Results: Choice was low for the entire sample, but particularly for those receiving pureed texture modified diets. Evidence of menu planning to facilitate the inclusion of choice and alternatives was limited. Discussion: Regulation and monitoring of the Australian Aged Care Accreditation Standards needs to be strengthened to mandate improvement of the choice and variety offered to residents, particularly those on pureed texture modified diets. Further research on how menu choice and a lack of variety in meals affects the quality of life residents is needed in this context, but current evidence suggests the effect would be detrimental and undermine resident autonomy and nutritional status.Background: Choice of food is an imperative aspect of quality of life for residents in Residential Aged Care Homes (RACHs), where overall choice and control is diminished upon entering a home to receive care. The purpose of this study was to examine the current strategies of menu planning in a range of RACHs in Australia, and whether this facilitated appropriate levels of choice for residents receiving texture modified and general diets. Methods: The study comprised a National Menu Survey using a new survey instrument collecting general information about the RACH and foodservice system, menu information and staffing information ( Providing food choice is very important in the aged care sector, where quality of life is paramount . The actResidents in RACHs need to have the ability to exercise food choice in order to maintain some autonomy over their food environment. This has previously been demonstrated as a very important aspect of foodservice provision in RACHs . The natIncreased levels of foodservice satisfaction of up to 30% have occurred when residents have the power to choose from the menu . It was The meal environment is defined as the system that underpins the menu, meal production and meal delivery. It needs to support food choice in terms of the timing when residents can select food items from the menu. It is not acceptable that choices be made days before the actual meal, as residents may not remember what they have selected when service takes place ,18. ThisIn the aged care setting, menus are often cyclic with limited alternative choices . RotatinAged care menu planning must include quality planning for textured modified meals. Quality menu planning includes ensuring that the non-texture modified and texture modified menus, particularly the most texture modified pureed meals, include the same number of options for residents ,22. The The extent of menu choice available in Australian RACHs is unknown. The expected outcomes from the Australian Aged Care Accreditation Standard 4.8  and Standard 2.10 (Nutrition and Hydration) do not provide guidance on the process for achieving adequate/any choice in menu planning. The standards are outcome-based rather than process-oriented . It is pi.e., variety) built into menu planning for both the general and texture modified menus in the Australian context.The aims of this study were to provide insight into: (i) how menus in residential aged care homes are planned to offer choice to residents; (ii) the options available for alternative choices; and (iii) the quality  to improve confidence in the results. The three studies were conducted over a three-year period (2010–2013). Ethical approval for study one and two was obtained from the University of Queensland Human Research Ethics Committee # HMS09/0212. Study three was approved by the University of Queensland Human Research Ethics Committee #12/1106.The critical realism framework was used to undertake this work, as it allows for in-depth causal explanation taking into account a wide variety of information from various sources . Three sThe National Menu Survey was designed to ascertain the variety of menu planning practices used to facilitate resident meal choice in Australian RACHs. The survey was mailed to the Hotel Services/Catering Manager or equivalent at 2664 RACHs across Australia. Homes were identified using the computerised Australian Aged Care Database on CDROM (2010) . InformeThe survey contained three main sections: (i) general information about the RACH and foodservice system; (ii) menu information and (iii) staffing information. In part one, participants were asked to provide information on the type of home, the location, facility size, care classification, type of foodservice production system (and whether in-house or outsourced), meal delivery system/s, the role classification of the person responsible for managing food services and the level of dietetic involvement in foodservices operations. Respondents were asked to indicate from a set list of possible time frames, when residents had to choose their meals. This ranged from on the day of service to greater than 24 hours prior to service. In part two, participants were asked to indicate the menu cycle length and seasonal menu changes, in addition to the meal components used for breakfast, lunch, evening meal, mid-meals and fluids. There was space for adding the types of texture modified mid-meal snacks provided. This section also included questions about the types of special diets catered for, and how additional foods and supplements  were used.Homes were asked if they undertook food fortification menu strategies and to detail the type and frequency of these strategies. To gain a sense of how menus were planned, homes were asked to provide information on how the obtain feedback from residents about the menu, barriers to menu planning and what guides their selection of food items for their menus. Homes were also asked to provide information about portion sizes for the general and texture modified meals, either through standardised recipes or actual portion size in grams. In part three, homes were asked to provide information on resident per day per meal costs, which comprised both labour and food. This data will be presented elsewhere.A detailed review of the menus provided with the returned surveys was completed for both the texture modified and non-texture modified diets. Menus were analysed for the amount of choice, meal alternatives offered, and instances where choice was included in the menu across the entire day’s menu pattern.i.e., dining room) across 36 RACHs in New South Wales and South Australia. Recruitment of homes continued until data saturation was reached. The Foodservice and Meal Environment Tool (FAMET) was developed to facilitate structured observation, as no complete meal environment tool could be found. A reflective journal was also kept for each site to record other observations, impressions and reflective thoughts. Each home provided a copy of their menu and portion size specifications. A total of 36 aged care meal environments were observed for all three main meals and morning and afternoon tea snacks . Of those 36, 30 meal environments included residents who had been prescribed texture modified diets.Case study methodology was used to complete detailed observations within the meal environment  was also noted for each meal observed. All data was coded numerically, de-identified and entered into SPSS . For all three studies frequency analysis was undertaken and Pearson Chi-square tests were used to examine statistical differences between proportions, with statistical significance (two-tailed) assessed at the 5% level (p < 0.05).Homes were selected based on their differences in production systems, location  and private The most frequent menu cycle throughout RACHs was four weeks in length. The typical production system was cook-fresh, and the most common meal delivery systems were decentralised bulk or tray services.Main meals on the menus provided were examined for the types of meals and the extent of choices offered. Results for the general menu and texture modified diet menus obtained are shown side by side for comparison purposes. Only three (2%) of menus offered a hot breakfast for those on texture-modified diets. Similarly, for all meals apart from the hot entrée, homes offered no choice to residents on texture modified diets. This presents a distinct disparity between the choices available for those on the general menu compared to those with special needs with regard to texture modification. A large number of homes failed to indicate on their written menu how they were integrating texture modifications into their menu. This is an aspect of menu planning which requires further investigation to ascertain how this impacts upon the quality of meals being planned in aged care homes.Choice of meals was not made on the day of service in over half the RACHs. Around 45% of residents were able to choose their meals close to point of service, either at the time or a few hours prior.Data obtained through observations in 36 meal environments was mostly consistent with that resulting from the National Menu Survey. Of those 36 meal environments observed, 30 provided data on texture modified diets. No hot breakfasts were provided at any of the meal environments observed. The general menu had many choices for cereal and the texture