The control of pre-analytical-factors in human biospecimens collected for health research is currently required. Only two previous reports using post-mortem brain samples have tried to address the impact of cold-ischemia on tissue pH. Here we report pH variations according to time  in mice for liver, kidney and lung samples. Tissue alkalosis in cold-ischemia time may be an underlying mechanism of gene expression changes. Therefore, tissue-pH regulation after organ removal may minimize biological stress in human tissue samples. There is a consensus that high-quality, data-rich samples are essential for future research; however, their collection and storage is not as straightforward as many researchers think3. When it comes to transcriptional analysis, this concern is especially true, since gene expression can be modulated by various factors4. In terms of high-throughput transcriptional analysis, the highest RNA quality is recommended. However, the real impact of tissue specimens’ pre-analytical factors on the transcriptional regulation, besides the RNA integrity, is not well understood. While technical issues such as preparation, quality and concentration of RNA are of great concern for the use of analytical tools and platforms, those related to tissue collection, such as cold ischemia, have been recently considered10. An increasing number of reports have demonstrated that inadequate human biospecimen handling can have an adverse impact on assay results and biomarker discovery, with a negative effect on the clinical practice. However, the underlying mechanism leading to such changes in gene expression is not wholly defined12. The RNA Integrity Number (RIN), a measurement of ribosomal RNA integrity, has been used to evaluate the overall RNA quality in biospecimens13. Additionally, RIN measurement fails to reflect the influence of the external factors that may have an impact on the modulation of gene expression, while not interfering with the overall RNA degradation. Specific and uncertain surgical factors, such as warm-ischemia time, are hard to be controlled. Moreover, the length of time a tissue sample could stay at room temperature after excision and before freezing is currently a matter of debate. The impact of cold-ischemia time may possibly vary by organ, with some of the organs requiring a much faster freezing process14. In order to adequately investigate the underlying mechanism involved, a strict control of tissue ischemia times before cryopreservation is also required.Biospecimens collected by tissue banks may provide valuable insights into the mechanisms of a wide range of diseases, thus leading to the discovery of biomarkers of diagnosis, prognosis and therapeutic guidance15. In addition, working with neoplastic disease-free mice allows avoiding the introduction of disease variables, which could not be bypassed with cancer patients’ tissues. Finally, a quality control marker identified in an animal model, particularly in mice, may be applied to human biospecimens, provided that the corresponding molecules or pathways are represented in this animal model11. Therefore, we investigated the impact of devitalization on tissue quality using a mouse model, to find out its effect of pH and RNA integrity after a cold ischemia insult.Mice are the vertebrates most widely used in scientific research, due to their genetic similarities with men. Approximately 99% of the human genes have been mapped into mice, making these vertebrates suitable for comparative studies16. This time interval was chosen considering the maximum acceptable cold ischemia time reported in the literature17. Complete model description and results are described in equations  or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalizedOur work could be considered a hypothesis generator study. Was the blood acidosis detected in a closed corporeal entity in hypoxia a consequence for the compensation of the tissue alkalosis produced in the first instance in the hypoxic tissues? This hypothesis should be further explored since high-throughput molecular assays are playing an ever-growing role in research and into the understanding of the pathobiology of human and animal diseases.In conclusion, this work found significant differences in pH values between organs at the same cold-ischemia time, and in the same organ at different times in an experimental animal model, although no differences were seen in the RNA quality assessed by its integrity number or absorbance ratios. Our results suggest that pH is high in tissues undergoing ischemia, a finding that has already been reported in previous studies, and the regulation of tissue pH after organ removal may minimize biological stress in human tissue samples. The results from this study suggest that although the RIN is a powerful tool to inspect the ribosomal profile to infer RNA quality from fresh and frozen tissues , it is not sufficient to predict the integrity of mRNA transcripts and describe the real biological conditions. Therefore, the role of pH as an underlying mechanism of gene expression changes after an ischemic insult warrants further investigation.Fifty-two mice (Mus musculus) C57BL/6-SPF from the Animal Facility at the CIPE were used. The study was approved by the Ethics Committee in the Use of Animals (CEUA) at Antonio Prudente Foundation (N°049/12). All experiments were performed in accordance with relevant guidelines and regulations. Animals were divided into 5 groups according to the time of tissue cryopreservation. All mice were pathology free (data not shown). The 26 male and 26 female mice were carefully selected in order to have a similar weight (24.89 ± 5.21 gr) and time of life for each gender . Three μm sections were cut in a −28 °C cryostat. Excess of Tissue-Tek® O.C.T. compound was separated and tissue was saved in the corresponding cryotube for subsequent macromolecules extraction. The histoarchitecture was studied using standard hematoxylin-eosin staining, to assess the morphological changes induced by cold-ischemia.20. The effects of pH and ionic strength have shown that the change in the A260/280 ratio is primarily due to a decrease in the absorbance at 280 nm, when the ionic strength or pH is increased. This effect was achieved irrespective of the method of purification. However, the elution method can interfere with the A260/280 ratio. Thus, RNA diluted into distilled-DEPC-treated-water has usually a lower ratio compared with Tris-EDTA pH 8.0.The ratio of absorbance at 260 nm and 280 nm (A260/280) was used to assess RNA purity. A ratio of 2.0 or greater is accepted as pure RNA . The RNA was isolated from up to 30 mg of disaggregated solid frozen tissue, which was homogenized with Precellys®24  for 4 cycles (10 seconds at 6500 rpm and 5 minutes in ice), and centrifuged 3 minutes at 13000 rpm. Supernatant was separated for total RNA purification, and pellet was saved at −20 °C for DNA extraction, if necessary. The RNA was purified using RNeasy Mini Qiacube Kit – Animal tissue and cells – Large Samples  on the automated Qiacube . RNA concentration and purity were measured with NanodropTM ND-1000 . RNA integrity was determined with Agilent RNA 6000 Nano chip  and measured on Agilent Bioanalyzer-2100 . Agilent Bioanalyzer showed a numerical value (RIN-RNA Integrity Number) which varies from 1 to 10, with 1 referring to degraded RNA and 10 to full RNARNA concentration over 0.1 µg/µl was considered the minimum concentration required for sample acceptance .The pH was measured at room temperature in technical duplicates for both lungs and kidneys, and for the anterior and posterior portions of the liver ; the j-th linear , quadratic (γj = βj2) or third (δj = βj3) time degree; the double time by organ interactions terms ; the k-th gender indicator , and the l-th organ side indicator (ρl for right or left). The random effects were the m-th animal am ~ N, i.e. normally distributed with zero mean and identity the variance matrix (I) times, the σa2 parameter; the n-th technical replicate within animal effect bn(m) ~ N, the o-th sample extraction day co ~ N, the p-th surgery day dp ~ N, and the random error function g(ε)ijklmnop which was modeled as independent errors, i. e., g(ε)ijklmnop = I(ε)ijklmnop = εijklmnop ~ N or dependent errors, with the lack of heterocedasticity being corrected with a constant variance function grouped by animals, formally, g(ε)ijklmnop = varIdent ijklmnop. Using the former annotations, the different experimental data measurements (yijklmnop) were modeled starting from the maximal model of equation  and Bayes information criterion (BIC) under the parsimony criterion. The corresponding model assumptions were verified using residual vs. predicted values and normal quantile-quantile plots. When required, outlier values were removed and models were estimated again. For the final model assessment, marginal hypothesis test was used with type-III sum of squares for fixed effects. A posteriori analyses included Fisher’s least significant difference (LSD) using Bonferroni adjusted p-values at each time period or main effect. All statistical tests, unless otherwise specified, were two-sided with a cut-off at 0.05 for statistical significance. Equations –9 and SuIschemia time evolution for pH in the three different organs studied is depicted in Fig. Supplementary Data


