-8 mutant worms eliminate similar number of synapses between 18 and 30 hours, i.e., about fifty DD synapses (RAB-3 puncta) disappear from the ventral cord. These results seem to indicate that synapse removal remains fully active without UNC-8. Would it be possible that the defects  in unc-8 mutants are due to ectopic synapse formation or false deposit of synaptic proteins? Indeed, more RAB-3 puncta exist in unc-8 mutants prior to remodeling  used several presynaptic markers to conclude that their interaction reflects the change of the entire presynaptic structure, instead of specific effect on individual protein/marker expression or subcellular localization. I like the vigor of this approach. I did notice  Presynaptic Marker analyses: Authors accessed the phenotype and genetic interactions between unc-8 and unc-2/tax-6/tom etc. (unc-8. These sections (the unc-8 pathway) could be extensively condensed and shortened as it employs very similar assays as those in unc-8 analyses.3) This manuscript may benefit from reorganization and editing. The second part of the story, the analyses between UNC-55 and IRX-1, almost stands alone as a separate study. Having it inserted between that of tom etc.  cuts theunc-55; unc-8 etc. has been extended and complemented with the hyperosmotic responses to pinpoint a potential functional defect in vesicle priming or fusion. This applies similarly to the much more substantial marker analyses on unc-55; unc-8 mutants.4) This study significantly extends from the 2011 paper. This does not at all reduce my enthusiasm to support it publication, but I think it is important for authors to refer in those places to the previous study so that they can clearly describe and discuss the importance of the extended analyses. For example, the electrophysiology analyses of ventral GABA minis on eLife. Your revised article has been favorably evaluated by Gary Westbrook as the Senior editor, a Reviewing editor, and two reviewers.Thank you for resubmitting your work entitled "The DEG/ENaC Cation Channel Protein UNC-8 Drives Activity-Dependent Synapse Removal in Remodeling GABAergic Neurons" for further consideration at eLife if these three remaining questions are addressed. We expect a quick turn-around for this last set of questions.Both reviewers found that the revision significantly improved the manuscript and found that the manuscript is suitable for publication in unc-55; unc-8 double mutants, both DDs and VDs contributed to the ventral residual synaptic marker expression, shouldn't DD in 1) Because the authors now demonstrate that in unc-8 mutants removed? These data are critical for readers to understand the phenotype of unc-8 mutants. We think that the 2) Why was the electrophysiology data of 3) The UNC-49 results were verbally described in the rebuttal letter, but the actual data were not shown in the revised manuscript. To convincingly show synapse elimination/removal, it is important to demonstrate that remaining presynaptic markers have had postsynaptic partners prior to remodeling. […] Both reviewers found the manuscript contains a lot of data, and two semi-related studies that address two UNC-55-dependent mechanisms that regulate synaptic remodeling. Both reviewers are impressed with the comprehensive EM and electrophysiological analyses. They found that the manuscript is appropriate for publication after the authors address the following questions. As you will see, many of the comments are about writing and the strength of the conclusions. Some of the comments need to be addressed experimentally.unc-55 mutants. In fact, our goal is to understand the native synaptic remodeling mechanism (in DD neurons). We have exploited the unc-55 remodeling phenotype to facilitate experimental analysis and because ample evidence supports the conclusion that the endogenous DD neuron remodeling pathway is activated in unc-55 mutant VD neurons. For example, multiple components of the DD remodeling mechanism including genes either reported in previous papers  or in this work  also promote synaptic removal in unc-55 mutant VD neurons. Moreover, both DD and VD remodeling programs are activity-dependent. We have emphasized these points in additional text now included in the Introduction and Discussion.We thank reviewers for their appreciation of this work and for many insightful suggestions for improving the manuscript. Of particular note, we have sought to clarify the impression that our results are limited to the ectopic synaptic removal mechanism that occurs in Essential revisions:1) Most importantly, both reviewers raised concerns about the interpretation of "synapse elimination" phenotypes in the unc-8 mutants. One reviewer wrote: "In several places, the manuscript made strong statements along the line that 'UNC-8 drives presynaptic structure removal'. I feel that authors may want to soften these statements and may need additional data to enforce such statement, for following reasons: a) the rescuing effect of removing UNC-8, UNC-2, TAX-6 and CED-4 is not only partial, and appears to reflect morphological, but not functional rescue of unc-55's GABAergic neuron defects. Compared to the effect of removing IRX-1 , the role UNC-8 pathway is less prominent.”unc-8  in synaptic removal is less prominent that of the transcription factor irx-1. We have modified the text to acknowledge this point and to more carefully describe our interpretation of these findings. In particular, we have re-worded statements to the effect that “unc-8 is required for synaptic removal” to say “unc-8 promotes synaptic removal.” This alternative description reflects our finding that unc-8 functions in parallel to other pathways that also promote synaptic removal. In fact, other known components of the remodeling mechanism are also not absolutely required. For example, the apoptotic pathway that removes DD synapses in young larvae is complemented by an additional unknown mechanism that eventually dismantles these connections by the adult stage . Together, these findings indicate that the synaptic remodeling mechanism is complex and likely involves multiple pathways. We have included the results of an additional experiment that validates the role of unc-8 in synaptic removal. We interpret our genetic results to indicate that wild-type unc-8 is required for the removal of a significant fraction of presynaptic domains in