modified diet only offered porridge or Weetbix™ in some cases, or no choice in other cases. No choice was provided for morning tea, lunch, afternoon tea, evening meal (soup), hot entrée, evening dessert or supper for either residents on the general menu or the texture modified menu. Two choices were offered at over half of the meal environments for the lunch dessert for those on the general menu (56%).For the texture modified menu, morning and afternoon teas were always made up on the day, and were inconsistent with the written menu. In around 30% of observations, different meat was used at lunchtime compared with that indicated on the menu, and in 40% of observations the lunch dessert was different. A different dinner meal or meals, left over from the previous day, were provided to residents on the texture-modified menu in around 20% of observations. It was noted consistently that in any meal environment, the written menu for those on texture modified diets was often not followed, meals were repeated on the same day, or repeated from previous days. The menu choice for this group was therefore decreased and the quality of the menu choice was poorer than for those on the general menu. A large proportion of RACHs in the observational study planned the textured modified menu on the day of service and were not using the prepared menu, resulting in the use of left overs and repeated meals. An interesting observation was that some homes restricted food items such as jelly and ice cream for residents on a pureed diet even though most residents were not on thickened fluids and the restriction was unnecessary, further reducing the quality of menu items offered.The primary outcome of this study was the collation of evidence from menus from RACHs indicating a low level of choice of meals for residents on both general and texture modified diets, and significantly less choice for the latter group. This is contradictory to the quality care principles that state that all services should be of the same quality to meet the needs of all . This isInternationally, choice has been reported to be one of the primary reasons for improved food intake in RACHs ,30. ChoiAlthough many homes in this study had cook fresh production systems with either a bulk or tray meal delivery system, there was little evidence of point of service choice, meaning the production system needed to forecast many days before to fill menu requirements. The homes in the study relied heavily on tray meal delivery systems (59%) that are known to reduce flexibility, resulting in residents having to pre-order for these to be set up correctly. In addition, other supporting systems for the menu such as tray meal delivery services limit the choice to residents and can further hinder optimum delivery of the menu. While residents may be able to choose certain parts of their meals, it is not at the point of service, and frequently, cognitive decline can lead to forgetting the choices made, leading to reduced satisfaction with meals .The Australian Aged Care Standard 3.9 is the expected outcome on choice, but there are no foodservice indicators translating this choice into menu options or guidelines for menu planning. Similarly, the Australian Aged Care Standards make no allowances for any choice in texture modified diets. The expected outcomes only make reference to texture modification being made available, and preferences being taken into consideration. How this is supposed to translate into practice is not discussed or suggested. The evidence provided by our studies suggests that this rarely occurs, and it was clear that adherence to residents’ preferences was not achieved. The core issue with the Australian Aged Care Standards and expected outcomes is that they are outcome based rather than process oriented and are open to interpretation by individual RACHs. Linking the meal environment to standards and guidelines to support and provide the structure in which aged care homes should operate, especially in terms of quality of care of residents on a texture modified meal, is imperative. There is evidence indicating that the more restricted food choice becomes, the greater the threat to the resident’s nutritional status . ServingWith the menu system issues identified here and the lack of menu design statements from the Australian Aged Care Standards, it is easy to understand why homes do not have a stronger menu planning focus when it comes to choice options for residents. The Australian Aged Care Standards are significantly more general than those utilised internationally. For example, the Ministry of Health, Canada, includes Standard 71 Menu planning (c) “alternative choices of entrees, meats, vegetables and desserts at lunch and dinner”, and this is required for both the general and texture modified menu plan . AustralThe written information on menus collected in this study was poor in detail and therefore, it was not easy to determine if residents had a choice as to meal make up, for example the type of sandwich, or just received a mixed sandwich. This case study model may lead to reduced objectivity as only one observer was used; however, this was reduced by only spending one day in each meal environment. The overall sample size was small, but there was good representation of a range of facility types. Despite this, it is unclear if all findings are generalisable to the broader range of aged care homes, as it is possible that facilities with higher compliance with the guidelines were more likely to respond, introducing the potential for non-response bias. Financial implications are an important consideration in the strengthening of implementation of quality standards and choice; however, there is no data on this. Further work building on the results of this study is therefore urgently needed.This study highlights the need for changes to the Australian Aged Care Standards to protect residents’ rights to have meal choice and ensure equality in menu planning for all. Regardless of diet type, all residents in homes should have access to the same quality of service provision. Residential aged care in Australia has seen a shift over the past 10 years to caring for residents who are older and frailer with lower cognitive function . The abi


Small non-coding regulatory RNAs control cellular functions at the transcriptional and post-transcriptional levels. Oral squamous cell carcinoma is among the leading cancers in the world and the presence of cervical lymph node metastases is currently its strongest prognostic factor. In this work we aimed at finding small RNAs expressed in oral squamous cell carcinoma that could be associated with the presence of lymph node metastasis.in silico analysis to investigate possible new molecules, not previously described, involved in the metastatic process.Small RNA libraries from metastatic and non-metastatic oral squamous cell carcinomas were sequenced for the identification and quantification of known small RNAs. Selected markers were validated in plasma samples. Additionally, we used Global expression patterns were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Eighteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic samples. Nine of these microRNAs had been previously detected in human plasma, eight of which presented consistent results between tissue and plasma samples. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients showing no metastasis. MiR-181 and miR-296 were detected in metastatic tumors and the expression of miR-296 was confirmed in plasma of patients presenting metastasis. A novel microRNA-like molecule was also associated with non-metastatic samples, potentially targeting cell-signaling mechanisms.We corroborate literature data on the role of small RNAs in cancer metastasis and suggest the detection of microRNAs as a tool that may assist in the evaluation of oral squamous cell carcinoma metastatic potential.The online version of this article (doi:10.1186/s12920-015-0102-4) contains supplementary material, which is available to authorized users. Small noncoding RNAs are regulatory molecules that have recently emerged as important players in several aspects of cellular biology. They are approximately 18 to 30 nucleotides in length and act mostly through the inactivation of complementary sequences. MicroRNAs (miRNAs) and PIWI interacting RNAs (piRNA), for instance, are involved in sequence-specific and chromatin-dependent gene silencing , 3.Caenorhabditis elegans [Among these molecules, miRNAs have been extensively studied. MiRNAs reduce mRNA stability and/or translation due to full or partial sequence complementarity within target mRNAs. They are transcribed as large pri-miRNA, which are then folded into stem-loop structures, and transported to the cytoplasm where they undergo additional processing generating a double-stranded RNA , 5. In g elegans , miRNAs  elegans , 9. Desp elegans ), and th elegans .More recently, genome-wide analysis following The Cancer Genome Atlas , showed Head and neck squamous cell carcinomas (HNSCC) arise from epithelial cells in the lining of the upper aerodigestive