Cancer development involves the stepwise accumulation of genetic lesions that overcome the normal regulatory pathways that prevent unconstrained cell division and tissue growth. Identification of the genetic changes that cause cancer has long been the subject of intensive study, leading to the identification of several RNA-binding proteins (RBPs) linked to cancer. Cross-reference of the complement of RBPs recently identified by RNA interactome capture with cancer-associated genes and biological processes led to the identification of a set of 411 proteins with potential implications in cancer biology. These involve a broad spectrum of cellular processes including response to stress, metabolism and cell adhesion. Future studies should aim to understand these proteins and their connection to cancer from an RNA-centred perspective, holding the promise of new mechanistic understanding of cancer formation and novel approaches to diagnosis and treatment. Current Opinion in Genetics & Development 2018, 48:112–120Cancer genomicsThis review comes from a themed issue on Fátima Gebauer and Omar Abdel-WahabEdited by Issue and the EditorialFor a complete overview see the Available online 5th December 2017https://doi.org/10.1016/j.gde.2017.11.006http://creativecommons.org/licenses/by/4.0/).0959-437X/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license  such as RNA-recognition motifs (RRMs)  cluster under normal iron levels, forming the catalytically active protein. However, under low iron concentrations [Fe–S] is no longer available, causing ACO1 to remain as an apoprotein. In this state, ACO1 binds the 5′ and 3′ UTRs of mRNAs involved in iron homeostasis and regulates their fate . RNA binIDH enzymes have well established roles in glioma, secondary glioblastoma and acute myeloid leukaemia (AML). The mechanistic role of IDH in these cancers has been determined by analysing point mutations at specific arginine residues  and glutaredoxin 3 (GLRX3), along with catalase (CAT), peroxiredoxin 1 and 3 (PRDX1/PRDX3), were identified as candidate cancer-associated RBPs , providing a potentially interesting link between antioxidant metabolism, RNA binding and cancer.All three members of the ezrin-radixin-moesin (ERM) protein family were identified as RBPs by RNA-IC , 20•• (Fin silico, in vitro and in vivo approaches for identification of RBPs and RBDs have recently been developed 7[Recently developed global approaches for identification of RBPs have uncovered a new universe of RNA-binding activities, many of which have been linked to tumorigenesis, cancer progression, invasion and metastasis. We have provided here an overview of the diversity of non-canonical RBPs and their potential links with oncogenesis. However, the precise roles of most of these unorthodox RBPs in cancer, and the relevance of their RNA-binding activities, remain to be elucidated. In this review, we have limited our discussion to human RBPs that have been experimentally identified by RNA-IC and RBDmap. However, the task of uncovering the cancer-related RNA-binding proteome is by no means complete. Other veloped 7, 59•, 60Nothing declared.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


A crucial stage in the origin of life was the emergence of the first molecular entity that was able to replicate, transmit information, and evolve on the early Earth. The amyloid world hypothesis posits that in the pre-RNA era, information processing was based on catalytic amyloids. The self-assembly of short peptides into β-sheet amyloid conformers leads to extraordinary structural stability and novel multifunctionality that cannot be achieved by the corresponding nonaggregated peptides. The new functions include self-replication, catalytic activities, and information transfer. The environmentally sensitive template-assisted replication cycles generate a variety of amyloid polymorphs on which evolutive forces can act, and the fibrillar assemblies can serve as scaffolds for the amyloids themselves and for ribonucleotides proteins and lipids. The role of amyloid in the putative transition process from an amyloid world to an amyloid–RNA–protein world is not limited to scaffolding and protection: the interactions between amyloid, RNA, and protein are both complex and cooperative, and the amyloid assemblages can function as protometabolic entities catalyzing the formation of simple metabolite precursors. The emergence of a pristine amyloid-based in-put sensitive, chiroselective, and error correcting information-processing system, and the evolvement of mutualistic networks were, arguably, of essential importance in the dynamic processes that led to increased complexity, organization, compartmentalization, and, eventually, the origin of life. It is generally believed that life on Earth passed through an RNA-world era in which RNA or an RNA-like polymer performed both informational and catalytic functions –4. It isWithin the framework of origin-of-life research, the question of the mechanism of replication and information transfer is crucial. Given the stability and functionality problems connected with the RNA-world hypothesis –7, 9, thRecent research has clarified many aspects of the molecular mechanisms underlying the amyloid-mediated information system. It has become evident that the coding element is the steric zipper structure of the amyloid motif and that recognition occurs by amino acid side chain complementarity , 18. In A schematic representation of the replication cycles is shown in Fig. A distinctive feature of amyloid formation is that the same peptide monomer can generate functionally and structurally different amyloid conformers of which one or several can propagate and make new copies of itself/themselves , 36. ThiThe amyloid polymorphs differ from each other in the architecture of the β-strands within and between the β-sheets and in the stacking of the β-plates, as well as in filament length. The number of protofilaments per fibril and the degree of twisting of the fibril can also vary. The β-sheets are tightly packed without water molecules between them; hydrogen bonds, van der Waals forces, and electrostatic polarization hold the zippers together , 18, 37.The catalytic activity of the peptide-based β-sheet assemblies is an important aspect of the amyloid world model. Amyloids not only catalyze their own formation, but they are able to catalyze other chemical reactions too. Rufo et al.  showed tThe prebiotic relevance of the β-sheet networks and assemblies was recently highlighted by Tena-Solsona et al.  who provThe amyloid model fulfils key criteria of a valid origin-of-life theory, i.e., the requirements of replication, information transfer, and variation. By repeated replication cycles, the amyloid conformers can generate a variety of polymorphic fibrillar networks Fig.  and struThe role of amyloid in the putative transition of the pristine amyloid world to an amyloid–RNA–protein world is not limited to scaffolding and protection; the interactions of amyloid with ribonucleotides and protein are both cooperative and dynamic. Fibrillar amyloid can act as an auto-catalyzing surface , 60–62 aThe transition from an amyloid world to an amyloid–RNA–protein world is likely to have evolved over a long period of time. However, the amyloid model , 11, 74 Prions are self-propagating infectious protein-based agents that can cause severe neurodegenerative disease. The prion hypothesis postulates that a misfolded form of the prion protein is the causative and transmissible agent of prion disorders . Most, iThe amyloidogenic regions of prions are evolutionarily conserved, and in many instances represented by short, low-complexity sequences enriched in glutamine/asparagine domains , 90. ImpInformation transfer on the early Earth for about 4000 million years ago occurred, according to the amyloid hypothesis, by means of a β-sheet peptide-based prion-like amyloid system in which environmentally derived information encrypted in the β-sheet zipper structure was transmitted by a templated conformational self-replication mechanism to “daughter” amyloid entities , 11, 74.The amyloid model emphasizes the importance of the cooperative interactions between amyloid and ribonucleotides and of the transition from a primitive β-sheet world to a more complex amyloid–RNA–protein world, a transition that was necessary from both an evolutionary and informational point of view: The information content of the β-sheet system, though potentially large, is very limited when compared to the virtually unlimited information content of a nucleic acid-based genetic system . The β-sThough based on amino acids and peptides, there are fundamental differences between the amyloid world hypothesis , 11 and It is well documented that under conditions simulating early Earth conditions, amino acids and short peptides are readily formed –105. A hA crucial stage in the origin of life was the emergence of the first informational molecular entity that was able to self-organize and evolve on the primordial Earth. The amyloid model, a hybrid replication-metabolism model of the origin of life, posits that, in the pre-RNA era, information storage and transfer was based on peptide-based catalytic amyloids. The template-assisted conformational replication cycles generate a variety of amyloid polymorphs on which evolutive forces can act. Amyloid, RNA, and protein interact dynamically and cooperatively, and the amyloid assemblages can function as primitive metabolic entities catalyzing the formation of simple metabolite precursors.In conclusion, the emergence of an amyloid-based pristine in-put sensitive, chiroselective, and error correcting information-processing system, and the evolvement of mutualistic networks were, arguably, of essential importance in the dynamic processes that led to increased complexity, organization, compartmentalization, and, eventually, the origin of life.