remodeling GABAergic neurons. To test the hypothesis that unc-8 function is also sufficient to drive synapse removal, we have used a transgenic strategy to over-express unc-8 in ventral cord GABAergic neurons. In this experiment, we observed that ventral synapses are substantially reduced in VD neurons that express ectopic unc-8, whereas neighboring VD neurons that do not express unc-8 retain the wild-type complement of ventral presynaptic domains. This result supports the model that UNC-8 drives presynaptic removal. This experiment has been included in We agree that the role of “b) if UNC-2 is to be stated as 'required for remodeling', it would be useful to analyze the remodeling phenotype in unc-2 single mutants , as previously reported in Petersen et al. (2011), the number of ventral GABA synapses declines in unc-55 mutants. In contrast, the number of ventral synapses in the unc-55; unc-8 animals remains steady (through 96 hours post lay) and does not decline. These results are consistent with the idea that the residual ventral synapses that we detected by fluorescence and electron microscopy in unc-55; unc-8 adults are likely to correspond to GABA synapses that are not properly removed as opposed to new synapses which might otherwise appear in this experimental approach as a spike in ventral puncta during later development of unc-55; unc-8 animals. Additionally, it is unlikely that the GABA synapses we see by EM in the ventral nerve cords of unc-55; unc-8 animals are due to ectopic protein trafficking, as they include organized presynaptic structures with active zones, synaptic vesicles, dense core vesicles, and juxtaposed postsynaptic muscle arms.Thank you for this observation and experimental suggestion. We performed time-course analysis in both mals See . At earl“In addition, do these presynaptic puncta have postsynaptic UNC-49::GFP? Because the authors mentioned that UNC-49::GFP clusters remain unchanged, would it be possible that these ventral synapses have lost their postsynaptic partners? If these ventral synapses had postsynaptic receptors prior to remodeling, that could be an indication for failed synapse removal in presynaptic neurons.Finally, given that no GABA transmission is recovered in unc-55, unc-8 mutants, the physiological consequence of remaining presynaptic components is unclear.”unc-55; unc-8 GABA neurons co-localize with postsynaptic UNC-49::GFP. These results (described below) are not included in the revised version of this paper, however, because we have also removed electrophysiological data indicating that residual ventral synapses in unc-55; unc-8 animals are not functional and will publish these findings in a separate paper separate manuscript featuring the parallel roles of unc-8 and irx-1 during synaptic remodeling. We captured images of wild-type, unc-8, and unc-55; unc-8 animals co-expressing a presynaptic GABA marker (punc-25::mCherry::RAB-3) and a postsynaptic GABA receptor marker (punc-49::UNC-49::GFP) and analyzed these images for co-localization using ImageJ software. Although, co-localization of mCherry::RAB-3 and UNC-49::GFP in unc-55; unc-8 animals (r2= 0.53) is decreased in comparison to wild-type and unc-8 animals , we attribute this difference to the observation that there is less punc-25::mCherry::RAB-3 ventral puncta in unc-55; unc-8 animals compared to wild type. This observation suggests that these synapses have not lost their postsynaptic partners.Thank you for this suggestion. We have performed experiments to test whether the ventral presynaptic puncta in unc-55; unc-8 animals. We agree that these apparently non-functional presynaptic GABAergic domains are not physiologically competent. However, the existence of these presynaptic structures in an unc-55; unc-8 mutant whether functional or not provides clear evidence that wild-type UNC-8 promotes the removal of presynaptic components including proteins that are required for neurotransmitter release . The non-functionality of the presynaptic regions that are not removed in unc-8 mutants suggests that an additional parallel pathway, which is still active in an unc-8 mutant, must account for this deficit. At the recommendation of the reviewers (see below) who felt that our analysis of a second parallel acting remodeling pathway detracted from the main point of this work  these data have been removed from the paper and will be featured in a separate manuscript that documents the parallel roles of the unc-8 and irx-1-dependent remodeling pathways.Reviewers are correct to note that GABA synaptic function is not restored in 2) Presynaptic Marker analyses: Authors accessed the phenotype and genetic interactions between unc-55 and unc-8 (and some other suppressors) used several presynaptic markers to conclude that their interaction reflects the change of the entire presynaptic structure, instead of specific effect on individual protein/marker expression or subcellular localization. I like the vigor of this approach. I did notice (e.g. compare unc-55 mutant VD neurons). The data in DD::SNB-1::GFP or DD::GFP::RAB-3” rather than using the less descriptive nomenclature “pflp-13::..”. Additionally, in C. elegans gene names “punc-25/pttr-39”. Finally, these figures now include additional schematics depicting the type of remodeling GABA neuron (DD or VD) to help readers recognize each of these specific experimental paradigms. We have also modified the text to clarify this point.Thank you for this comment. We apologize for the confusion and have sought to clarify this point by noting that we used two different paradigms to monitor synapse removal  This manuscript may benefit from reorganization and editing. The second part of the story, the analyses between UNC-55 and IRX-1, almost stands alone as a separate study. Having it inserted between that of unc-8 and unc-2/tax-6/tom etc.  cuts theirx-1 detracts from the main thrust of the paper and that it could stand alone as a separate study. Accordingly, we have removed these data from the paper for a separate manuscript.Thank you for this suggestion. We agree that our analysis of 4) This study significantly extends from the 2011 paper. This does not at all reduce my enthusiasm to support it publication, but I think it is important for authors to refer in those places to the previous study so that they can clearly describe and discuss the importance of the extended