tract, comprising the nasal cavity and paranasal sinuses, the nasopharynx, larynx, pharynx, the oral cavity and the oropharynx. HNSCC is one of the leading cancer types by incidence worldwide, with approximately 500,000 new cases a year worldwide and a five-year survival rate of about 40-50 % . Tumors Oral squamous cell carcinoma (OSCC) is a deadly disease and, when grouped with pharyngeal cancer, it is the sixth most common cancer in the world . This tuin silico analysis to investigate possible new molecules, not previously described, involved in the metastatic process.In this work we aimed at finding small RNAs expressed in OSCC that could be associated with the presence of lymph node metastasis. We selected patients with tumors at early stage of development (T1 or T2) with neck metastases, and later stage tumors (T3 or T4) with no neck lymph node metastases for high-throughput sequencing aiming at the quantification of small RNAs. Selected markers were validated in plasma collected from HNSCC patients before surgery and in additional tumor samples at various pathological stages. The identification of a biomarker in plasma is of great use to the clinical practice due to the minimally invasive characteristic of this kind of assay. Additionally, we used The small RNA fraction of 18 OSCC was sequenced. Clinical and pathological data associated with the samples are described in Table Template RNA for library sequencing was either total RNA or the small-RNA fraction, according to the availability in our specimen repository at the time of the experiment. In order to evaluate differences in the small-RNA fraction associated with the kind of RNA template, we assessed the expression levels of two endogenous controls (RNU48 and U6) in each sample. Figure The total number of sequenced and mapped reads is reported in Additional file The 10 most expressed miRNAs corresponded to at least 50 % of the total number of expressed miRNAs  and non-metastatic (N0) samples. Table Two microRNAs were significantly more expressed in N0 when compared to N+ samples: miR-31 and miR-130b. In agreement with our data, miR-31 was previously described as a potent inhibitor of breast cancer metastasis , and wasMiR-130b was also found up regulated in HNSCC when compared to cancer-free tissues , 24 and p < 0.05). The most expressed molecule in N+ samples, miR-181, has been previously associated with metastasis and, in fact, it has been considered as a marker for lymph-node metastasis in OSCC [Six miRNAs were found to be up regulated in metastatic OSCC with statistic significance . DespitSmall RNAs other that miRNAs may also be implicated in cancer progression/metastasis. Piwi-interacting RNA (piRNA) pathway, for instance, is known for its role in germ cell maintenance and is currently being studied in the context of cancer , and snoIn order to search for small RNAs other than miRNAs in our dataset we used BLAST to match reads that did not correspond to sequences deposited in miRBase against a dataset containing sequences from public non-coding RNA databases. A total of 197 reads had good hits. Of these, 68 reads were associated with specific ncRNA categories, in particular: piRNA (Piwi-interacting RNA), snoRNA , snRNA , Y RNA, easRNA , rasRNA  or pasRNA   . All patients were smokers at the time of cancer diagnosis and had a history of chronic alcohol use. Primary tumor tissue samples were collected from patients submitted to surgical resection of primary tumor at Hospital das Clinicas and Hospital Heliopolis, in Sao Paulo, Brazil, and plasma samples were collected from patients before surgery for tumor resection at Hospital das Clinicas and Hospital Heliopolis. All patients provided written informed consent, and the research protocol was approved by review boards of the institutions involved and by the National Committee of Ethics in Research (CONEP 1763/05). Tumor samples were snap-frozen in liquid nitrogen immediately after surgery and stored. Analysis of hematoxylin and eosin-stained sections by the study pathologists confirmed at least 70 % of tumor cells in all OSCC samples.Eighteen patients with OSCC were selected for small RNA sequencing and 30 patients for the identification of circulating miRNAs in plasma. Table RNA was prepared from OSCC tissue samples using AllPrep DNA/RNA/Protein Mini Kit (Qiagen) in compliance with the manufacturer’s protocol. RNA integrity and miRNA population concentration were assessed using the RNA 6000 Nano Assay kit and the Small RNA Assay kit, respectively, with Agilent 2100 Bioanalyzer according to the manufacturer's instructions .Eighteen small RNA libraries were constructed, one for each OSCC sample: 10 samples presented lymph node metastasis at the time of diagnosis and 8 samples did not present metastasis. Small RNA library construction followed the SOLiD Total RNA-Seq Kit for Small RNA Libraries protocol  and the SOLiD RNA Barcoding System  was used for library multiplexing. One μg of total RNA was used as template. The SOLiD 5500 Genetic Analyser  was used to generate 35 bp-long reads. Default parameters were used at all instances during sequencing. Sequencing was performed at GENIAL , CEFAP-USP (Centro de Facilidades de Apoio à Pesquisa da Universidade de São Paulo).http://www.mirbase.org/).For miRNA annotation we used the Small RNA Analysis Tool    with theAlignments were restricted to 5 hits and, aiming to identify molecules most likely to represent a miRNA, multiple hits were only considered when representing different positions within of a single miRNA family. For instance, reads could map identically to hsa-mir-24-1 and hsa-mir-24-2 but for further analysis both such hits were counted as “hsa-mir-24” and only reads matching the mature miRNA sequence were counted as known miRNAs.To visualize better the differential expression in clinical samples we clustered the candidates based on the global expression patterns. This clustering was performed using the Principal Components Analysis (PCA) implemented within Partek Genomics Suite (v6.6).Due to concerns regarding public sharing of patient sequence dataset and the anonymity of patients, raw sequencing results are available upon request but, depending on the scope of the study, it will have to be submitted to the Ethics Committee approval.For the analysis of small RNA expression levels between N+ and N0 samples we used the EdgeR Bioconductor software package . In EdgePlasma was separated from 5 ml of EDTA whole blood using a centrifugation step of 3600 rpm for 10 min. The miRNA population was isolated from 200 μl of plasma using the miRCURY RNA Isolation Kit – Biofluids (Exiqon), following the instructions provided in the manual. For the identification of miRNAs we used the Locked Nucleic Acid (LNA™)-based miRNA qPCR platform from Exiqon. Briefly, 4 μl of RNA were used for cDNA synthesis using MiRCURY LNA™ Universal RT kit microRNA PCR. The cDNA was diluted following the manufacturer’s protocol and real time PCR was carried out using specific, pre-defined microRNA primer pairs and the ExiLENT SYBR Green Master Mix (Exiqon), following the manufacturer’s protocol, using a ABI7500 instrument (Lifetechnologies). MiRNAs evaluated in plasma were those identified as differentially expressed between N+ and N0 tumors following small RNA sequencing in this study but which had been previously detected in plasma by a large scale study : miR-20bTo validate the sequencing data, 3 miRNAs were subjected to quantitative Real Time-PCR using the Locked Nucleic Acid (LNA™)-based miRNA qPCR platform from Exiqon. Briefly, 5 ng of RNA were used for cDNA synthesis using MiRCURY LNA™ Universal RT kit microRNA PCR. Real time PCR was carried out using specific, pre-defined miRNA primer pairs and the ExiLENT SYBR Green Master Mix (Exiqon), following the manufacturer’s protocol and an ABI7500 instrument (Life Technologies). The expression data was normalized to the small RNA SNORD48 expression levels (Exiqon 203903) and for relative quantification we used the comparative ∆Ct method .In order to search for small RNAs other than miRNAs we took the trimmed and filtered sequences that did not match miRBase  and matched them to sequences downloaded from public databases containing small RNA sequences: Cogemir ; DeepBasHits were restricted to those with 100 % similarity, 100 % coverage of our query or of the subject sequences of the databases, and with identical coordinates in the genome.Sequences that did not match known miRNAs or other small RNAs could be part of novel small RNA-like molecules. In order to evaluate this possibility we used a discovery process based on ab initio classification, similarity search and structural search.miRNA-candidates. We extracted the three candidates from each original read due to the negative impact extra nucleotides can have in ab initio prediction methods based on previous folding.If our differentially expressed reads corresponded to mature miRNAs, they should be part of a larger precursor miRNAs. Characterizing this putative precursor miRNA was the goal of our ab initio classification and structural search. At the time of this publication miRNA precursor sequences deposited in miRBase v.20 presented a maximum of 180 nucleotides in length Fig. . ConsideTo detect candidates we applied a pipeline that included: (i) similarity search against the sequences from the 11 databases described in the previous section; (ii) structural search performed against the RFAM  using thAfter excluding all candidates where the orginal read mapped to known ncRNAs of other families, we considered putative miRNAs those candidate sequences that were classified as miRNAs by RFAM, or those that were classified as miRNAs by HMMIR and did not present positive scores by SnoScan or SnoReport.Additionally, we required that strong miRNA-candidates should have the original read mapped to the stem region of the predicted secondary structure. When this was the case we looked for a possible mature sequence within the sequenced read using default parameters from the algorithm MatureBayes . Mature