The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction  and solid-phase separation with/without affinity resin . We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes.We identified differences between each of the RNA extraction methods in the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. For the purposes of consistency in quantity, quality and high recovery of miRNAs from tissues, we identified that Phenol:chloroform phase separation combined with silica column-based solid extraction method was preferable (miRVana microRNA isolation). We also identified a method that is not appropriate for miRNA analysis from tissue samples (Bioline Isolate II). For target gene expression any of the kits could be used to analyse mRNA, but if interested in analysing mRNA and miRNA from the same RNA samples some methods should be avoided.Different methods used to isolate miRNAs will yield different results and therefore a robust RNA isolation method is required for reproducibility. Researchers should optimise these methods for their specific application and keep in mind that “total RNA” extraction methods do not isolate all types of RNA equally.The online version of this article (10.1186/s12896-018-0421-6) contains supplementary material, which is available to authorized users. They act via binding to specific sequences in the 3′-untranslated region (3′-UTR) of mRNAs, leading to either translational repression or message decay. miRNAs have been linked to distinct biological processes in normal development and their altered expression is implicated in many diseases including neurological disorders, immune diseases and cancers . miRNA pWith the increasing interest in miRNAs, particularly as diagnostic and prognostic biomarkers, a number of platforms have been developed to analyse miRNA expression. Current detection methods of miRNAs involve cloning, in situ hybridisation, microarray, bead-based flow cytometry, northern blot, next generation sequencing and quantitative real time PCR (qRT-PCR) , 6. ThesThe development of a miRNA for therapeutic use also requires knowledge of its specific gene targets, a field of research which is in its infancy. Therefore, it may be of specific importance for researchers to analyse both miRNA and target gene expression in the same RNA sample. However, different extraction methods may bias towards isolation of either long or short RNAs, potentially impacting the findings of the study. This research used five commonly used RNA isolation methods to extract total RNA including miRNAs from solid tissues, and compared the extraction efficiency, quality and yield of resultant RNAs. We analysed miRNA and target gene expression using qRT-PCR from brain, liver and lung tissues from mice. The data presented herein could serve as a useful reference for researchers interested in analysing both miRNA and target gene expression in animal tissues for diagnostic and prognostic use or miRNA validation.The overall workflow for the study is outlined in Fig. n = 3). Mice were euthanised by cervical dislocation, then tissue samples were harvested and immediately snap frozen using 100% ethanol and dry ice, and stored at − 80 °C until used. The use of mice was in accordance with local institutional ethical guidelines.Brain, liver and lung tissue samples were from 8 to 10 week old male C57Bl/6 mice , miRVana microRNA isolation kit , Total RNA isolation kit , miRNeasy mini kit , and TRIzol Reagent (Thermo Fisher Scientific). Frozen tissue samples were weighed and processed on ice to prevent thawing. In preparation for RNA extraction 5–10 mg of tissue  was added to 700 μl of the appropriate lysis buffer. Samples were homogenised using a TissueLyser II (Qiagen) with 5 mm stainless steel beads for 3 × 1 min cycles at 30 Hz, with resting on ice in between. Homogenates were subjected to a single freeze/thaw cycle to aid cell lysis and then cleared by centrifugation at 10,000 x g for 5 min at 4 °C. RNA extractions were performed according to manufacturer’s instructions, with the exception of an additional chloroform extraction step for the TRIzol reagent protocol as illustrated in Fig. RNA samples were quantitated using the Qubit HS RNA kit (Thermo Fisher Scientific) with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific), following manufacturer’s instructions. The 260/280 and 260/230 ratios of absorbance values were used to assess the purity of RNA using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). A 260/280 ratio of ~ 2.0 and 260/230 ratio in the range of 2.0–2.2 was accepted as “pure” for RNA. Lower ratios may indicate the presence of protein, phenol, EDTA, carbohydrates or other contaminants that absorb at or near 260, 230 or 280 nm. The miRNA content was quantitated using a Qubit microRNA assay kit (Thermo Fisher Scientific) with a Qubit 3.0 Fluorometer, according to manufacturer’s recommendations. The integrity of the RNA was determined for all samples using an Agilent Bioanalyzer 2100  with an Agilent RNA 6000 kit (Agilent Technologies). RNA Integrity Numbers (RINs) were used to evaluate the integrity of the RNA samples with > 7.0 considered intact and < 7.0 considered degraded. The quality assessment of RNA samples is provided in Additional file For miRNA detection, 10 ng of the RNA samples were reverse transcribed in technical duplicates using a TaqMan microRNA reverse transcription kit (Thermo Fisher Scientific) with Taqman microRNA primers specific for the miRNAs of interest, following manufacturer’s instructions , taking PCR efficiency into account. GeNorm and Normfinder were used to select appropriate reference genes for normalisation. One-way ANOVA was used to compare the different extraction methods for each tissue and Student’s t-tests  used to detect differences in normalised gene expression with For miRNA expression analyses the quality of the RNA is critical. In addition, if there is a limited amount of biological material from which to extract the RNA, researchers may be interested in the yield produced by different RNA extraction methods. Comparison of the yield, quality and integrity of the RNA samples revealed discrete differences with different extraction methods Table . With remiRNA expression can be analysed by a number of methods including qRT-PCR, microarray hybridization platforms , high throughput sequencing using libraries for small RNAs and nanostring technology based on the nCounter analysis system. However, the gold-standard for analysing miRNA expression is qRT-PCR using specific primers such as Taqman miRNA primers (Thermo Fisher Scientific) (most common method used), locked nucleic acid primers (Exiqon) and poly(A) tailing . In this study, miRNA expression was analysed using Taqman miRNA assays which employ a sequence specific stem-loop primer. We chose to look at six miRNAs  and snoRNA-234 are commonly used for normalisation of mouse tissue samples –27. FurtIn this study we hypothesised that different methods of RNA isolation will impact results when analysing gene expression in tissues. RNA extraction methods can be broadly characterised into either phenol:chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction  and solid-phase separation with/without affinity resin . These methodologies were primarily developed for the extraction of long mRNAs and have been based on the assumption that all RNAs are purified equally. Moreover, there are a number of considerations for choosing a particular RNA extraction method such as the quality, quantity, price, and ease of use (time to extraction). Herein, we present data that shows the method used to extract the RNA will also produce variable results compared to different methods in downstream applications and therefore is another important consideration.later (ThermoFisher Scientific), which allows unfrozen tissue samples to be processed later. It may help ensure the integrity of the RNA, but it is not compatible with all RNA isolation procedures and users should check their specific method prior to performing extractions. The addition of a peak after 60 s in some samples in the Bioanalyser analysis suggests gDNA contamination. An additional DNase treatment can be performed on columns with some kits, however a gDNA elimination step is highly recommended during the reverse transcription step. The inclusion of this step in our methods may explain why there was no interference when analysing mRNA expression of the Norgen lung samples, but it did impact miRNA expression analysis as the Taqman miRNA assay protocol does not include gDNA removal. Furthermore, the purity of RNA samples is a critical factor as even though samples with RIN > 7 should perform okay in most downstream applications, those that involve enzymatic reactions such as qPCR can be inhibited by nucleases, metal ions or organic contaminants. In general, the Bioline Isolate II RNA samples had lower 260/230 ratios and contaminants could be contributing to its poor performance. Finally, differences were observed in the enrichment for miRNAs in the total RNA preparation. As miRNAs represent a small fraction of the overall RNA repertoire, it can be difficult to determine their contribution from the integrity analysis. The Qubit microRNA assay offers highly selective detection of low amounts of small RNAs even in the presence of common contaminants. High percentages of miRNAs were commonly detected with miRVana and Norgen extraction methods, however given the integrity analysis for Norgen total RNA preparations, it is possible that the miRNA enrichment may be overestimated as smaller RNA fragments are being detected following degradation or oxidation of larger RNAs  or in the case of the lung samples DNA contamination perturbing the Qubit results. Hence, the quantity, quality and purity of RNA samples are highly important factors for choosing a particular RNA extraction method and further research for how to optimise these methods for your tissue of interest can help improve these.This study demonstrates that RNA isolation methods vary in terms of the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. We chose organs that can be difficult to isolate RNA from for different reasons. For example, RNA isolation from brain is often noted to result in low yields due to its high lipid content. This was particularly evident with the Bioline Isolate II kit. The manufacturers’ protocol suggests that up to 5 μg of RNA should be purified from 10 mg of