analyses. For example, the electrophysiology analyses of ventral GABA minis on unc-55; unc-8 etc. has been extended and complemented with the hyperosmotic responses to pinpoint a potential functional defect in vesicle priming or fusion. This applies similarly to the much more substantial marker analyses on unc-55; unc-8 mutants.unc-8 as an unc-55-regulated transcript and by demonstrating that RNAi knockdown of unc-8 suppresses unc-55 . We have summarized these results here and emphasized additional experiments in this paper that extend our original findings. For example, the first Results section uses an unc-8 promoter-GFP reporter gene to confirm the prediction that unc-8 is negatively regulated by unc-55 in VD neurons. Next, we report experiments with a new unc-8 deletion allele that confirm the initial RNAi results and which show, for the first time, that unc-8 also functions in DD neurons to promote synapse removal in the native GABA neuron remodeling pathway. Electrophysiological data  have been removed from this paper for a separate manuscript that will test a specific molecular model to explain this effect.Thank you for noting that this work significantly extends our previous findings. Our earlier paper  provided the initial evidence of a role for UNC-8 in synaptic remodeling by detecting Both reviewers found that the revision significantly improved the manuscript and found that the manuscript is suitable for publication in eLife if these three remaining questions are addressed. We expect a quick turn-around for this last set of questions.1) Because the authors now demonstrate that in unc-55; unc-8 double mutants, both DDs and VDs contributed to the ventral residual synaptic marker expression, shouldn't DD in unc-8 in the removal of both DD and VD ventral synapses, and the schematic in Yes, we agree that this work demonstrates a role for 2) Why was the electrophysiology data of unc-8 mutants removed? These data are critical for readers to understand the phenotype of unc-8 mutants. We think that the unc-55; unc-8 synapses are non-functional also required us to suggest that an additional pathway, acting in parallel, likely accounts for this effect .We have restored the original electrophysiology data  and cont3) The UNC-49 results were verbally described in the rebuttal letter, but the actual data were not shown in the revised manuscript. To convincingly show synapse elimination/removal, it is important to demonstrate that remaining presynaptic markers have had postsynaptic partners prior to remodeling.unc-8 animals  co-localize in larval animals prior to remodeling and are also juxtaposed after remodeling.These data are now included . We show


Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection.Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations  isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells.Using the uropathogenic These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process. Escherichia coli (UPEC) strain 536, the pathogenic bacterium used in this study, the MVs have been reported to contain the protein toxin hemolysin [Bacteria communicate to one another using diffusible signals  and alsoCaenorhabditis elegans and E. coli [Botrytis cinera and plant Arabidopsis [To date the bacterium-to-host signaling focus has been largely on the protein and lipids as the effector molecules, particularly in the study of MV cargos. However it has recently been noted that ‘bacterial silencing suppressors may derive from regulatory RNAs instead of proteins…’ . There h E. coli  and betwbidopsis . In thesProchlorococcus to non-pathogenic E. coli to Vibrio cholerae, the water borne bacterium that causes Cholera. In parallel timeframes we studied, and now present here, the existence of MV associated RNA population derived from uropathogenic E. coli strain, 536.Recently, three groups have reported that a variety of bacteria release MVs that are associated with RNA –12. Thesin vitro.In this study we have focussed on the analysis of the RNA content and uptake of UPEC MVs. We provide the first report confirming that UPEC MVs are associated with RNA as well as using a new method to provide a comprehensive sequence profile of this cargo. We also demonstrate the uptake of MVs and their associated RNA into target human bladder epithelial cells Escherichia coli (UPEC) strain 536 (O6:K15:H31) [3 (RF), at 37°C with shaking at 200 r.p.m. and then diluted 1:100 in RF (OD600≈0.015) to be grown to stationary phase for ~16 hours overnight. After overnight incubation (OD600≈2.5) the culture was assessed for viability using the LIVE/DEAD BacLight kit (Thermo Fisher Scientific) using the manufacturers recommended protocol. OD readings were taken and cuvettes with a 1 cm path length.Uropathogenic K15:H31)  was growg for 10 min at 4°C, after which any residual cells were removed from the supernatant by filtration using 0.22μm PES filter (Merck Millipore). Supernatants were concentrated using 100 kDa Vivaflow 200 cassettes (Sartorius AG), which removes proteins/molecules under 100 kDa, and the vesicles pelleted by centrifugation at 75,000 xg for 2.5hr at 4°C. Vesicles were resuspended in 20mM HEPES or PBS, filter sterilised using a 0.22μm PES filter syringe and again concentrated using 100 kDa Vivaspin 500 columns (Sartorius AG) and stored at -80°C.Bacterial cells were removed by centrifuging twice at 7,000 xg for 16 hours at 4ᵒC . Resulting fractions were determined by visual inspection and removed from the top and diluted in ~ 50 mL of PBS. Fractions were then concentrated with 100 kDa Vivaspin 20 columns to volumes of ~600–900 μL. Aliquots for estimates of protein and RNA content and particle analysis were taken from these fractions and the remainder was stored at -80ᵒC.The crude MV preparations were further purified by density gradient centrifugation (DGC). A 6 layer  OptiPrep density gradient medium (Sigma-Aldrich) was set up in 12 mL ultracentrifugation tube . The crude preparation adjusted to the 45% medium was loaded and the tube was centrifuged at 100,000 xVesicle preparations were quantified for protein content using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), according to the manufacturer’s instructions and protein profiles assessed with 10μg sample loaded onto NuPAGE Bis-Tris 4–12% gradient gels (Thermo Fisher Scientific) and Coomassie Blue staining.MV preparations were analysed