Caenorhabditis elegans. We showed that the transmembrane receptor, UNC-40 (DCC), whose canonical ligand is the UNC-6 (netrin) guidance cue, is required for extension towards MADD-4. Here, we demonstrate that MADD-4 interacts with an EVA-1/UNC-40 co-receptor complex to attract cell extensions. EVA-1 is a conserved transmembrane protein with predicted galactose-binding lectin domains. EVA-1 functions in the same pathway as MADD-4, physically interacts with both MADD-4 and UNC-40, and enhances UNC-40's sensitivity to the MADD-4 cue. This enhancement is especially important in the presence of UNC-6. In EVA-1's absence, UNC-6 interferes with UNC-40's responsiveness to MADD-4; in UNC-6's absence, UNC-40's responsiveness to MADD-4 is less dependent on EVA-1. By enabling UNC-40 to respond to MADD-4 in the presence of UNC-6, EVA-1 may increase the precision by which UNC-40-directed processes can reach their MADD-4-expressing targets within a field of the UNC-6 guidance cue.We recently discovered a secreted and diffusible midline cue called MADD-4 (an ADAMTSL) that guides migrations along the dorsoventral axis of the nematode  During animal development, cells and cell extensions migrate along stereotypical paths to their target destination by interacting with guidance cues in their environment. The guidance receptors on the surface of these cells can each interact with several different cues, and many of the cues can each interact with multiple receptors. How a migrating cell can reach its target amid this apparent receptor-cue promiscuity is poorly understood. Here, we extend our earlier investigation of how the UNC-40 receptor, which is known to interact with the UNC-6 guidance cue, mediates attraction towards the MADD-4 guidance cue. We show that another transmembrane protein called EVA-1 increases the sensitivity of UNC-40 to MADD-4. This increase in sensitivity allows UNC-40 to respond to MADD-4 in the presence of UNC-6. Without EVA-1, UNC-6 dominates UNC-40 function and restricts its response to MADD-4. Hence, the presence of EVA-1 acts like a switch to change UNC-40's sensitivity from UNC-6 to MADD-4, and in turn allows UNC-40-expressing cells to migrate towards the source of MADD-4 within a field of the UNC-6 guidance cue. Caenorhabditis elegans simplifies this problem because of its facile genetics and a limited number of paralogous guidance cues and receptors.A migrating cell or growth cone will likely encounter many different spatial cues as it advances towards its destination. For example, motor axons in the developing spinal cord encounter more than six different guidance cues as they exit the spinal column C. elegans, called muscle arms, as a model to better understand guided migration Our group uses specialized membrane projections from the body wall muscles of C. elegans ortholog of the poorly understood non-enzymatic ADAMTSL superfamily of proteins, is the diffusible cue that is secreted by motor neurons to attract extending larval muscle arms Muscle arm extension has proven to be a useful model of guidance because its study has led to the discovery of previously uncharacterized components that also play less obvious roles in axon guidance. We recently discovered that MADD-4, which is the sole In addition to guiding muscle arms, MADD-4 and MADD-2 also play roles in guiding the AVM mechanosensory axon. The AVM cell body is located near the lateral midline and extends a single pioneer axon that is guided to the ventral midline by two pathways that function in parallel Previous work has established that UNC-40's attractive response to the UNC-6 cue can be manipulated in at least two ways to achieve different outcomes. First, the UNC-5 receptor can subvert UNC-40's attractive response to UNC-6 to repulsion Here, we report a third mechanism by which UNC-40's activity is modulated. We found that EVA-1 is part of an UNC-40 co-receptor complex that functions to sensitize UNC-40 to the MADD-4 cue. By doing so, EVA-1 counteracts UNC-6's interference of UNC-40-mediated attraction towards MADD-4-expressing targets. This mechanism may enhance the ability of cell extensions to reach MADD-4-expressing targets within a field of the more globally distributed UNC-6 guidance cue.madd-4 null mutation in the screening strain as it increases the number of processes directed towards laterally-expressed ectopic MADD-4 and therefore creates a sensitized background . Given our previous characterization of these gene classes eva-1 that we call tr301. tr301 mutates eva-1's Q86 codon to non-sense. We subsequently found that all known eva-1 alleles are suppressors of muscle arm extension towards ectopic MADD-4  EVA-1 is a single-pass transmembrane protein that functions in muscle to direct muscle arm extension, and that ii) our genetic analyses indicate that MADD-4 and EVA-1 function in the same pathway, we hypothesized that EVA-1 may be a receptor for MADD-4. We therefore investigated whether MADD-4 might interact physically with EVA-1. We expressed luciferase-tagged MADD-4 and SLT-1 (as a control) from HEK293 cells and collected the conditioned medium . We overproteins . We thenproteins . By contproteins . Consistproteins  and S1b.in vivo. When expressed from the nervous system, MADD-4::YFP localization appears restricted to neurons  EVA-1 and UNC-40 are needed for muscle arm extension towards endogenous and ectopic sources of MADD-4; ii) EVA-1 likely functions within an UNC-40 co-receptor complex; iii) In the absence of MADD-4, UNC-6 has demonstrable activity in attracting muscle arms to the ventral nerve cord; and iv) UNC-6 is capable of interfering with the MADD-4-UNC-40 interaction. Together, these observations led us to hypothesize that EVA-1's role might be to counteract the UNC-6 interference by sensitizing UNC-40 to the MADD-4 cue. We tested this hypothesis in several ways.eva-1 null mutant background. Indeed, we observed restoration of lateral muscle arm extension towards ectopic MADD-4 in the eva-1; unc-6 double null mutants , the VL11 muscle arm defects of the eva-1 unc-6 double null mutant are rescued , but requires EVA-1 to attract muscle arms from the muscle that is further from it (VL11) . This suAbove, we demonstrated that EVA-1 is necessary to facilitate UNC-40 sensitivity to MADD-4, and that UNC-40's enhanced sensitivity to MADD-4 is especially important in the presence of UNC-6. We took two approaches to determine whether EVA-1 is also sufficient for this activity. First, we tested whether EVA-1 can counteract UNC-6's interference of the MADD-4-UNC-40 interaction that we observed in cell culture. Upon co-expressing EVA-1 with UNC-40 in HEK293 cells, we are indeed able to counteract UNC-6's interference of UNC-40's interaction with MADD-4 .C. elegans development is led by the migratory distal tip cell at the leading edge of each gonad lobe unc-5 mutants allows the migrating cell to respond to dorsally-expressed MADD-4 despite the presence of endogenous UNC-6 . Genetic removal of UNC-40 in the background of the eva-1; unc-6 double reduces AVM axon attraction to the levels seen in the eva-1 single mutant, demonstrating that guidance towards MADD-4 remains dependent on UNC-40 in the absence of EVA-1 and UNC-6  EVA-1 functions cell-autonomously; ii) EVA-1 localizes to the plasma membrane of muscle arm extensions; iii) EVA-1 is necessary and sufficient to enable UNC-40-mediated attraction towards MADD-4 in the presence of UNC-6; iv) EVA-1 functions in the same genetic pathway as MADD-4; v) muscle-expressed EVA-1 is sufficient to recruit neuronally-expressed MADD-4; vi) EVA-1 co-immunoprecipitates MADD-4; and vii) EVA-1 interacts with MADD-4 via its two galactose binding lectin-like domains.Our second main conclusion is that EVA-1 and UNC-40 function within a co-receptor complex to mediate attraction towards MADD-4. We also provided numerous lines of evidence in support of this second conclusion, including: i) Both EVA-1 and UNC-40 are necessary to mediate attraction towards MADD-4; ii) In the presence of UNC-6, neither EVA-1 nor UNC-40 alone is sufficient to mediate attraction towards MADD-4; iii) EVA-1 and UNC-40 likely function in the same pathway; iv) EVA-1 