rat/mouse brain. However, the miRNeasy handbook suggests that 5–20 μg of RNA should be achievable from brain with the same amount of input material. Troubleshooting on the manufacturer’s website describes one problem with lipid rich tissues is clogging of the column and suggests decreasing the sample amount or increasing the lysis buffer volume. Our extractions were performed within the suggested limits of the protocol. Subsequently, they suggest performing pre-extraction using TRIsure reagent  then column-based separation with the kit to clean-up the aqueous phase. This method would then be comparable to miRVana and miRNeasy RNA extraction methods and may result in better yields for this tissue. Additionally, the RNA quality correlates to tissue-specific responses to physiological stress both prior to and following tissue death. Tissues such as lung and liver characteristically have low RINs and a small 28S peak as these tissues are prone to faster degradation of RNA by high levels of nucleases. To inactivate nucleases the tissue is rapidly frozen and thawing prevented until the weighed material is added to the extraction buffer. Although, thawing of tissue was prevented, we cannot rule out the time from euthanasia of the animal and excision of the organs to rapid-freezing as a contributing factor to the overall low RIN values and some degradation products observed with all lung samples, irrespective of isolation kit used. An alternative method to inactivate nucleases is using a RNA stabilising solution such as RNAProfiling of miRNAs can give insight as biomarkers for diagnostic or prognostic applications. For example, panels of miRNAs can be used to classify different cancer phenotypes, predict recurrence or response to therapies , 29–31. . Currently, no reports have directly compared miRNA and target gene detection from total RNA samples using different extraction methods. We show that RNA extraction methods differ in their efficiency in isolating both short and long RNAs and therefore careful consideration must go into choosing an appropriate method if you wish to detect both from the same sample.The biological functions and targets of very few miRNAs have been experimentally validated, but this is critical to realising the potential of miRNA-based therapy. Furthermore, uncovering tissue-specific biological functions will help identify their therapeutic action and potential off-target effects in normal tissues. Validation of miRNA-mRNA interactions is primarily performed in cell culture assays, which involve artificial manipulation of endogenous miRNAs. However, the levels achieved through cell culture manipulation (e.g. transfection of mimics) are not usually at physiological levels observed in vivo therefore, it is important to recapitulate the results in appropriate animal modelsPrevious research has commonly assumed that all kinds of RNA are purified equally. However, several reports have emerged indicating differences in extraction efficiency depending on the method used for RNA isolation. Kim et al. (2011) retracted their paper in Molecular Cell as they found that their conclusions about differences in miRNA expression from cells grown at different confluencies (high density versus low density) and when they were detached from the culture dish (adherent versus suspension) were actually accounted for by differences in RNA extraction efficiency using the TRIzol method . They foThere are several steps during the work-flow that can introduce experimental variation. Beginning with the method for fixing or freezing the tissue, storage of the material, RNA extraction method, method used for reverse transcription and qPCR amplification or other downstream platforms. In this study we have attempted to control for some of these variables by flash-freezing the tissues, storage at − 80 °C, preventing thawing before extraction and using the most recommended practices for miRNA expression analysis. For instance, the use of sequence specific RT primers as opposed to a universal RT primer has been shown to be superior for specific product amplification . qPCR isComparative analysis of five commonly used RNA extraction methods revealed the miRVana kit was the best performer for producing consistently high yield and quality RNA without compromising miRNA and target gene detection levels. Although this kit was the most expensive, its high quality output and ease of use makes it a preferable method for RNA extraction, particulary if researchers wish to use the same RNA preparation for miRNA and gene expression analyses, as the other methods had lower detection levels and more variability. Empirically, this needs to be optimised for a specific application. A final caution to researchers is that just because a kit is described as isolating total RNA and is appropriate for mammalian tissues, cultured cells or biological fluids; it does not mean that it yields all types of RNAs equally.Additional file 1:Figure S2. RNA integrity analysis of samples isolated from brain tissues. RNA was isolated using five different methods and integrity analysed using an Agilent Bioanalyser with Bioanalyser RNA 6000 Nano Assay. RIN, RNA integrity number. (EPS 4419 kb)Additional file 2:Figure S3. RNA integrity analysis of samples isolated from liver tissues. RNA was isolated using five different methods and integrity analysed using an Agilent Bioanalyser with Bioanalyser RNA 6000 Nano Assay. RIN, RNA integrity number. (EPS 3085 kb)Additional file 3:Figure S4. RNA integrity analysis of samples isolated from lung tissues. RNA was isolated using five different methods and integrity analysed using an Agilent Bioanalyser with Bioanalyser RNA 6000 Nano Assay. RIN, RNA integrity number. (EPS 3137 kb)Additional file 4:Table S1. Primers used for qRT-PCR. (DOCX 32 kb)Additional file 5:Figure S1. GC content and predicted secondary structure of miRNAs used in this study. GC content was calculated based on the mature sequence provided in miRBase.org. Secondary structure and thermodynamic stability was determined using Mfold web server for nucleic acid folding and hybridization prediction. (EPS 794 kb)


Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a ‘model hopping’ approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode  C. elegans but it is now becoming apparent that this is not a good predictor for many parasitic species. In particular we have found that the LEV-8 subunit which is involved in levamisole sensitivity in C. elegans, is not present in many levamisole-sensitive parasitic species. Here we used heterologous expression systems and gene silencing to provide the functional in vivo demonstration that the ACR-8 subunit, which is not an essential component of the levamisole receptor in C. elegans, has a critical role in the levamisole sensitivity of parasitic nematodes. This has important significance for understanding the molecular targets of cholinergic anthelmintics and addresses the increasing challenge of drug resistance.Parasitic nematodes have global health and economic impacts. They infect animals, including livestock, humans, and plants including all major food crops. Their control in human and veterinary medicine is reliant on anthelmintic drugs but this is now challenged by resistant worms especially in livestock. Importantly, for anthelmintics such as levamisole and other cholinergic agonists, resistance appears to be less frequent stressing the need to investigate their molecular target in parasitic nematodes. The levamisole receptor was first identified in the free-living model nematode  However, resistance against these drugs compromises the sustainable control of highly pathogenic nematode species infecting humans, livestock and companion animals –7. In or targets –18.C. elegans, screenings for levamisole-resistant mutants led to the identification of the five genes encoding the L-AChR subunits  [et al. demonstrated that combination of these five AChR subunits with a set of three ancillary proteins  gives rise to a functional L-AChR when co-expressed in the Xenopus laevis oocyte heterologous expression system [C. elegans L-AChR, whereas this receptor is insensitive to nicotine [C. elegans body wall muscle is conferred by a homomeric receptor comprised of ACR-16 [C. elegans L-AChR provided a strong basis to investigate its parasitic nematode counterparts. Studies of parasitic nematode L-AChRs highlighted some striking differences with the C. elegans L-AChR [C. elegans L-AChR, searches in the genomic databanks from Brugia malayi (Clade III) and Trichinella spiralis (Clade I) failed to identify a lev-8 ortholog in these parasites [lev-8 orthologs was also reported in parasitic nematode species phylogenetically closely related to C. elegans (Clade V) for which levamisole is successfully used as an anthelmintic  and has known spastic potency [C. elegans L-AChR. This has important ramifications for understanding the mode of action of the widely used anthelmintic levamisole and the emergence, monitoring and management of resistance.Cholinergic agonists bind to acetylcholine receptors (AChRs) which are members of the pentameric Cys-loop ligand-gated ion channels super-family. These receptors play a pivotal role in fast neurotransmission and are widely used at important neuromuscular synapses that control motility and feeding behavior in nematodes. Anthelmintics such as levamisole and pyrantel induce a spastic paralysis of the worm by activating the levamisole-sensitive AChR (L-AChR) expressed in the body-wall muscle cell –21. Takid lev-8) –26. In 2n system . Levamisnicotine . The latf ACR-16 . This fis L-AChR , 12. Not potency , 31. TakH. contortus, comparative transcriptomic analysis performed on levamisole-susceptible and levamisole-resistant isolates led to the identification of an AChR subunit (Hco-ACR-8) closely related to the ACR-8 and LEV-8 subunits from C. elegans [C. elegans acr-8, like lev-8, is expressed in body wall muscle, although it is not required for a functional levamisole receptor [Xenopus oocyte expression system, we previously demonstrated that Hco-ACR-8 is able to associate with the Hco-UNC-63, Hco-UNC-29.1 and Hco-UNC-38 subunits to form a functional AChR (referred to as Hco-L-AChR-1) highly sensitive to levamisole, but relatively insensitive to nicotine and pyrantel [et al. reconstituted two novel L-AChRs of H. contortus: Hco-L-AChR-1.3 and Hco-L-AChR-1.4. [H. contortus UNC-29 paralogs, contained