using Nanosight NS300 system  and data analysed using NTA software version 3.0. Each sample was diluted 100–10000 times, administered at constant flow with a syringe pump at 25°C and recorded in sets of three videos of 30 s with 5 s delay between recordings.g for 10 min at 4°C then fixed in 2.5% (w/v) glutaldehyde in 0.1M phosphate buffer (pH 7.4) for 1hr at room temperature. Fixed pellets were post-fixed for 1hr at room temperature in 1% (w/v) osmium tetroxide in 0.1M phosphate buffer, dehydrated in a graded series of ethanol and propylene oxide, embedded in Agar 100 epoxy resin  and polymerised at 60°C for 48 hr. Ultrathin sections of 80nm were cut on a Leica Ultracut UCT ultramicrotome  and collected on copper grids . Sections were stained with uranyl acetate and lead citrate.Bacterial cultures (1mL) were pelleted by centrifuging at 7,000 x2 Spirit TWIN transmission electron microscope  at 120 kV accelerating voltage. Images were captured using a Morada digital camera .For negative staining, MV preparations were adsorbed onto Formvar coated copper grids for 2 min. Excess sample was blotted off with filter paper. The grid was then transferred to a drop of 2% (w/v) aqueous uranyl acetate for 2 min before blotting off the excess and air drying the grids. All grids were viewed in a Tecnai GrrsG .DNA was isolated from vesicle preparations using a GES method . DNA cong for 10 min at 4°C. The aqueous phase was removed and mixed with an equal volume of 100% ethanol, and purified using a Purelink RNA mini column (Thermo Fisher Scientific) according to the manufacturer’s protocol for total RNA. Yields and purity were determined by spectrophotometry and RNA integrity was assessed using a TapeStation (Agilent Technologies) or Agilent Bioanalyser (Agilent Technologies). RNA for sequencing was separated into the small and large RNA fractions (><200bp) with an Ambion miRvana RNA isolation kit (Thermo Fisher Scientific) following the procedure for RNA clean-up.For RNA, vesicle preparations were resuspended in up to 1mL of TRIzol (Thermo Fisher Scientific), with 20μg of glycogen (Thermo Fisher Scientific) and 200μL of chloroform. Samples were vortexed for 15 sec, incubated for 10 min at room temperature and centrifuged at 17,000 xMV RNA was isolated from UPEC 536. Libraries were prepared using a TruSeq small RNA library kit (Illumina), retaining the fractions from the gel isolation step that correspond to 15-50bp and 50-200bp. These were run on an Illumina MiSeq sequencing machine , one library per MiSeq lane.Libraries of longer RNAs were prepared using an Illumina TruSeq stranded mRNA kit, excluding the poly-A selection step and without ribo-zero depletion . The ribo-zero step was deliberately excluded in order to provide us with a full understanding of the RNA content of UPEC MVs, including these highly abundant ribosomal RNAs. 60ng of total RNA was used as an input for the fragment, prime and finish step of the protocol. One library was run on each lane of a HiSeq 2500 sequencing machine (2x100bp). RNA-sequencing was performed as a contract service by New Zealand Genomics Ltd.http://hannonlab/cshl/edu/fastx_toolkit/) then quality and length filtered  using SolexaQA [Data was linker trimmed using fastq-clipper (SolexaQA . Reads wSolexaQA ) using BSolexaQA  with sta2 at 37°C. The cells were seeded  in full growth media and incubated overnight for 15 hr before addition of membrane vesicles and further incubation at 37°C for various timeframes, up to 96 hr. At specified timepoints cells were washed twice with PBS and placed into TRIzol (Thermo Fisher Scientific) for RNA isolation.5637 bladder carcinoma cells were purchased from the American Type Culture Collection  and cultured in RPMI 1640 containing 10% vol/vol foetal bovine serum  in humidified air with 5% vol/vol COxg for 1 hour to re-pellet the labelled MVs and to remove unincorporated dye.UPEC MVs containing 120μg of protein were labelled with 2μM of PKH67 (Thermo Fisher Scientific) in 2mL final volume by following manufacturers instructions. The labelled MV pellet was washed with RPMI and re-centrifuged at 75,000 5637 bladder carcinoma cells were seeded  into Ibidi culture plates (Ibidi GmbH) in full growth media and incubated for 15hr at 37°C. 10μg of PKH67-labelled MVs were added to the cells and incubated for 2–15 hr at 37°C. Treated cells were stained with 10μM CellTracker™ Red CMTPX (Thermo Fisher Scientific), according to manufacturer’s instructions, fixed in 4% formaldehyde for 15 min and then permeabilised in 0.25% Triton for 20 min. Cells were further stained for 5 minutes with 600nM DAPI (Thermo Fisher Scientific), immersed in Citifluor AF3 anti-fading agent (Citifluor Ltd.) and imaged using confocal microscopy ZEISS LSM 710 Inverted Confocal Microscope.To label MV RNA, UPEC 536 were grown with 100μM 5-ethynyl uridine  for 6h at 37°C, shaking at 200rpm, prior to MV isolation. 5637 bladder carcinoma cells were treated with 5EU-labelled membrane vesicles (10μg protein) and stained with CellTracker Red as above. Cells were washed twice with 1% BSA in PBS, fixed and permeabilised as above before being incubated in Click-iT® reaction cocktail  containing 2.5μM azide-containing Alexa Fluor 488 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The nucleus was highlighted with DAPI and visualised as prior.A standard curve was also generated by spiking different protein amounts of MV into 5637 cells lysed in trizol prior to RNA extraction. The spiking method ensured that the PCR product amplified was specific to the MV RNA and not due to background amplification of cellular transcripts.csrC with 100nM each F (5’- GAGGCGAAGACAGAGGATTG) and R (5’- TTTTTCCATTAGCCGGAACA) primers and ddPCR EvaGreen master mix (Bio-Rad) in a 22μL mix. Droplets were prepared by mixing 20μL PCR mix with 70μL EvaGreen generation oil in a QX100 droplet generator and then 40μL transferred to C1000 thermal cycler to cycle as per the manufacturers instructions . After PCR amplification, droplets were read using a QX200 Droplet Reader and analysed using QuantaSoft software.Equal volumes of RNA from MV treatments of 5637 cells and the standards were used for cDNA synthesis using qScript cDNA supermix (Quanta Biosciences) following the manufacturers conditions. A ddPCR mix was prepared using cDNA diluted 1:5 for 9 colony forming units/mL and 96.9±0.02% viable cells. Extracellular