co-immunoprecipitates with UNC-40 and does so via its transmembrane domain; and v) EVA-1's transmembrane domain cannot be substituted with another transmembrane domain and preserve EVA-1 function, suggesting that EVA-1's interaction with UNC-40 via its transmembrane domain is functionally relevant.in vivo; and iii) EVA-1 can counteract UNC-6's interference of UNC-40's interaction with MADD-4 in HEK293 cell culture experiments.Our third main conclusion is that EVA-1 sensitizes UNC-40 to the MADD-4 cue and that this heightened sensitivity is necessary to counteract UNC-6's interference of UNC-40-mediated attraction towards MADD-4. Again, we have presented numerous lines of evidence in support of this conclusion, including: i) EVA-1 is necessary to prevent UNC-6's interference of UNC-40-mediated muscle arm, axon, and distal tip cell extension towards endogenous and/or ectopic MADD-4; ii) EVA-1 is sufficient to enable UNC-40-mediated attraction towards MADD-4 in the presence of UNC-6 madd-4 null mutant. Because the eva-1 null mutation fails to enhance the madd-4 mutant, EVA-1 must not have a role in muscle arm extension that is independent of UNC-40. Fourth, in special circumstances , MADD-4 attraction can be mediated by UNC-40 in an EVA-1-independent manner. This suggests that UNC-40 has all of the signaling elements necessary to transduce the MADD-4 signal. Together, these observations suggest that EVA-1's role in mediating MADD-4 attraction must be UNC-40-dependent.Several lines of evidence suggest that EVA-1's role in transducing MADD-4 activity is entirely dependent on UNC-40. First, in the absence of UNC-40, muscle arm extension and axon extension towards MADD-4 is abolished, suggesting that EVA-1 cannot mediate attraction towards MADD-4 without UNC-40. Second, the suppression of muscle arm extension and axon guidance towards ectopic MADD-4 in animals that lack UNC-40 cannot be further suppressed by removing EVA-1. This suggests that EVA-1 and UNC-40 function together to mediate attraction towards MADD-4. Third, in normal circumstances, MADD-4 activity is dependent on both EVA-1 and UNC-40. Hence, if EVA-1 had an UNC-40-independent role in muscle arm extension, then genetic removal of EVA-1 would enhance the muscle arm defects of the eva-1 madd-4 double mutant, making it unlikely that EVA-1 antagonizes UNC-6's interaction with UNC-40 in a MADD-4-independent fashion. Furthermore, ectopically expressing EVA-1 in the distal tip cells in an otherwise wild type background failed to induce distal tip cell migration defects, providing further evidence that it is unlikely that EVA-1 reduces UNC-40's sensitivity to UNC-6 in any direct way. Instead, we infer that EVA-1 may limit UNC-6's access to UNC-40 by increasing the local concentration of MADD-4 and presenting it to UNC-40 within a ternary complex. This idea is consistent with our observation that in animals lacking UNC-6, EVA-1 facilitates MADD-4 function in muscles that are further away from the ventral nerve cord (and the source of endogenous MADD-4), but is superfluous in muscles that are closer to the ventral nerve cord.A model by which EVA-1 may counteract UNC-6's interference of the MADD-4-UNC-40 interaction emerges upon considering all of the data: EVA-1, UNC-40 and MADD-4 form a ternary complex that limits the ability of UNC-6 to engage UNC-40. It is unlikely that EVA-1 allosterically modulates UNC-40 to limit its interaction with UNC-6. If this were true, then removing EVA-1 should suppress some of the ventral muscle arm defects observed in MADD-4's absence because UNC-6 would then be free to engage UNC-40. However, suppression is not observed in the EVA-1's role within the complex is unlikely to be limited to the presentation of MADD-4 to UNC-40 because an EVA-1 mutant protein lacking its cytoplasmic tail is non-functional, despite being able to bind UNC-40 and MADD-4. We speculate that EVA-1's cytoplasmic tail is needed to recruit or engage factors or processes that facilitate UNC-40-mediated signal transduction.We have shown that EVA-1 functions with UNC-40 to direct muscle arm and AVM axon extensions towards the MADD-4 guidance cue. However, upon comparing lateral AVM axon guidance errors to dorsal AVM guidance errors in response to dorsally-expressed MADD-4, it is evident that EVA-1 plays two distinct roles in AVM axon guidance. Considering only the dorsal guidance of the AVM axon in response to dorsally-expressed MADD-4, EVA-1 functions with UNC-40 and independent of the SLT-1 pathway to mediate extension towards MADD-4. By contrast, when we consider only lateral AVM axon guidance errors, EVA-1 functions with SLT-1 in a pathway that is distinct from UNC-40. Together, these results suggest that EVA-1 can play simultaneous roles in distinct guidance pathways within the AVM neuron during axon extension.EVA-1 was first identified as a co-receptor for SAX-3 (robo) within the SLT-1 (slit) pathway that helps guide the AVM and PVM axons ventrally Why are multiple pathways needed to guide migrations along a single trajectory? One answer might be to provide increased precision. UNC-6 can be considered a global cue because ventral sources of UNC-6 can guide migrations along the entire circumference of the animal trIs30I or trIs70II, both of which express membrane-anchored YFP from the him-4 promoter in select distal body wall muscles along with other markers of commissural motor axons trIs integrated transgenes. For transgenic strains, muscle arms were visualized either in the background of trIs30I or trIs70II, or directly from fluorescence associated with the expression of the transgenic array. To visualize YFP-tagged proteins, we used the Leica 513867 filter set; to visualize CFP-tagged proteins, we used the Leica 513866 filter set; to visualize RFPs, we used Leica's 513868 filter set. Muscle arms were counted as previously described muIs32II madd-4(ok2862) or slt-1(ok255), which cannot be followed using obvious behavioral phenotypes, were instead followed using PCR and/or sequence of 5 or more individual progeny from a single cloned animal.All strains were cultured and maintained at 20°C according to standard protocol All statistical analyses were performed using a one-tailed, two-sample equal variance Student's t-test. We used a one-tailed test because for all analyses we hypothesized that the effect would be unidirectional. For example, when comparing muscle arm extension in mutants with wild type worms, we hypothesize that the mutant mean would be smaller than the wild type mean. Lastly, we chose a test that compared two independent samples of equal variance because all counts performed fall under normal distributions.madd-4(ok2854); trIs70II; trIs63X animals in 50 µM ethyl methanesulfonate (EMS) for 4 hours. trIs63 drives expression from the ceh-23 promoter in the bisymmetrical pair of CAN neurons that extend axons along the lateral length of the worm. See below for details on the trIs integrated transgenes. Resulting F1s were synchronized as L1s A forward genetic screen for mutants that suppress muscle arm redirection towards CAN-expressed MADD-4 was performed by incubating a mixed stage population of RP1912 C. elegans Genetic Centre or from us: trIs30I; trIs34X; trIs41II; trIs63X; trIs65; trIs66X; trIs69; trIs70II; trIs73[pPRKC638.2(him-4p::UNC-40::MYC::3XFLAG::YFP)(100 ng/ul)]; trIs77; trIs78IV; trIs89. The constructs were built using standard protocols. Note that expression of MADD-4 and EVA-1 at relatively high concentrations can induce dominant muscle arm extension defects.The following chromosomally-integrated transgenic arrays were made using standard techniques 500 µL of mixed stage packed worms were washed thrice with M9 solution and once with PBS, and re-suspended in 1 mL ice cold solubilization buffer  per 10 ml of solution). The samples were incubated on ice for 30 minutes, and lysed through three cycles of flash freezing in liquid nitrogen, partial thawing and sonnication for 5 seconds at 8 Watts. Next, the samples were incubated at 4°C with agitation for 30 minutes and then centrifuged . A BCA assay (Thermo Scientific) was used to measure the protein concentration in the supernatant. 