Hco-ACR-8 and displayed high sensitivity to acetylcholine and levamisole. Using a similar approach, the involvement of the ACR-8 subunit in levamisole sensitivity was further confirmed in the pig parasitic nematode Oesophagostomum dentatum recombinant L-AChR [in vitro and in vivo.In  elegans . In C. ereceptor . Using tpyrantel . StrikinChR-1.4. . Both ret L-AChR . This suacr-8 orthologs are widely distributed in parasitic nematode genomes, which is in sharp contrast to lev-8 orthologs that could be only identified in a subset of parasitic species. We expressed the ACR-8 subunits from a range of animal and plant parasitic nematode species in Xenopus oocytes and showed that they functionally complement the C. elegans L-AChR missing out the LEV-8 subunit. We expressed acr-8 from the parasitic nematode H. contortus in a C. elegans levamisole resistant lev-8 null mutant and report that H. contortus ACR-8 fully rescued the levamisole sensitivity of the transgenic worms. Finally, we optimized the use of RNAi in H. contortus and demonstrate that AChR subunits from the parasite are amenable targets for gene silencing. Taking advantage of this approach, we provide the first functional demonstration of the ACR-8 subunit involvement in the levamisole sensitivity in vivo. Intriguingly, acr-8 gene silencing in H. contortus reduced the levamisole but not the pyrantel sensitivity providing new evidence for a discrete difference in their mode of action. Overall, our findings strongly support the hypothesis that in contrast with the model species C. elegans, the ACR-8 subunit from parasitic nematodes plays a critical role in anthelmintic sensitivity. This has major importance for understanding the molecular targets of the widely used cholinergic anthelmintics and will facilitate drug discovery in this arena.In the present study, we report that C. elegans ACR-8 amino-acid sequence as a query, tBLASTn searches were performed against 68 nematode genomic databases available in WormBase-ParaSite  . Strikingly, when a similar search was performed using the closely related C. elegans LEV-8 amino-acid sequence as a query, lev-8 homologs could only be identified in a subset of 21 species out of 68, including 17 Clade V species and the five Ascaridae species from Clade III. Noticeably, no lev-8 homolog could be found in the 17 genomes of Clade IV species studied. Moreover, no acr-8/lev-8 homolog could be clearly assigned in any Clade I nematode species, although some “acr-8 related” sequences were identified.Using the se.org/) . This reC. elegans acr-8/lev-8 with their counterparts from other nematode species representing Clade I, III, IV and V. This analysis also highlighted that in addition to acr-8 orthologs, some parasitic nematode species from Clades III (i.e. Toxocara canis) and V (i.e. O. dentatum) possess a lev-8 ortholog. Importantly, the Clade I nematode acr-8 related sequences clustered apart from the acr-8 and lev-8 sequences from species belonging to Clades III, IV and V. This suggests that a gene duplication leading to the acr-8 and lev-8 genes occurred early in nematode evolution, soon after divergence from the Clade I nematodes.acr-8 sequences from the Clade V species C. elegans, H. contortus and O. dentatum , in the present study full-length acr-8 cDNAs were obtained from other distantly related parasitic nematode species such as the vertebrate parasites Ascaris suum  and Dirofilaria immitis  and the plant parasite Meloidogyne incognita . Based on their respective orthologous relationship with Cel-ACR-8  in combination with cRNA encoding three ancillary proteins  [Xenopus oocytes. Three days after injection, each of these combinations produced robust responses to 100μM ACh in oocytes , we did not observe any modulation of the ACh EC50 values (The d RIC-3) . Noticead RIC-3) , 36. The oocytes . Similar oocytes . In comp0 values . TherefoThese data suggest that ACR-8 subunits from parasitic nematodes harbor important structural determinants of the Lev response.C. elegans L-AChR [C. elegans ACR-8 subunit remained unclear. Here, we investigated the putative functional redundancy of the C. elegans LEV-8 and ACR-8 subunits using the Xenopus oocyte as an expression system. The substitution of LEV-8 by ACR-8 in the recombinant C. elegans L-AChR led to the robust expression of levamisole-sensitive AChRs . Strikingly, the omission of lev-1 cRNA in the injected mix also led to the robust expression of functional L-AChRs three days after micro-injection . In contrast, the omission of UNC-63, UNC-38 or UNC-29 resulted in the absence of functional receptor  that is resistant to Lev [Hco-acr-8 promoter and mCherry driven by the muscle cell specific C. elegans myo-3 promoter was monitored in C. elegans N2 (wild type). Both PHco-acr-8 and Pmyo-3 showed a similar expression pattern in body wall muscle cells  was very similar, as scored from the thrashing rate in liquid ; cePmyo-3::hco-acr-8) showed a drastic reduction in thrashing rate  suggesting that this anthelmintic could target the L-AChR [lev-8 null mutants. Therefore, we compared the thrashing rate of N2 and lev-8 null mutants after 10, 20, 30 or 40 min exposure time to Pyr concentrations ranging from 10 to 250μM  labeled with the fluorochrome Alexa-594  null mutants [Hco-acr-8 did not impact on the motility of H. contortus larvae  after 25 min. Therefore, we were able to automate the measurement of larval migration, and the effect of anthelmintics on this behaviour, by recording the increase in fluorescence against time in the absence or presence of drug. Paralysis assays performed on H. contortus L2 stage with Lev or Pyr confirmed the suitability of this approach to parameterize the effects on motility   . Strikinol worms . These dC. elegans mutants that resist to Lev have been instrumental to identify the 5 AChR subunits constituting the L-AChR  [lev-8 homologs in closely related parasitic species for which Lev is widely used as an anthelmintic raised the question of an alternative L-AChR subunit composition in these nematodes. In the present study we demonstrate that ACR-8, the closest homolog of the C. elegans LEV-8 subunit, can play a pivotal role in vitro and in vivo in the composition and pharmacological properties of L-AChR from parasitic nematodes.Screens for d LEV-8)  and laidacr-8 and lev-8 homologs within the Nematoda. The phylogenetic analyses show that the acr-8 and lev-8 genes result from an early gene duplication event that occurred after divergence of the Clade I with the other nematode clades. It is noteworthy that Clade I species such as Trichinella spiralis, Trichuris suis or Trichuris muris are sensitive to Lev [C. elegans L-AChR, the LEV-8 subunit is not involved in the functional Lev receptors of these parasitic species.The analysis of genomic data from 68 nematode species raised the question of the origin of e to Lev –46 provilev-8 must have been present in the common ancestor of Clade III, Clade IV and Clade V nematodes, the absence of lev-8 in species from these clades strongly suggests independent events where lev-8 was lost. Even though we cannot rule out that incomplete genomic data could explain the failure to identify lev-8 orthologs in some species from Clade III, Clade IV and Clade V, the systematic identification of lev-8 homolog in species harboring an acr-8 homolog as well as common amino acid signature shared by the C. elegans LEV-8 and ACR-8 sequences from parasitic species support the hypothesis that lev-8 loss was mediated by distinct evolutionary events.Since C. elegans LEV-8 subunit by ACR-8 from animal or plant parasitic nematode species in the recombinant C. elegans L-AChR (Cel-L-AChR) led to the robust expression of functional receptors. Interestingly, we showed that ACR-8 from A. suum and O. dentatum, species that also possess a lev-8 homolog, also functionally complement the Cel- L-AChR lacking the LEV-8 subunit. These results highlight the need for future research in order to investigate the putative functional redundancy between ACR-8 and LEV-8 in these two distantly related species. Interestingly, such redundancy is apparent in C. elegans as we demonstrated that the C. elegans ACR-8 subunit can functionally substitute the LEV-8 subunit in the recombinant Cel-L-AChR. In accordance with previous molecular and electrophysiological data that support the involvement of Cel-ACR-8 in a putative subset of L-AChR channels in C. elegans, here we report the first functional evidence for novel L-AChR subtypes from C. elegans (Cel-L-AChR-2.1 and Cel-L-AChR-2.2) containing the ACR-8 subunit [The substitution of the  subunit , 47. The subunit .H. contortus and O. dentatum expressed in Xenopus oocytes, we reported that ACR-8 is a critical subunit for the Lev response of Hco-L-AChR-1 and Ode (38-29-63-8), respectively [C. elegans LEV-8 subunit was substituted by either Hco-ACR-8 or Ode-ACR-8 in the C. elegans L-AChR, Lev acted as a full agonist on the composite receptors as previously reported for Hco-L-AChR-1 and Ode (38-29-63-8), respectively [C. elegans recombinant L-AChR, Lev acts only as a partial agonist. Indeed, Lev Imax values of the composite receptors containing Hco-ACR-8 (109.5±20.7) or Ode-ACR-8 (112.4±19.0) are within the same range as for Hco-L-AChR-1 (119.9±16.8) and Ode (38-29-63-8) (119±3.7) respectively [max for the Cel-L-AChR is 49.1±10.2. Interestingly, when either Cel-UNC-63 or Cel-UNC-38 subunit was substituted by its H. contortus counterpart, the Lev Imax value was not modified in comparison with the “native” recombinant Cel-L-AChR. Similarly, the replacement of Cel-UNC-29 by UNC-29 paralogs from H. contortus in the Cel-L-AChR does not modulate the Lev response of the resulting composite receptors [In previous studies performed on recombinant L-AChRs from ectively , 36. In ectively , 36. In eceptors . TherefoA. suum for which co-expression in Xenopus oocyte of UNC-38 and UNC-29 subunits led to functional Lev-sensitive receptors [A. suum functionally complements the Cel-L-AChR lacking LEV-8, resulting in a Lev-sensitive AChR. In A. suum, in vivo electrophysiological studies revealed three distinct AChR subtypes responsive to Lev [A. suum L-AChRs that incorporate the ACR-8 subunit would be of particular interest in order to decipher the potential L-AChR subtype diversity for A. suum.Of note, is the particular case of eceptors . In the e to Lev . In thatTaken together, our results provide novel functional evidence