RNA was quantified at approximately 40ng/mL in these supernatants following passage through 0.22μm filters to remove intact bacteria.If our hypothesis that RNA signals are released by UPEC is correct we would expect to find RNA in cell-free supernatants from bacterial culture. Spent supernatants were collected from UPEC 536 cultured overnight to stationary phase. Cultures contained approximately 2x109 MV per mL of culture. Protein banding patterns were also consistent between MV preparations and differ distinctly from donor bacterial cells  genome with annotation in SeqMonk for RFAM and RefSeq genes, it was found that each size library had a distinct content of RNAs , rather than the medium size (0.23%) in which we would have expected these mature ~90nt RNAs to reside. We also identified varying proportions of reads derived from ncRNA and mRNA genes in each of the sized libraries. The protein coding RNAs  associated with MVs  were notThe RNA we sequenced was isolated from an initial MV preparation using a simple filtration and ultracentrifugation protocol and may arguably have included RNAs associated with non-MV contaminants in the sample. To exclude this possibility we quantified the RNA after further DGC fractionation for MVs isolated from three independent cultures. The fact that UPEC MVs associate with specific RNAs led us to investigate whether it could be directly delivered to cells, to play a proposed role in cell-cell communication with respect to human infection. By applying lipid PKH67 stained MVs to cultured human bladder cells we confirmed that from 2 hr post-application stained vesicles could be detected within the cytoplasm of the treated cells , often lWe then looked at whether we could track uptake of MV RNA cargo into treated host cells by labelling UPEC nascent RNA in the bacteria before it was packaged with MVs. This was achieved by adding an excess of a modified uracil, 5EU (5-ethynyl uridine), which was incorporated into the newly synthesised and MV associated RNA. The level of 5EU incorporation and packaging was low because we used the least 5EU possible for nascent RNA labelling, but this was sufficient to show the uptake of MVs into a selection of bladder cells by 15–16 hrs. The cellular localisation was observed in multiple replicates to be both in the cytoplasm and intriguingly in the nucleus . Due to csrC ncRNA into treated bladder cells by droplet digital PCR. This validation showed a rapid uptake of bacterial RNA into the cells, seen even at the first 5 minute timepoint, which peaked at 1 hr and then declined to almost undetectable by 48 hrs  assessed the mRNA content of vesicles released by Prochlorococcus, a marine picoplankton[Vibrio cholerae [The roles of bacterial MVs are under increasing scrutiny, particularly in terms of their protein and lipid cargo, and especially in infection scenarios. The potential of MVs to deliver a bioactive RNA to a target cell has been reported only five times, three of these in the past two years, highlighting the very recent but now growing interest in this phenomenon. In 1989, Dorward orrhoeae , 18. Sinoplankton. This wacholerae . In the et al. [E. coli grown in Luria-Bertani broth, a medium that does not claim to mimic the host environment [all the RNA content without selection bias. As a result we are able to form the first comprehensive picture of the RNA content of MVs from a human pathogen, beyond that destined for translation.In the final paper to report MV-RNA, Ghosal et al.  performeironment , identifin vitro [E. coli K-12 [et al. [Chlamydia and Legionella can be incorporated in the host RNA-induced silencing complex (RISC) and thus could be involved in direct subversion of host gene translation and mRNA stability.By splitting the RNA into three different ‘sized’ libraries we have assessed the complete profile of MV associated RNA from UPEC 536. Each library differed in its contents but all were dominated by abundant ribosomal RNA (71.5–99.4% annotated reads) which is not seen so commonly in eukaryotic vesicles . Once thin vitro  and mostoli K-12 . Our MV  [et al.  have recenrichment specifically for RNAs transcribed from the genomic regions of each the five major UPEC 536 pathogenicity islands [hlyA) and S-fimbrial-adhesin A (sfaA) [ompA, were from a ~42kb region encompassing ECP_1132 to ECP_1186, mostly prophage-derived genes. These prophage regions can interestingly be classed as further pathogenicity islands in UPEC [E. coli (EHEC) also makes detectable small RNAs from such phage genomic regions [After the rRNA and tRNA the next most common reads matched to protein coding mRNAs. These varied in abundance across the sized libraries (0.44–1.6%) but read coverage suggests that they are full-length mRNAs. Although present in our sequencing data we did not detect an  islands . HoweverA (sfaA) , 26, wer in UPEC . Small R regions  with posoxyS, ryhB and csrC. Based on their presence in specific ‘sized’ libraries and read coverage, these also appear to be packaged in their full-length forms. Cellular uptake of readily ‘active’ full-length RNA has been reported for mammalian mRNAs and regulatory RNAs transported in exosomes [The final class of RNAs found in MVs were small non-coding RNAs. The annotation of this class of RNAs in the UPEC 536 genome is limited but includes stress responsive genes such as exosomes  but not We also confirmed that the major proportion of RNA we isolated for our sequencing was co-fractionated with the MVs and protein using density gradient centrifugation and subsequent molecular analyses. Therefore the presence of ‘contaminating’ RNA in our simpler isolation method for the sequenced MVs, for example that associated with just protein aggregates was negligible. Thus the sequenced RNA can be considered as representative of that associated with the expected normal heterogenous population of MVs produced by UPEC.ompA, lpp and tonB. Furthermore, some of the proteins reported as packaged into E. coli MVs also bind RNA such as the protein chaperone GroEL, tRNA synthetases and ribosomal proteins [There are several possibilities for RNA association with MVs. Firstly the RNA may be simply tightly attached to the outside of the MVs, associating in this way after secretion from the bacterium, and comprising extracellular RNA released by general bacterial cell lysis. Secondly this could be an active and selective