6 mg/ml of protein lysate was incubated with either 30 µl of packed Antiflag M2 agarose coupled antibody (Sigma) or 30 µl of packed Protein A/G agarose beads (SantaCruz) along with 2–4 µg of rabbit polyclonal anti-GFP antibody (GenScript) for 3 hrs with agitation at 4°C. The agarose beads were centrifuged and washed 5 times with 1 ml of cold solubilization buffer. Samples were resuspended in 2× Laemlli buffer and analyzed by western blot. The FLAG immunoprecipitated samples were analyzed using rabbit anti-GFP polyclonal antibodies (Genscript), and the GFP immunoprecipitated samples were analyzed using mouse anti-GFP monoclonal antibodies (SantaCruz).http://imagej.nih.gov/ij/). Normalized relative luciferase signal was obtained by expressing relative luciferase signal values as a fraction of protein level expression.Normalization of luciferase signal to protein level expression was performed by first subtracting control luciferase signal values from experimental luciferase signal values to obtain relative luciferase signal values. Next, protein level expression was measured by quantifying the number of pixels in a given protein band as visualized on film, using the publicly available software ImageJ (trIs41II; trIs69; trIs73[pPRKC638.2(him-4p::UNC-40::MYC::3XFLAG::YFP)(100 ng/ul)]; trIs89.The integrated transgenes used in the co-immunoprecipitation experiments are as follows: trIs41 integrated strain, and by following the aforementioned co-immunoprecipitation protocol: pPRGS382(myo-2p::mCherry), pPRKC793(him-4p::EVA-1::MYC::3XFLAG::CFP), pPRJZ866(him-4p::EVA-1(GAL1+2)::MYC::3XFLAG::CFP), pPRJZ867(him-4p::EVA-1(CYTO)::MYC::3XFLAG::CFP), pPRJZ868(him-4p::EVA-1(GAL1)::MYC::3XFLAG::CFP), pPRJZ869(him-4p::EVA-1(GAL2)::MYC::3XFLAG::CFP), pPRAVS927.5(him-4p::EVA-1(PAT-2-TM)::MYC::3XFLAG::CFP). EVA-1 truncation constructs were all injected at 10 ng/ul together with pPRGS382 at 2 ng/ul.The ability of EVA-1 truncations to co-immunoprecipitate UNC-40 was performed by establishing extrachromosomal transgenes in the For protein level analysis of MADD-4 transgenes, total protein was obtained by sonnication and flash freezing worm lysates of respective integrated strains as described above. 20 ul containing 50 ng of total protein in 2× Laemmli sample buffer was analyzed by western blot. Blots were analyzed using rabbit anti-MADD-4 antibodies (which we made in conjunction with Genscript Inc. by injecting rabbits with MADD-4 peptides) and mouse anti-tubulin antibodies (Sigma) for the loading control.pPRGS861(CMV8-SS::3XFLAG::EVA-1); pPRGS831(CMV8-SS::3XFLAG::UNC-5(no death domain- see pPRGS757(pCDNA3-SS::3XFLAG::PAT-2). CMV3-SS:SAX-3::3XFLAG and CMV3-SS::3XFLAG::UNC-40 were obtained from Joe Culotti pPRGS827(pCDNA3-SS::HA::MADD-4B::Gaussia luciferase (Gluc)); and pPRGS854(pCDNA3-SS::HA::SLT-1::Gluc)). All expression plasmids have been sequence verified.The constructs for expressing the five receptors in HEK293T cells are as follows: HEK293T cells were maintained and transfected in Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% FBS (DMEM-10% FBS). All transfections were performed using JetPrime transfection reagent (Polyplus Transfection). Cell surface Gaussia luciferase binding assays were performed as previously reported To obtain concentrated conditioned culture media containing HA::MADD-4B::Gluc or Gluc::SLT-1::HA, 10 ug of respective expression plasmid was transfected into HEK293T cells, and culture media was changed to DMEM-0.2% FBS. After 24 hours of culture, the conditioned media was collected and centrifuged to remove cellular debris, and subsequently concentrated 10 times using Amicon Ultra-15 centrifugal filter units (Millipore). When necessary, ligands were immunoprecipitated from transfected cells by using an anti-HA affinity matrix (Roche) and analyzed by western blot using rabbit anti-HA antibodies (Sigma).C. elegans receptors (previously described) were transfected into HEK293T cells. 48 hours post-transfection, culture media was removed and cells were incubated with concentrated conditioned media for 4 hours at 4°C. Cells were then harvested in a solution containing PBS, 0.2% BSA and protease inhibitors (Roche), and lysed in TNTE buffer  containing protease inhibitors. C. elegans receptors were immunoprecipitated using mouse anti-FLAG antibodies (SIGMA), and were washed once in lysis buffer, and then in TNTE buffer containing 0.1% Triton X-100 plus protease inhibitor; five times for HA::MADD-4B::Gluc and 3 times for Gluc::SLT-1::HA ligands. One third of the immunoprecipitate was taken for Gaussia luciferase assays by using the BioLux Kit (NEB) and MicroLumat Plus LB96V luminometer (BERTHOLD). Remainder of the immunoprecipitates were analyzed by western blot using rabbit anti-Flag antibodies (NEB).For the cell surface binding assays, 2 ug of individual plasmids expressing the various trIs66 array) to localize to EVA-1::CFP expressing muscles (from the trIs89 array), transgenic muscles were first identified by fluorescence under the CFP filter, and then re-examined under the YFP filter for presence of MADD-4::YFP. To assay the presence of MADD-4/EVA-1 vesicles, muscles were first identified for co-expression of both MADD-4::YFP (from the trIs78 array) and EVA-1::CFP (from the trIs89 array). These muscles were then examined for the presence of vesicles and were considered a positive score if containing more than two vesicles.To assay the ability of MADD-4::YFP (50 ng/ul); pPRGS382(myo-2p::mCherry)(5 ng/ul)]X; trIs78IV; trIs89; huIs91[myo-3p::mCherry::RAB-5]; huIs97[myo-3p::mCherry::RAB-11]Integrated transgenes used in these colocalization studies are as follows: emb-9 promoter similar to previous work unc-5(e53) trIs78IV; trIs30I to generate unc-5(e53) trIs78IV; trIs30I; trEx948 #1(or#2) lines. Distal tip cell migration defects were scored as previously described only in transgenic animals. RNAi-by-feeding experiments were performed as previously reported To test sufficiency of EVA-1, we used the pPRGS394(him-4p::EVA-1::CFP)(10 ng/ul) and pPRGS382(myo-2p::mCherry)(2 ng/ul). Muscle expressed UNC-40::YFP came from the trIs34 transgene.Localization of EVA-1::CFP and UNC-40::YFP were examined under 63× magnification. Muscle expressed EVA-1::CFP was from an extrachromosomal array harboring Dataset S1Contiguous dataset for figures presented throughout manuscript. Dataset S1 contains all relevant data with regards to (XLSX)Click here for additional data file.Dataset S2Contiguous raw dataset for figures presented throughout manuscript. Dataset S2 contains all relevant raw data with regards to (XLSX)Click here for additional data file.Figure S1A. Quantification of VL11 muscle arm extension in young adults of the indicated genotype. Statistical significance (p<0.001) is indicated with a solid asterisk which is matched in colour with a dot above the data point to which the comparison was made. An outlined asterisk indicates a lack of significance. B. Analysis of the requirement of EVA-1's predicted galactose-binding lectin-like domains in binding luciferase-tagged MADD-4. The indicated version of FLAG-tagged EVA-1 was expressed in HEK293 cells, incubated with luc-tagged MADD-4-conditioned media, immunoprecipitated using antibodies against FLAG, and the resulting co-immunoprecipitated luciferase signal was measured (see the p<0.01) is indicated with a solid asterisk which is matched in colour with a dot above the data point to which the comparison was made. An outlined asterisk indicates a lack of significance. C. Representative images of the ability of the indicated versions of muscle-expressed EVA-1::CFP to recruit neuronally-expressed (from the trIs66 transgenic array) MADD-4::YFP to muscle cells. The orange arrows indicate MADD-4::YFP recruitment to EVA-1::CFP-expressing muscle cells. The red asterisks indicates gut auto-fluorescence. The scale bar represents 50 micrometers. D. A western blot showing an analysis of which EVA-1 domains are necessary to maintain an interaction with UNC-40 as determined through co-immunoprecipitation of muscle-expressed proteins. All versions of EVA-1 shown are able to co-immunoprecipitate UNC-40. Only the substitution of EVA-1's transmembrane domain for that of PAT-2 abolishes EVA-1's interaction with UNC-40 (see Related to C-40 see .(TIF)Click here for additional data file.Figure S2A. A western blot of lysates of five strains harbouring the indicated transgenic arrays probed and with anti-MADD-4 antibodies. The MADD-4A and MADD-4B isoforms are indicated with arrowheads. β-tubulin is used as a loading control. Only trIs66  and trIs78  are of relevance here. A quantification of the relative abundance of the proteins  is shown above the blot. B. A strain expressing muscle specific MADD-4::YFP (from the trIs78 array), EVA-1::CFP (from the trIs89 array) and mCherry::RAB-11 (from the huIs97 array). Arrows indicate vesicles in which all three markers co-localize. C. A strain expressing muscle specific MADD-4::YFP (from the trIs78 array), EVA-1::CFP (from the trIs89 array) and mCherry::RAB-5 (from the huIs91 array). Arrows indicate randomly chosen vesicles in which all three markers co-localize. The scale bar represents 50 µM.Related to (TIF)Click here for additional data file.Figure S3A. Muscle-expressed EVA-1::CFP  remains localized to muscle arm termini (arrows) in the unc-40(n324) null mutant. B. Muscle-expressed UNC-40::YFP (from the trIs34 transgene) remains localized to muscle arm termini (arrows) in the eva-1(tr301) null mutant. C. A western blot showing that EVA-1 will not co-immunoprecipitate with PAT-2. D. A western blot showing that UNC-40 will not co-immunoprecipitate with PAT-2. The scale bar represents 50 µM.Related to (TIF)Click here for additional data file.Figure S4trIs78 transgenic array) for the indicated genotype. The posterior distal defects are shown in emb-9 promoter as was previously done for UNC-5 trEx948#1 ([DTC-EVA-1]#1 on the graph) and trEx948#2 ([DTC-EVA-1]#2 on the graph). p<0.05) is documented as described for Related to (TIF)Click here for additional data file.