for a critical role of the ACR-8 subunit in determining the Lev sensitivity of the parasitic nematode L-AChR and further validate the composite AChR approach to delineate the discrete role of parasitic nematode subunits in receptor function and pharmacology.C. elegans lev-8 null mutant genetic background for the following reasons:In the present study, in order to investigate the specific function of ACR-8 subunit from parasitic nematodes, we decided to use C. elegans both lev-8 and acr-8 null mutants show no significant changes in thrashing rate with respect to N2 (wild type) worms [lev-8 null mutants are partially resistant to Lev, acr-8 mutants are not resistant to this drug [C. elegans with UNC-29/LEV-1 as baits resulted in the identification of ACR-8 as a co-assembled subunit [First, in e) worms . Howeverhis drug , 33. Sec subunit .C. elegans L-AChR expressed in Xenopus oocyte led to functional rescue of the receptor.Third, in the present study we demonstrated that substitution of LEV-8 by ACR-8 from parasitic nematodes in the C. elegans lev-8 null mutants could represent a relevant model to investigate the functional role of ACR-8 subunit in parasitic nematodes. Even though we cannot exclude that Hco-ACR-8 could combine with a different set of native muscle AChR subunits, this result strongly supports the hypothesis that ACR-8 from H. contortus is able to assemble into a functional L-AChR in C. elegans. In C. elegans, L-AChR has been proposed as the pharmacological target of Pyr as unc-63, unc-38 and unc-29 mutants are resistant to 1mM of this drug [C. elegans is also resistant to 250μM Pyr, but only partially. This partial Pyr resistance in lev-8 null mutant could be explained by the expression of functional Cel-L-AChR-2.1/Cel-L-AChR-2.2 in the worms. Indeed, we demonstrated that both of these L-AChR subtypes (that do not include the LEV-8 subunit) are responsive to Pyr when expressed in the Xenopus oocyte. An analysis of Pyr sensitivity in a double null mutant, lev-8/acr-8 would be required to test this hypothesis. Surprisingly, lev-8 null mutants expressing the Hco-ACR-8 were more susceptible to Pyr than wild-type C. elegans. Because the recombinant C. elegans L-AChR expressed in Xenopus oocyte is less responsive to Pyr than ACh and Lev, we reasoned that the C. elegans L-AChR in which lev-8 was substituted by Hco-ACR-8 could be more responsive to Pyr, providing a potential explanation for the increased sensitivity to Pyr in the transgenic worms. However, we found that relative responses to Pyr were similar in both native and composite L-AChRs  [unc-38, unc-63, unc-29, lev-1, lev-8 and acr-8 null mutants suggested that the main functional L-AChR in wild type worms is made of UNC-38, UNC-63, UNC-29, LEV-1 and LEV-8 [unc-63, unc-29 and unc-38 mutants Lev-activated muscle currents are abolished, in lev-1 and lev-8 mutants they are only reduced [C elegans L-AChR subtype subunit composition including UNC-63/UNC-38/ UNC-29 as core components in combination with LEV-1 and/or LEV-8. In addition, if single channel recording experiments indicated that Cel-ACR-8 might not be a component of the main L-AChR subtype, analysis of the double null mutant lev-8/acr-8 strongly suggested that ACR-8 is able to replace LEV-8 in the heteropentamer when this subunit is absent [Xenopus oocytes. These results highlight the need to further explore the diversity of C. elegans L-AChR subtypes as they could contribute to a better understanding of the synapse biology but also might be used as reference for comparative studies with parasitic nematode L-AChRs lacking LEV-8 and/or LEV-1 subunits. In comparison with wild-type C. elegans, electrophysiological experiments revealed an even greater functional and pharmacological diversity of L-AChRs in parasitic nematode species. For example, in O. dentatum (Clade V) single channel recordings revealed up to four distinct L-AChR conductance states [H. contortus and O. dentatum [Xenopus oocyte as an expression system, combination of UNC-38, UNC-63, UNC-29 and ACR-8 subunits led to the functional expression of L-AChRs preferentially activated by Lev  for O. dentatum) whereas the same subunit combination lacking ACR-8 led to the functional expression of another AChR subtypes preferentially activated by Pyr  for O. dentatum). These recombinant experiments reinforce the potential for distinct subtypes of L-AChR assembled from distinct subunit combinations in animal parasitic nematodes.Single channel recordings performed on wild-type el (Mor) . The sysnd LEV-8 . Importa reduced , 33, 56.e states . The divdentatum , 36. ForHco-acr-8 gene reduced Lev sensitivity of H. contortus L2 without impacting their Pyr sensitivity. This observation supports the notion of distinct subtypes of muscle L-AChR and the hypothesis that Lev and Pyr act on distinct AChR subtypes in H. contortus. Recently, we described a novel muscle AChR subtype made of ACR-26/ACR-27 subunits that is specific to parasitic nematodes. The co-expression of ACR-26/ACR-27 from H. contortus in C. elegans N2 conferred an increased sensitivity to both Pyr and Mor in the transgenic worms [Hco-acr-8 gene silencing is underpinned by the presence of alternative functional Pyr-sensitive-AChRs which might include the putative Hco-L-AChR-2 and/or Hco-26/27 receptors. In conclusion, whereas C. elegans remains a highly valuable model for deciphering the pharmacological targets of cholinergic anthelmintics, the differences between the prototypic L-AChR of C. elegans and its counterparts in parasitic nematodes highlight the need to further explore AChRs in target parasitic species. This is particularly pertinent in view of the pivotal role these targets play in drug treatment and resistance. In the present study, we used a set of complementary approaches to investigate specific functions of AChR subunits from nematodes and paved the way for a better understanding of anthelmintic mode of action in a wide range of species. Such information will be of major importance for the rational development of drug combination as well as the improvement of target-based drug screening.In accordance with this, the present work demonstrated that silencing of the ic worms . Thus, wHaemonchus contortus to extract nematode's eggs from fresh fecal material and collect adult worms after necropsy. For the three other animal parasitic nematode species used in the present study , adult worms were obtained from already-existing collections.All animal care and experimental procedures were conducted in strict accordance with the European guidelines for the care and use of laboratory animals and were approved by French ministry of teaching and research and the regional Val de Loire ethics committee (no 19) as a protocol registered under the number 00219.02 in the experimental installations (n° agreement: C371753). For the purpose of this study, three-month-old sheep were infected with 6000 infective larvae (L3) from Haemonchus contortus experiments were performed on the Weybridge isolate as previously described [Dirofilaria immitis adult male worms were supplied by the Filarial Research Reagent Resource Center, University of Georgia [Meloidogyne incognita nematodes (Morelos strain) were cultured on greenhouse-grown tomato plants at the French National Institute for Agricultural Research  and collected as described previously [Ascaris suum and Oesophagostomum dentatum levamisole-sensitive isolate (SENS) were obtained from the French National Institute for Agricultural Research; UMR 1282 ISP (Nouzilly) nematode collection [Caenorhabditis elegans experiments were carried out on the Bristol N2 wild-type and lev-8(ok1519) strains obtained from the Caenorhabditis Genetics Center (CGC).escribed . Dirofil Georgia . Meloidoeviously . The adullection . CaenorhH. contortus, cross-section (5mm thick) from the mid body region of an individual adult worm of Ascaris suum, 5 adult males of D. immitis, 50μl of pelleted J2 stages of M. incognita, 10 adult males of O. dentatum or 100μL of pelleted mixed stages of C. elegans respectively. Frozen samples were ground in liquid nitrogen and homogenized in Trizol reagent  and total RNA was isolated according to the manufacturer’s recommendations. RNA pellets were dissolved in 25 μL of RNA secure resuspension solution  and DNase-treated using the TURBO DNA-free kit (Ambion). RNA concentrations were measured using a Nanodrop spectrophotometer . First-strand cDNA synthesis was performed on 1μg of total RNA using the superscript III reverse transcriptase  according to the manufacturer’s recommendations.Total RNA was prepared from the distinct nematode species using: 10 adult males or 50μL of pelleted eggs, L2 or L3 larvae of Asu-acr-8, Cel-acr-8, Dim-acr-8 and Min-acr-8, were obtained using first-strand cDNA as PCR template. Amplifications were performed with the Phusion High fidelity Polymerase (New England Biolabs) with a forward primer including the first ATG codon in combination with a reverse primer including the first stop codon. Amplicons were inserted in the transcription vector pTB207 [Asu-acr-8, Dim-acr-8 and Min-acr-8 were deposited to GenBank under the accession numbers: KY654347, KY654349 and KY654350, respectively.Complete coding sequences corresponding to r pTB207  using thhttp://www.ebi.ac.uk/Tools/psa/emboss_needle/). Signal peptide predictions were performed using the SignalP 4.1 server [https://github.com/stephaneguindon/phyml-downloads/releases) as previously described in Duguet et al. 2016 [Ancylostoma caninum; Ace: Ancylostoma ceylanicum; Acs: Angiostrongylus costaricensis; Asu: Ascaris suum; Avi: Acanthocheilonema viteae; Bma: Brugia malayi; Bxy: Bursaphelenchus xylophilus; Cbr: Caenorhabditis briggsae; Cel: Caenorhabditis elegans; Cjp: Caenorhabditis japonica; Cre: Caenorhabditis remanei; Dim: Dirofilaria immitis; Dme: Dracunculus medinensis; Dvi: Dictyocaulus viviparus; Eel: Elaeophora elaphi; Eve: Enterobius vermicularis; Gpu: Gongylonema pulchrum; Hba: Heterorhabditis bacteriophora; Hbk: Heligmosomoides polygyrus; Hco: Haemonchus contortus; Llo: Loa loa; Mfl: Meloidogyne floridensis; Mha: Meloidogyne hapla; Min: Meloidogyne incognita; Nam: Necator americanus; Nbr: Nippostrongylus brasiliensis; Ode: Oesophagostomum dentatum; Ovo: Onchocerca volvulus; Ppa: Pristionchus pacificus; Ptr: Parastrongyloides trichosuri; Rcu: Romanomermis culicivorax; Rhb: Rhabditophanes kr3021; Smu: Syphacia muris; Spa: Strongyloides papillosus; Sra: Strongyloides ratti; Sst: Strongyloides stercoralis; Svz: Strongyloides venezuelensis; Tca: Toxocara canis; Tci: Teladorsagia circumcincta; Tco: Trichostrongylus colubriformis; Tmu: Trichuris muris; Tna: Trichinella nativa; Tsp: Trichinella spiralis; Tsu: Trichuris suis; Ttr: Trichuris trichiura; Tzc: Thelazia callipaeda; Wba: Wuchereria bancrofti.Database searches were performed with the tBLASTn or BLASTP algorithms . Deduced1 server  and memb1 server . Phyloge1 server . Regionsal. 