mechanism of RNA incorporation into MVs; thirdly it may just represent RNA in the cytoplasm being non-specifically enveloped within vesicle blebs; finally it may be due to RNA being passengers on MV-bound proteins. All of these could be operational in bacterial culture. Prokaryotic RNA is non-randomly distributed with at least four patterns of mRNA localisation recognised . Bacteriproteins . EF-Tu iproteins , linked proteins , 34 and proteins .csrC RNA delivered by MVs in to cultured bladder cells confirmed that RNA is internalised, with approximately 1% of the cargo delivered into the cells. This relatively low rate of RNA delivery by MVs unfortunately limited our ability to perform a full time-course microscopy to localise and track the uptake of MV-RNA. However, the finding of labelled bacterial MV RNA in the cell nucleus after 16 hrs in some of the cells is intriguing, particularly with small RNAs now reported to be involved in epigenetic regulation by alteration of chromatin state through methylation of histones and DNA [The packaging of molecules into MVs allows protection from external degradation and specific delivery to target host cells. Indeed our own MV preparations carried a range of RNAs including long intact RNAs. These RNA components, if active, could confer a selective advantage to the bacteria, either through RNA exchange signalling between different bacteria, or similarly, directly between the bacterium and host. Focusing on the latter, we have shown that MVs are internalised into cultured human epithelial cells. Although technically limited by the amount of 5EU labelled RNA that could be packaged into MVs, visualisation of 5EU MV-RNA by confocal microscopy confirmed cargo internalisation in the host cell. Further ddPCR of the  and DNA , 37.Some of the proposed actions of packaged RNAs have been discussed above; with hijack of the RNA-inhibition system by tRNAs and miRNA-like RNAs a possibility for some of the MV associated content. These mechanisms are proposed to ultimately lead to a change in host gene expression that is beneficial to the pathogen and its survival.This study provides further important new support for the plausible role of bacterial MV-RNA as a novel bacteria-host signaling molecule and adds to the growing literature characterising prokaryotic MVs. We have confirmed here that UPEC 536 releases MVs that associate with RNA. This RNA comprises much of the bacterial transcriptome in a largely intact form. Furthermore we have found that the MVs are capable of delivering their associated RNA cargo into treated host cells which poses an interesting hypothesis of whether this RNA could act as a novel signaling molecule in uropathogenesis.S1 FigScale bars are shown and the sizes of some identified vesicles are labeled.(PDF)Click here for additional data file.S1 TableMatched sequencing reads listed by annotated gene feature in each biological sample. Columns with reads (K-S) are split into libraries, small (<50nt), med (50-200nt), large (>200nt) by replicate and also presented as the sum of these .(XLSX)Click here for additional data file.


RNA editing is a dynamic mechanism for gene regulation attained through the alteration of the sequence of primary RNA transcripts. A-to-I (adenosine-to-inosine) RNA editing, which is catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family of enzymes, is the most common post-transcriptional modification in humans. The ADARs bind double-stranded regions and deaminate adenosine (A) into inosine (I), which in turn is interpreted by the translation and splicing machineries as guanosine (G). In recent years, this modification has been discovered to occur not only in coding RNAs but also in non-coding RNAs (ncRNA), such as microRNAs, small interfering RNAs, transfer RNAs, and long non-coding RNAs. This may have several consequences, such as the creation or disruption of microRNA/mRNA binding sites, and thus affect the biogenesis, stability, and target recognition properties of ncRNAs. The malfunction of the editing machinery is not surprisingly associated with various human diseases, such as neurodegenerative, cardiovascular, and carcinogenic diseases. Despite the enormous efforts made so far, the real biological function of this phenomenon, as well as the features of the ADAR substrate, in particular in non-coding RNAs, has still not been fully understood. In this work, we focus on the current knowledge of RNA editing on ncRNA molecules and provide a few examples of computational approaches to elucidate its biological function. While in the past researchers mainly focused on DNA mutations in order to further elucidate molecular pathways involved in numerous cancers, in the last decade focus has shifted to the analysis of post-transcriptional modification events, such as RNA editing. Concurrently, it has been estimated that only 1% of mammalian genome codes for protein, while the vast majority of the transcriptome is composed of non-coding RNAs crucially involved in gene expression pathways, such as transcription, translation, and gene regulation , potentially located several hundred to several thousand nucleotides upstream or downstream of the edited A. This requires experimental validation and represents one critical issue with the detection of editing sites.RNA editing is a type of post-transcriptional modification that takes place in eukaryotes. Several forms of RNA editing have been discovered, but nowadays A-to-I RNA editing is considered the predominant one in mammals    molecules.In the early 2000s, the ADAR enzyme family was observed to play an important role during embryonic development , things radically improved. In 2009, Li et al. . It was Table The birth of the first computational methods for the identification of RNA editing events  and biochemically-based , increasing the rate of siRNA and miRNA processing and facilitating RISC loading and RNA silencing, instead of an antagonistic role in RNAi by an ADAR1–ADAR1 homodimer complex . In recent years, HTS analyses have led to the identification of thousands of lncRNAs, many of which have revealed to be transcripts deriving from the antisense strand of protein coding genes. lncRNAs, due to their stable long double-strand regions, often originating from the presence of repetitive elements, such as Alus, can be affected by A-to-I RNA editing  in eukaryotes, can possibly be a result of adenosine deaminases acting on tRNA enzyme family (ADATs) (Su and Randau, As seen above, currently Alon’s pipeline is the only HTS-based method to systematically identify A-to-I editing sites in pre- and mature microRNAs. There is a current and urgent necessity for new HTS-based methodologies to emerge in order to not only accurately identify and analyze editing events in other categories of ncRNA molecules, such as tRNAs, lncRNAs, and so on, but also to investigate through functional enrichment analysis, the biological outcomes that a single editing event can generate. Concurrently, it could be interesting to analyze how the editing phenomenon can influence a biological pathway within a temporally changing cellular condition, such as starvation or hypoxia, considering that a single editing site in a ncRNA molecule could drastically modify its function.