C9orf72 gene. The repeat-containing intron should be degraded after being spliced out, however GGGGCC repeat-containing RNA species either accumulate in nuclear foci or are exported to the cytoplasm where they are translated into potentially toxic dipeptide repeat proteins by repeat-associated non-AUG-initiated (RAN) translation.The most common forms of amyotrophic lateral sclerosis and frontotemporal dementia are caused by a large GGGGCC repeat expansion in the first intron of the C9orf72 transcripts in lymphoblasts from C9orf72 expansion carriers (n = 15) and control individuals (n = 15). We have identified polyadenylated C9orf72 RNA species retaining the repeat-containing intron and in which downstream exons are spliced correctly resulting in a C9orf72 mRNA with an enlarged 5’-UTR containing the GGGGCC repeats. Intron-retaining transcripts are produced from both wild-type and mutant alleles. Intron-retaining C9orf72 transcripts were also detected in brain with a 2.7 fold increase measured in the frontal cortex from heterozygous expansion carriers (n = 11) compared to controls (n = 10). The level of intron-retaining transcripts was increased 5.9 fold in a case homozygous for the expansion. We also show that a large proportion of intron 1-retaining C9orf72 transcripts accumulate in the nucleus.In order to determine the mechanisms of repeat-containing intron misprocessing, we have analyzed C9orf72 mRNA can potentially explain nuclear foci formation as well as nuclear export of GGGGCC repeat RNA and suggests that the misprocessing of C9orf72 transcripts initiates the pathogenic process caused by C9orf72 hexanucleotide repeat expansions as well as provides the basis for novel therapeutic strategies.Retention of the repeat-containing intron in mature The online version of this article (doi:10.1186/s40478-016-0289-4) contains supplementary material, which is available to authorized users. For example, in the United Kingdom, the G4C2 repeat expansion accounts for 20–50 % of familial or 5–10 % of sporadic cases of ALS [4C2 repeat expansion is located either in intron 1, between two 5’-untranslated region (5’-UTR) exons, or in the promoter region according to whether an upstream or a downstream transcription start site is used.Amyotrophic lateral sclerosis (ALS), a devastating degenerative disease of motor neurons and frontotemporal dementia (FTD), the second most common form of presenile dementia after Alzheimer’s disease, show considerable overlap clinically and genetically. Pathologically, ALS and FTD patients display abundant cytoplasmic inclusions of the DNA and RNA-binding protein, TDP-43, suggesting that both neurodegenerative conditions are likely to represent two ends of a single pathological continuum. The most common forms of familial and sporadic ALS and FTD, referred to as c9ALS/FTD, are caused by a non-coding GGGGCC G4C hexanucltrans-dominant toxic effects and repeat-associated non-AUG-initiated (RAN) translation of G4C2 RNA repeats into potentially toxic dipeptide repeat proteins (DPRs) [4C2 repeats are transcribed from both sense and antisense strands of the C9orf72 gene [C9orf72 repeat RNA can form nuclear foci that have been detected in brain and spinal cord tissue from affected individuals [4C2 repeats and co-localizing with foci identified to date include hnRNP A3 [Drosophila [In addition to possible haploinsufficiency, two, not mutually exclusive, mechanisms have been proposed to explain the pathogenesis of c9ALS/FTD: RNA s (DPRs) –7. The Gf72 gene , 8–11. Sividuals , 9–13 anividuals , 14, 15.hnRNP A3 , Pur α [hnRNP A3 , ADARB2 hnRNP A3 , hnRNP HhnRNP A3  and nuclhnRNP A3 . Sense aosophila  or are tosophila –24.C9orf72-linked pathogenesis. For instance, components of the nuclear pore complex have been identified as modifiers of pathogenesis in several model systems [Impairment of nucleocytoplasmic transport appears to be a key mediator of  systems –28 and t systems .C9orf72 intron 1, where G4C2 repeats are located, should be degraded after being spliced out during the processing of pre-mRNA into mature mRNA. However, in c9ALS/FTD, expanded G4C2 repeats fail to be degraded and form nuclear foci or are RAN translated into DPRs. The mechanisms of defective nuclear degradation as well as of nuclear export of C9orf72 G4C2 repeat sequences are presently unknown but are central to the pathogenesis of c9ALS/FTD. Interestingly, treatment with antisense oligonucleotides (ASOs) targeting C9orf72 exons downstream of intron 1, up to exon 11, promotes degradation of expanded C9orf72 transcripts [C9orf72 intron 1 in cells derived from expansion carriers.nscripts , 15. As C9orf72 RNA species retaining the repeat-containing intron and in which downstream exons are spliced correctly resulting in a C9orf72 mRNA with an enlarged 5’-UTR containing the G4C2 repeats. Generation of intron 1-retaining RNA species potentially explains a number of pathological features of c9ALS/FTD and opens the way to novel therapeutic strategies.Here we identify polyadenylated C9orf72 expansion carriers (n = 15) were generated using a standard protocol where the Epstein-Barr virus is used to immortalize B-lymphocytes or were obtained from the UK MND DNA Bank. Control lymphoblasts from individuals free from neurological disease (n = 15) were generated as above or were from the European Collection of Animal Cell Cultures (ECACC). Cells have been genotyped for the C9orf72 G4C2 hexanucleotide repeat expansion by repeat-primed PCR [Lymphoblasts from iers n = 5 were gen = 11) and control cases from individuals free from neurological disease (n = 10) were obtained from the MRC London Neurodegenerative Diseases Brain Bank , and were collected in accordance with local and national research ethics guidelines  or frontotemporal lobar degeneration with motor neuron disease (FTLD-MND) n = 1 and conC9orf72 G4C2 repeat expansion [+ RNA was selected from total RNA using oligo(dT) conjugated to magnetic beads (Dynabeads® Oligo(dT)25, Ambion) according to the manufacturer’s protocol. RNA concentration was determined with a Nanodrop spectrophotometer (Thermo Scientific). RNA integrity number (RIN) was measured with an Agilent RNA 6000 analyzer .Total RNA was extracted from whole lymphoblasts or from cytoplasmic and nuclear fractions (see below) using TRIzol (Life Technologies). Frozen brain tissue samples were homogenized in matrix lysing-D tubes  in conjunction with the Fastprep sample preparation system and total RNA was extracted using the RNeasy lipid tissue kit (Qiagen). RNA from an FTLD case homozygous for the xpansion  was obta2, 0.5 % Nonidet P-40, 1 mM dithiothreitol and 100 U/ml RNasin). The suspension was centrifuged at 1000 g for 3 min at 4 °C and the supernatant recovered as the cytoplasmic fraction. Nuclear pellets were resuspended in lysis buffer with 3.3 % (w/v) sodium deoxycholate and 6.6 % (v/v) Tween 40. The samples were vortexed and incubated on ice for 5 min. Nuclei were re-pelleted by centrifugation at 1000 g for 3 min at 4 °C. Pooled supernatants were centrifuged at 1000 g for 5 min at 4 °C and transferred to fresh tubes. Nuclei were lysed in thiocyanate buffer  sarkosyl). RNA was extracted from cytoplasmic and nuclear fractions using TRIzol, as above. Final RNA volumes from each fraction were adjusted to represent cell-equivalent concentrations [Nuclear and cytoplasmic fractionation was carried out using a modification of earlier protocols –33. Lymptrations .4C2 repeats, reactions were supplemented with betaine (Sigma), DMSO and 7-deaza GTP (New England Biosystems) [RNA was reverse transcribed using the TaqMan RT kit or the Superscript III kit (Life Technologies) with oligo(dT) or random hexamers according the manufacturer’s protocols. Reverse-transcribed RNA was amplified by PCR using GoTaq polymerase (Promega) using primers and conditions detailed in Additional file systems) . No RT cE. coli. Concentrations of plasmid DNA were determined using a Nanodrop spectrophotometer (Thermo Scientific) and copy numbers calculated. Serial dilutions were made to produce standard curves ranging from 101 to 107 molecules , 1.25 μM of each primer and 10 ng of cDNA. Primers  and cloned into the pGEM-T Easy vector (Promega) using the TA cloning system. Sequencing of both strands was performed commercially using SP6 and T7 primers (MWG Eurofins). For allele analysis, a 451 bp fragment spanning the intron 1-exon 2 boundary and the rs10757668 SNP was amplified