2016 . StandarH. contortus first strand cDNA prepared from eggs, L2 and L3 larvae. PCR reactions were carried out in a final volume of 20μl, containing 100ng of first strand cDNA, 1 unit of GoTaq polymerase (Promega), 0.25mM dNTPs each and 0.3μM of each primer. The reaction mixture was denatured by heating to 94°C for 5 min, followed by 34 cycles of 94°C for 45 sec, 56°C for 45 sec, 72°C for 45 sec. A final extension step was performed at 72°C during 5 min. H. contortus GAPDH (HM145749) was used as the reference transcript. Primer sequences are provided in PCR was carried out on A. suum, C. elegans, D. immitis, and M. incognita acr-8 cDNAs were PCR-amplified to be sub-cloned into the expression vector pTB207 that is suitable for in vitro transcription [H. contortus and O. dentatum acr-8 cDNA in pTB207 have been previously reported [C. elegans unc-63, unc-38, unc-29, lev-8 and lev-1 cDNA subcloned in pTB207 were kindly provided by Thomas Boulin [NheI restriction enzyme (Fermantas) and used as templates for cRNA synthesis using the T7 mMessage mMachine kit (Ambion). Defolliculated Xenopus laevis oocytes were obtained from Ecocyte (Germany). The oocytes were injected in the animal pole with a total volume of 36nL of cRNA mix containing 50ng/μL of each cRNA in RNase-free water using the Drummond Nanoject II microinjector. Microinjected oocytes were kept at 20°C in incubation medium  for 4 days to allow the receptors expression. Two-electrode voltage-clamp recordings were carried out as previously described [cription . Subclonreported , 36. C. s Boulin . The plaHco-acr-8 was PCR amplified on genomic DNA prepared from adult males of H. contortus using the NucleoSpin Tissue kit  following manufacturer recommendations. Primers were designed upon scaffold 5709 sequence available in H. contortus PRJEB506 Wormbase parasite database. Primer sequences are provided in C. elegans N2 gonad (50 ng μl-1). Expression of GFP was monitored on fixed wormed from the F1 progeny by fluorescence microscopy.A 2,9 kb region upstream the initiation codon of Hco-acr-8 coding sequence was sub-cloned into the pPD96.52 (Addgene) vector containing a myosin promoter Pmyo3, using the In-FusionHD cloning kit (Clontech). Primer sequences are listed in C. elegans lev-8 (ok1519) were injected with 30ng μl-1 of the resulting plasmid to drive the expression of H. contortus acr-8 in the body muscle cells. Transformed worms were identified by co-injecting pPD118.33 (Pmyo-2::gfp) plasmid at 50ng μl-1 (Fire lab vector kit), which drives expression of green fluorescent protein (GFP) from the pharyngeal muscle promoter Pmyo-2. The co-injected gfp transformation marker forms an extra-chromosomal array with the plasmids carrying the gene sequence and thus worms with fluorescent green pharyngeal muscle can be identified as carrying the plasmid of interest. For all the experiments, two independently transformed stable lines of transgenic C. elegans were assayed.C. elegans were picked into 900 μL of M9 buffer containing 0.1% BSA in a 12 well plate. The worms were left to settle for 10 min. The number of thrashes was counted for 1 min. Then 100 μL of either M9 (control), Lev or Pyr solution was added to the wells to give a final concentration of 10, 25, 50, 100 or 150μM Lev or Pyr. The number of thrashes was counted after 10, 20, 30 and 40 min (and 60 min for some concentrations) of exposure to Lev. The data were plotted as a mean ± standard error of the mean.Experiments were performed on age synchronized worms by picking L4 stage a day before the assay. The next day, one-day old adult hermaphrodite in vitro culture of eggs using the standard procedure described by Rossanigo and Gruner (1991) [gfp) siRNA labeled with the Alexa 594  was added to the culture medium. Each tube was incubated horizontally at 20°C on a rocking table. After 2 hours of incubation, L2 larvae were washed three times with tap water and ingestion was monitored under fluorescent microscopy. The absence of toxicity was confirmed by comparing the viability of dsRNA treated vs untreated L2 larvae after 72h of incubation. Double strand RNA targeting specifically Hco-unc-63, Hco-unc-38 and Hco-acr-8 respectively were synthesized by Eurogentec (Belgium). Soaking of larvae in 1μM dsRNA was performed for all the experiments as described for ingestion assays and putative phenotypes were visually checked on a daily basis (up to 96h). The RNAi experiments were performed at least three times in independent replicates. The custom siRNA duplex sequences are provided in gfp were purchased from Eurogentec.L2 larvae were obtained from r (1991) . ApproxiH. contortus L2 auto-fluorescence using a Quanta Master spectrofluorometer . Motility assays were performed using 7500 H. contortus L2 larvae. Worms were transferred into a 5mL glass tube and left for 15 min to concentrate by gravity. The supernatant was removed and replace by 2 mL of tap water or anthelmintic solution. After 5 min., the tube was inverted on a 20μm sieve. After a stabilization time of 60 sec, the fluorescence accumulation (correlated to the number of larvae migrating through the sieve) was measured during 25 minutes. The recording rates were either one measure /sec , (-)-tetramisole hydrochloride (levamisole), pyrantel citrate, were purchased from Sigma-Aldrich.The accession numbers of annotated cDNA sequences mentioned in this article are:Ascaris suum: ACR-8 KY654347; Caenorhabditis elegans: ACR-8 NP_509745; ACR-12 NP_510262; ACR-14 NP_495716; ACR-16 NP_505207; LEV-1 NP_001255705; LEV-8 NP_509932; UNC-29 NP_492399; UNC-38 NP_491472; UNC-63 NP_491533. Haemonchus contortus: ACR-8 EU006785; UNC-38 GU060984; UNC-63 GU060985; RIC3.1 HQ116823; UNC-74 HQ116821; UNC-50 HQ116822; GAPDH HM145749; Oesophagostomum dentatum: ACR-8 JX429921; Teladorsagia circumcincta: ACR-8 HQ215517; Trichostrongylus colubriformis: ACR-8 HQ215518; Meloidogyne incognita: ACR-8 KY654350. Dirofilaria immitis: ACR-8 KY654349.S1 FigC. elegans ACR-16 subunit sequence. Branch labels correspond to SH values. Scale bar represents the number of substitution per site. C. elegans AChR subunit groups were named as proposed by Mongan et al. [Ancylostoma ceylanicum, Ascaris suum, Bursaphelenchus xylophilus, Caenorhabditis elegans, Dirofilaria immitis, Haemonchus contortus, Meloidogyne incognita, Oesophagostomum dentatum, Toxocara canis, Trichinella nativa and Trichuris trichiura, respectively. ACR-8-related sequences are highlighted in purple, ACR-8 orthologs are highlighted in blue, LEV-8 orthologs are highlighted in red. The node corresponding to the putative duplication event is indicated by a green star.Tree was built upon an alignment of AChR subunit sequences excluding the predicted signal peptide and rooted with the n et al. . The thr(TIF)Click here for additional data file.S2 Figacr-8 deduced amino-acid sequences from C. elegans, H. contortus, O. dentatum, A. suum, D. immitis and M. incognita were aligned with ACR-8 and LEV-8 sequences from C. elegans using the MUSCLE algorithm [C. elegans ACR-8- are highlighted in red. The Cys-loop, the four transmembrane regions (TM1-TM4) and the primary agonist binding site (YxGCC) are noted above the sequences.The lgorithm . Predict(TIF)Click here for additional data file.S3 FigA. Representative recording traces of currents elicited by 100μM acetylcholine (ACh) or 100μM levamisole (Lev) application on the C. elegans L-AChR .B-F. 100μM ACh or 100μM Lev elicited currents on composite C. elegans L-AChRs including Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and the ACR-8 subunit from a parasitic nematode species: H. contortus ACR-8 (B), O. dentatum ACR-8 (C), A. suum ACR-8 (D), D. immitis ACR-8 (E) and M. incognita ACR-8 (F).G-H. 100μM ACh or 100μM Lev elicited currents on composite C. elegans L-AChRs with Hco-UNC-38 replacing Cel-UNC-38 (G) or Hco-UNC-63 replacing Cel-UNC-63 (H).I-J. Representative recording traces of currents elicited by 100μM acetylcholine (ACh) or 100μM levamisole (Lev) application on the C. elegans L-AChR-2.1 , (I) and L-AChR-2.1 , (J). The bars indicate the time period of the agonist application.(TIF)Click here for additional data file.S4 FigXenopus oocyte expressing Hco-UNC-63/Hco-UNC-38/Hco-UNC-29.1/Cel-LEV-8 (A), Hco-UNC-63/Hco-UNC-38/Hco-UNC-29.1/Cel-ACR-8 (B) or Hco-UNC-63/Hco-UNC-38/Hco-UNC-29.1 (C) challenged with 100μM ACh or 100μM Lev. The bars indicate the time period of the agonist application. D) Scatter plot (mean ± SEM) of currents elicited by 100μM ACh or 100μM Lev on Hco-UNC-63/Hco-UNC-38/Hco-UNC-29.1/Cel-LEV-8; Hco-UNC-63/Hco-UNC-38/Hco-UNC-29.1/Cel-ACR-8 or Hco-UNC-63/Hco-UNC-38/Hco-UNC-29.1, respectively. Number of oocytes is reported on the graph.Representative recording traces from a single (TIF)Click here for additional data file.S5 FigA), Cel-L-AChR-2.2 (B), Hco-L-ACHR-1 with Cel-ACR-8 replacing Hco-ACR-8 (C), Cel-L-AChR (D), Hco-L-AChR-1 (E), Cel-L-AChR with Hco-ACR-8 replacing Ce-LEV-8 (F).Representative recording traces of currents elicited by 100μM Pyr application on the Cel-L-AChR-2.1  are reported for each receptor above their respective representative recording traces.The bars indicate the time period of the agonist application. I(TIF)Click here for additional data file.S6 FigA. Expression patterns in C. elegans (N2) of GFP and mCherry driven by the H. contortus acr-8 and C. elegans myo-3 promoters respectively. B. Thrashing rate in M9 medium of C. elegans N2, lev-8(ok1519) and transgenic lev-8(ok1519) expressing Hco-ACR-8.A thrashing rate was established for wild type N2, lev-8(ok1519) and two lines of transgenic lev-8(ok1519); Pmyo-3::hco acr-8 C. elegans after 10, 20, 30 and 40 min in M9 medium. Data are the mean ± SEM.