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SCs) play a central role in the development of germ cells within functional testes and exhibit varying morphology during spermatogenesis. This present study investigated the seasonal morphological changes in SCs in the reproductive cycle of Pelodiscus sinensis by light microscopy, transmission electron microscopy (TEM), and immunohistochemistry. During hibernation period with the quiescent of spermatogenesis, several autophagosomes were observed inside the SCs, the processes of which retracted. In early spermatogenesis, when the germ cells started to proliferate, the SCs contained numerous lipid droplets instead of autophagosomes. In late spermatogenesis, the SCs processes became very thin and contacted several round/elongated spermatids in pockets. At this time, abundant endoplasmic reticulum and numerous mitochondria were present in the SCs. The organization of the tight junctions and the adherens junctions between the SCs and germ cells also changed during the reproductive cycle. Moreover, SCs were involved in the formation of cytoplasmic bridges, phagophores, and exosome secretions during spermatogenesis. Tubulobulbar complexes (TBC) were also developed by SCs around the nucleus of the spermatid at the time of spermiation. Strong, positive expression of vimentin was noted on the SCs during late spermatogenesis compared with the hibernation stage and the early stage of spermatogenesis. These data provide clear cytological evidence about the seasonal changes in SCs, corresponding with their different roles in germ cells within the Chinese soft‐shelled turtle Pelodiscus sinensis.Sertoli cells ( Testes are considered components of both the reproductive and the endocrine systems. The testis parenchyma consists of numerous seminiferous tubules and interstitial tissues. Each seminiferous tubule is composed of germ cells and Sertoli cells (SCs). SCs play a central role in the development of a functional testis and contribute to the expression of the male phenotype Mackay . SCs areSertoli cells play a key role in the regulation of spermatogenesis and the production of spermatozoa. Without the physical and metabolic support of the SCs, germ cells cannot proliferate and survive Sharpe . SCs areThe morphology of SCs is very complex due to continuous changes in cellular structure and size, and the ultrastructural features differ among species, ages, seasons, and stages of the spermatogenesis , spermatogenesis is active through late spring, summer, and fall (May to October), ending in a temporal rather than a spatial pattern, resulting in one massive release of sperm in late October or early November. It remains quiescent (hibernation period) throughout the rest of the year (December to April), so it is a potential model organism with which to determine seasonal effects in China , May (early spermatogenesis) and October (late spermatogenesis), five turtles during each time period. The animals were rendered comatose using intraperitoneally administered sodium pentobarbital  and were sacrificed by cervical dislocation. The testes were collected immediately and fixed (details below) for light, electron microscopy, and immunohistochemistry analysis. Sample preparation was conducted according to accepted international standards and was approved by the Ethics Committee for Animal Care and Use by the Science and Technology Agency of Jiangsu Province (SYXK (SU) 2010‐0009).Fifteen mature, male (3–4 years‐old) μm. These sections were stained with hematoxylin and eosin procedures (Harry's hematoxylin for 2 min and 1% eosin for 30 sec) for light microscopic analysis using an Olympus microscope (BX53), camera .The sample were placed in 10% neutral buffered formalin for fixation overnight and then embedded in paraffin wax, and wax blocks were prepared. Sectioning was performed at 5 3) and then immersed in 2.5% glutaraldehyde in PBS  for 24 h. Tissue was rinsed in the same PBS and then postfixed for 60 min. at room temperature in the same way using buffered 1% osmium tetroxide  and washed in the buffer. The samples were then dehydrated in ascending concentrations of ethyl alcohol, infiltrated with a propylene oxide–Araldite mixture and then embedded in Araldite. The blocks were then sectioned using an ultramicrotome , and the ultrathin sections (50 nm) were mounted on copper coated grids. The pieces were stained with 1% uranyl acetate and Reynold's lead citrate for 20 min. Finally, specimen were examined and photographed using a high resolution digital camera (16 mega pixel) connected to the TEM, Hitachi H‐7650 (Japan).The specimen were cut into small parts (1 mmμm) were placed on glass slides, which are already treated with by poly‐l‐lysine and were stained according to immunohistochemical standard techniques. Briefly, after deparaffinization and washing in phosphate‐buffered saline (PBS), these sections were enclosed with 3% hydrogen peroxide in PBS for 15 min at 37°C in order to block the further activity of endogenous peroxidase. The samples were blocked with 5% bovine serum albumin and incubated with rabbit anti‐vimentin (1:75) antibody  in a moisture chamber at 4°C for 24 h. After washing, the sections were incubated with biotinylated anti‐rabbit IgG  for 1 h at room temperature. The sections were then rehydrated in PBS (PH 7.2), incubated with avidin‐biotinylated peroxidase complex for 45 min at 37°C. After being washed with PBS, peroxidase activity was revealed using DAB  according to the