(TIF)Click here for additional data file.S7 Figlev-8(ok1519) and two lines of transgenic lev-8(ok1519); Pmyo-3::hco acr-8 C. elegans after 10, 20, 30 and 40 min of exposure to Lev 10μM (A), 50μM (B), 100μM (C), 200μM (D) or Pyr 10μM (E), 25μM (F), 50μM (G) and 100μM (H) respectively. Basal thrashing rate was established after 10 min acclimatisation in M9 buffer. Data are the mean ± SEM of n ≥8, ****p<0.0001, ***p<0.001, **p<0.01 and *p<0.05, one way ANOVA with Bonferroni post-hoc test between basal and after drug treatment thrashing rate for the same strain. In black: wild-type N2 strain, in dark red: lev-8 (ok1519), in blue: cePmyo-3::hco-acr-8 l.1, in purple: cePmyo-3::hco-acr-8 l.2.A thrashing rate was established for wild type N2, (TIF)Click here for additional data file.S8 FigA. Expression of Hco-unc-29.1, Hco-acr-8, Hco-unc-63 and Hco-unc-38 in the free-living stages of Haemonchus contortus. Transcription of Hco-unc-29.1, Hco-acr-8, Hco-unc-63 and Hco-unc-38 throughout the free-living stages of H. contortus was investigated by RT-PCR; ω: embryonated egg; L2: second stage larvae; L3: third stage larvae. Integrity of cDNA preparations was verified by PCR using primers designed to amplify a fragment of the H. contortus gapdh cDNA.B-C. Monitoring of labelled siRNA ingestion by Haemonchus contortus second stage larvae. H. contortus L2 larvae incubated during 2 hours in a culture medium containing 1μM of non-specific siRNA (targeting gfp) labelled with Alexa 594 (B). Scale bar: 75μm. Negative control without fluorochrome added in the culture medium(C). Scale bar: 35μm.D.H. contortus L2 larvae migration assay using auto-fluorescence quantification. Correlation between the fluorescence counting (counts/sec) and the number of L2 larvae that migrated through the 30μM sieve during 25min. Migration assays were performed using 1000, 2500, 5000, 7500 and 10000 L2 larvae respectively. Each data point represents mean± SE of three independent runs.E. Non-target siRNA does not modulate motility / levamisole sensitivity of H. contortus L2 larvae. The automated larval migration assay (ALMA) was used to determine the putative impact of siRNA targeting gfp on migration or Lev sensitivity (0.3μM) of H. contortus L2 larvae. A. Representative recording traces of the real-time fluorescence counting relative to the L2 migration during 25min. Each trace corresponds to the mean data from 3 runs performed with 7500 L2 larvae. The control corresponds to untreated L2 larvae.(TIF)Click here for additional data file.S1 TableWormBase Parasite (https://parasite.wormbase.org/). Nematode Clades as determined by Blaxter et al. [Deduced amino-acid sequences from Cel-ACR-8 (Genbank accession number: JF416644.1) and Cel-LEV-8 (Genbank accession number: NM_077531.4) were used to perform a tBlastn search against a set of genomic databanks from nematodes available in r et al. .When ava(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Video(MPG)Click here for additional data file.S2 Video(MPG)Click here for additional data file.S3 Video(MPG)Click here for additional data file.S4 Video(MPG)Click here for additional data file.S5 Video(MPG)Click here for additional data file.


RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross‐species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health.Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of  Bats and rodents are known reservoirs of highly diverse viral and bacterial populations, and a number of these viruses have been implicated in the emergence of novel infectious diseases in humans.The close proximity of humans to bat and rodent populations in South‐East Asia creates frequent opportunities for viral spillover and thus poses an unknown risk to human health.Viral surveillance in animal reservoirs is an important step to understanding the exposure of humans to potential zoonoses, the types of human–animal interaction that impact potential for spillover infection and the factors that determine the transmissibility and pathogenicity of viral zoonoses in humans.1A large proportion of the agents of emergent infectious disease have a zoonotic origin. These zoonotic pathogens fall into a wide spectrum of genera, but due to their high genetic variability and wide circulation, RNA viruses arguably pose the most significant threat to human health. Further, due to the close proximity between animals and humans in low‐ and middle‐income countries, human populations in these locations are disproportionally at risk of exposure from these viral pathogens , thus increasing the likelihood of exposure to infectious agents  rather than individual bats were targeted for screening; three bat guano farms were identified in three geographically distinct sampling locations in the south of Viet Nam. These farms were selected because they were the only bat guano farms identified and consenting to participate in regular sampling within the catchment area for Dong Thap Provincial Hospital, a primary site of ongoing human sampling for the identification of zoonotic infections under the VIZIONS project , Rattus argentiventer (n = 234), Rattus losea (n = 20) and Rattus tanezumi (n = 8).The rodent surveys and sampling were conducted as previously described by Van Cuong et al. . Briefly2.3Total nucleic acid was extracted from the bat urine, bat faecal and rat faecal samples using an automated extracted system  with MagNA Pure 96 Viral RNA Small Volume Kits (Roche). Following extraction, nucleic acid samples were screened for target viruses using pan‐family primers for paramyxoviruses, coronaviruses and filoviruses. The primer sequences of viral targets and the nested PCR conditions were as previously described , a 153‐sequence data set for alphacoronaviruses and a 93‐sequence data set for betacoronaviruses , using Seqotron  bat faecal samples and 11 of 222 (4.9%) urine samples tested positive for paramyxovirus RNA. The paramyxovirus‐positive faecal samples were identified in sampling round two on a single farm and in sampling round three on two farms. The positive urine samples were detected on three bat guano farms during the sixth round of sampling. Screening of bat faeces and urine for coronaviruses indicated that 55 of 248 (22.2%) of faecal samples were positive for coronavirus RNA; none of the urine samples contained detectable coronavirus RNA. Coronavirus RNA was detected in bat faecal samples isolated from all three sampled farms at all of the sampling time points. The bat urine and faecal samples were also screened for filoviruses, but no PCR amplification for filovirus RNA was detected despite amplification of appropriate controls. Specific information on coronavirus and paramyxovirus positivity by sampling site, time and species is indicated in Tables 3.2R. argentiventer.Of the 270 rat faecal samples screened for coronaviruses and paramyxoviruses, 12 of 270 (4.4%) were positive for coronavirus RNA, while none of the samples had detectable paramyxovirus RNA. The cDNA amplicons were sequenced and all twelve positive samples produced sequences suitable for phylogenetic analysis. Notably, all coronavirus‐positive faecal samples were obtained from rats of one species, 3.3Fifteen RNA sequences obtained from the bat paramyxovirus RNA‐positive samples   of 21 insectivorous and fruit‐eating bat species sampled . Our sampling was further limited by the use of faecal and urine samples, which are unlikely to be optimum materials in which to detect filoviruses. Thus, a much larger and more comprehensive sampling of multiple bat species using more direct sampling methods would be necessary to detect and understand the potential circulation of filoviruses in the region.Although none of the bat samples tested in this study indicate the presence of filoviruses, this does not provide conclusive evidence that these viruses are not circulating in Viet Nam. While fruit bats have been implicated as the primary reservoirs of filoviruses, a previous survey of multiple bat species in the Philippines confirmed Ebola Reston virus infection in one insectivorous bat species . These data do not exclude the possibility that other rat species carry this coronavirus virus, particularly as our sampling was biased towards R. argentiventer (234/270).In contrast to previous investigations (Woo et al., Despite a lack of appropriate control measures for zoonotic viruses in Viet Nam, viral spillover from bats and/or rats has not, as yet, been observed, although these preliminary findings suggest that both coronaviruses and paramyxoviruses may be endemic in Vietnamese bat populations. While we are unable to comment on the pathogenic potential of these novel viruses in humans, it is likely that if such infections do arise, they are rare and go undiagnosed. Paramyxoviruses in particular are known to cause infections in humans, and the spillover of Hendra virus and Nipah virus from bats to horses or pigs, respectively, and subsequently to humans has been reported in Australia and Malaysia (Edson et al., In conclusion, we have detected the presence of bat paramyxoviruses, bat coronaviruses and rodent‐associated coronaviruses within a limited geographic area and time frame in Viet Nam. Consequently, we suggest that larger‐scale surveillance is needed to fully understand the role played by rodents and bats in the evolution, emergence and dissemination of paramyxoviruses and coronaviruses in Viet Nam.The authors state that they have no competing interests. Click here for additional data file.


Scientific Reports6: Article number: 3074910.1038/srep30749; published online: 08022016; updated: 10032016The Acknowledgements section in this Article is incomplete.“We thank all the study participants. For technical assistance we thank Kuo Yang and members of the UNC Clinical Trials Unit, the UNC CFAR Immunology Core and the UNC Flow Cytometry Core Facility. The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center”.should read:“We thank all the study participants. For technical assistance we thank Kuo Yang and members of the UNC Clinical Trials Unit, the UNC CFAR Immunology Core and the UNC Flow Cytometry Core Facility. This study was supported by NIH grants to the Collaboratory of AIDS Researchers for Eradication  and the UNC Center for AIDS Research . This research was also supported by the Creative and Novel Ideas in HIV Research Program (CNIHR) through a supplement to the University of California at San Francisco (UCSF) Center For AIDS Research funding (P30 AI027763). This funding was made possible by collaborative efforts of the Office of AIDS Research, the National Institute of Allergy and Infectious Diseases, and the International AIDS Society. The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center”.