instructions of company.Paraffin sections (6 In January (hibernation period), SCs with irregular nuclei were observed and were located away from the basal membrane. A few layers of germ cells, typically spermatogonia and residual spermatozoa, were observed in the seminiferous tubules. The adluminal compartment appeared almost clear, and the decreased sperm number was indicative of the testes being cleared into the epididymis Fig. A and B. In January (hibernation period), weak immunoreactivity of the vimentin antibody was noted in the basal compartment of the SCs, but delaminating from the adluminal compartment of the seminiferous tubule Fig. A and B. In January (hibernation period), irregular nuclei and numerous degenerated germ cells were observed in the SCs. The cytoplasm of the SCs contained entotic vacuoles, which contained numerous autophagosomes Fig. . In May In October (late spermatogenesis), the SCs were in close proximity to the basal membrane, and irregular invaginations toward the basal membrane were observed Fig. A. The SCA double membrane, cup‐shaped structure known as the phagophore appeared within the cytoplasm of the SCs. This cup‐shaped structure invigilates the cytoplasm of SCs, inside the double membrane Fig. B. ElectrP. sinensis testes.Furthermore, the processes of the SCs were interconnected by well‐developed tight junctions between the basal and the adluminal compartment Figs. A,B and DSince the discovery of Sertoli cells (SCs) in the late 1800s, their morphology has been difficult to understand because they continuously change their structure and have three–dimensional contact with developing germ cells  around the nucleus of germ cells. Vimentin filaments also help to maintain the integrity of SC contact with the seminiferous epithelium around spermatogenic cells. Therefore, they play a key role in maturation of spermatogenesis and gap junction intercellular communication  within SCs around mature spermatids. TBCs are fascinating structures appearing mostly in late spermatogenesis, during spermiation  in late spermatogenesis within the Chinese soft‐shelled turtle The authors have no conflict of interest to declare.


Due to the increasing number of known microRNAs, these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA  ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the ribonuclease-specific degradation grade depending on the RNA class was determined.In this in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel electrophoresis. In addition, we have examined whether the integrity observed for ribosomal RNA is applicable to microRNA and mRNA. Depending on the kind of ribonuclease used, our results demonstrated a higher stability of microRNA relative to mRNA and a limitation of the relevance of ribosomal RNA integrity to the integrity of other RNA groups.In the present Our results suggest that the degradation status of ribosomal RNA is not always applicable to mRNA and microRNA. In fact, the stabilities of these RNA classes to exposure to ribonucleases are independent from each other, with microRNA being more stable than mRNA. The relative stability of microRNAs supports their potential and further development as biomarkers in a range of applications. MicroRNAs (miRNAs) are small, non-coding RNAs which are encoded within the genome. The primary transcript, pri-miRNA, contains one or more hairpin structures and is cleaved to pre-miRNA ,2. Upon Previous studies have suggested that mRNA stability is an important control point for gene expression regulation . The matA widespread interest has developed in small non-coding RNAs for cancer research and drug development. In particular, miRNAs appear to play a critical role in tumorigenesis and are thus promising candidates as potential therapeutic targets or as biomarkers ,10. Morein vitro conditions. For this reason, we decided to enhance general knowledge about the stability of mRNA and miRNA in response to ribonucleases commonly used in routine laboratory practice. We were interested in finding out how a hypothetical contamination with ribonuclease might affect mRNA and miRNA stability. Furthermore, we investigated possible differences in the stability of different classes of RNA . For this purpose, total RNA was treated in vitro with a range of ribonucleases under identical experimental conditions and their effects were analysed by RT-qPCR.Although miRNAs comprise an important class molecules used to uncover regulatory mechanisms in the cell, nearly nothing is known about their decay in comparison to mRNA under defined In contrast to mRNA, which represents only 0.5 to 3% of the transcriptome, ribosomal RNA is the most prevalent constituent of total RNA in mammalian cells . For thiRepresentative electropherograms of our samples that were analysed with the RNA6000 Nano labchip are illustrated in Figure f, even though the ribosomal peaks were not detectable by this method, the measured absorption was above the base line. This is caused by residues of ribosomal RNA. No specific changes in comparison to the untreated sample were observed in the electropherograms of the samples after treatment with RNase H, Exonuclease T or Exonuclease T7.In the case of treatment with benzonase and RNase IIn order to determine the decay of ribosomal RNA after the treatments with a higher sensitivity, a relative measurement of 18S RNA was performed with RT-qPCR . For treatment with RNase If, a statistically non-significant trend of reduction was observed.A similar effect could be observed after treatment with benzonase and RNase I7 showed no significant changes compared to the untreated sample. This confirmed the result obtained by microfluidic analysis in a quantitative manner.The treatments with RNase H, Exonucleases T and Exonucleases Tf treatment, comparison of sample electropherograms, which represented the 18S and 28S ribosomal RNAs  .Since reference genes are also influenced by the treatments, relative quantification was performed by normalization to the untreated control. For this purpose, the Cq value of 40 cycles was assumed to represent the total digestion of RNA (0% of RNA undigested) and the Cq value of the untreated control was assigned as not digested (100% of RNA undigested). The undigested part after treatment was calculated using the following formula: x = 100 * . The significance was calculated by the two-tailed unpaired