by PCR using standard procedures. Amplicons were directly sequenced with the same primers using Big-Dye Terminator v1.1 and products run on an ABI3130 Genetic Analyzer (Applied Biosystems).C9orf72 intron 1 could result in its retention in an otherwise mature mRNA. To determine whether polyadenylated RNA contained sequences derived from C9orf72 intron 1, we analyzed poly(A)+ RNA from cultured lymphoblasts established from heterozygous C9orf72 G4C2 repeat expansion carriers and control individuals. Lymphoblasts derived from C9orf72 G4C2 repeat expansion carriers display nuclear foci [Defective splicing of ear foci , thus, a+ RNA was purified from lymphoblasts and reverse transcribed using an oligo(dT) primer. The lack of contaminating genomic DNA was demonstrated by PCR using primers spanning a short intron in the HOXB4 gene [C9orf72 RNA, cDNA was analyzed by PCR using primers spanning the exon 1a-intron 1 and intron 1-exon 2 boundaries. PCR with a forward primer specific for exon 1a and a reverse primer annealing to intron 1, 5’of the G4C2 repeats, generated a 278 bp product from both control and expansion carrier cells XB4 gene   in lymphoblasts from expansion carriers (n = 15) and controls (n = 15). No significant differences were detected between the two groups  rs10757668 in 2 exon 2 . An intrele Fig. .C9orf72 transcripts between the nucleus and the cytoplasm in lymphoblasts from G4C2 repeat expansion carriers and control cases. Nuclear and cytoplasmic fractions were prepared and poly(A)+ RNA was extracted from each fraction and reverse transcribed. HOXB4 analysis confirmed the lack of contaminating genomic DNA in both fractions  showed a statistically significant 2.7 fold increase compared to control cases (n = 10)  Fig. . The levC9orf72 RNA that are unspliced at the 5’ and 3’ end of intron 1 and in which downstream exons are spliced correctly. As intron 1 is located between two 5’-UTR exons, its retention results in a C9orf72 mRNA with an enlarged 5’-UTR. Interestingly, higher levels of intron 1-retaining C9orf72 RNA species were detected in the frontal cortex of expansion carriers compared to control individuals. Two recent papers also reported increased levels of sense and antisense C9orf72 RNA containing intron 1 in the frontal cortex from expansion carriers [C9orf72 RNA that accumulates in the brain from expansion carriers is, at least in part, a fully processed mRNA retaining intron 1 within an enlarged 5’-UTR. This has important mechanistic implications for the pathogenic process, as discussed below. No significant difference was found in the levels of transcripts unspliced at the 3’ end of intron 1 in brain. This could be explained by the fact that RNA unspliced at the 5’ end of intron 1 originates from transcription variants that contains the hexanucleotide repeat sequence, whereas RNA unspliced at the 3’ end of intron 1 can, in addition, originate from transcription variants not containing the repeat sequence.Here, we have identified previously unrecognized RNA species derived from polyadenylated carriers , 39. Howcarriers , 40. HerC9orf72 RNA up to exon 11 [Drosophila [Intron 1 retention is consistent with the degradation of expanded repeat-containing transcripts induced by RNase H-sensitive ASOs targeting  exon 11 , 15. As osophila .C9orf72 polyadenylated RNA from both the wild-type and the expanded alleles and contains the G4C2 repeats , and, thus, is part of a normal process. This is not an unusual situation as intron detention (delayed splicing) or retention, either arising from defective splicing or as a regulatory process, is a common occurrence within the mammalian transcriptome [C9orf72 transcripts appears to be independent of the presence of an expanded G4C2 repeat sequence, this process results in the expanded G4C2 sequence from the mutant allele being included in the 5’-UTR of a fully processed mRNA. C9orf72 mRNA with an enlarged 5’-UTR that includes the G4C2 repeats, is similar to FMR1 transcripts associated with fragile X-associated tremor ataxia syndrome (FXTAS), caused by moderate (<200) CGG repeat expansions in the 5’-UTR of the FMR1 gene [Intron 1-retention was observed in criptome , 42, 43.MR1 gene .HTT gene, resulting in a truncated, aggregation prone, protein [In addition to transcripts retaining the entire intron 1 our analysis does not rule out the presence of shorter RNA species, for example resulting from the use of cryptic polyadenylation sites in intron 1. Use of cryptic intronic polyadenylation sites has been reported in Huntington’s disease, caused by a CAG repeat expansion in exon 1 of the  protein .C9orf72 transcripts unspliced at the 5’ end of intron 1 were found to be expressed at higher levels in the frontal cortex of expansion carriers compared to control individuals. This was particularly clear in the brain of a homozygous case albeit material from one case only was available for analysis. As suggested by Mori et al. [4C2 sequences could also reduce splice site usage, hence inhibiting intron 1 splicing. Of note, introns with a high GC content are prone to be retained [CNBP, formerly known as ZNF9) gene has also been reported in myotonic dystrophy type 2 (DM2) [4C2 repeat tracts might also slow down the rate of transcription of the C9orf72 gene in expansion carriers, hence promoting intron retention.No difference in the level of intron retention was detected between lymphoblasts from expansion carriers and controls. By contrast i et al.  the incrretained . Partial 2 (DM2) . Long G4C9orf72 transcripts accumulated in the nucleus. Nuclear retention of incompletely spliced transcripts has been demonstrated as a regulated process for the control of gene expression or as part of a surveillance pathway (for reviews see [C9orf72 mRNA retaining intron 1 could be the consequence of disrupted nucleocytoplasmic transport of mRNAs resulting from C9orf72 repeat expansion toxicity [C9orf72) are prevalent in the cytoplasm. Of note, huntingtin mRNA with expanded CAG repeats accumulates in the nucleus [We found that a large proportion of intron 1-retaining iews see –49). Coniews see . Nucleartoxicity , 50. How nucleus .C9orf72 mRNA may be targeted to such a pathway that would be unable to destabilize the G-quadruplex structure formed by the G4C2 repeat sequence [C9orf72 mRNA is targeted, it is important to note that RNA can be degraded through different pathways. Indeed, as mentioned above, long G4C2 repeat sequences can be degraded following ASO-mediated RNase H cleavage of C9orf72 RNA [mRNAs that fail to be exported to the cytoplasm are degraded in the nucleus by a surveillance pathway that has been studied in yeast and reported, but yet to be fully characterized, in mammals , 53. Thisequence , 57–59 arf72 RNA , 12, 15.C9orf72 transcripts accumulate in the nucleus, intron retention could also explain export of the expanded G4C2 repeat RNA to the cytoplasm where it would become template for RAN translation into DPRs. As intron 1-retaining C9orf72 transcripts have the structure of mature mRNAs, an, albeit small, proportion is exported to the cytoplasm through the conventional pathway of nuclear export of mRNA  [Finally, intron retention in the sense transcript might explain transcription of repeats from the reverse strand. GR-loops) . Intron R-loops) . R-loopsR-loops) .C9orf72 mRNA species with an enlarged 5’-UTR that includes the G4C2 repeat sequence that can explain a number of features of c9ALS/FTD. Interfering with intron 1 processing would offer novel ways of dissecting the cascade of events leading to neuronal dysfunction and death and represents an innovative and promising therapeutic strategy for c9ALS/FTD.We have identified Additional file 1:Supplementary material. (PDF 456 kb)


This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era.Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA)-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing) a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of  A microarray is typically defined as a collection of microscopic spots of biological solutions attached and arranged in a defined location on a solid surface that allows a massive number of parallel genotyping and/or phenotyping measurement. The fundamental principles of parallelized microspot (microarray) technology were conceived of on the basis of the ambient analyte model of Roger Ekins and colleagues in the late 1980s. However, the enormous interest evoked by microarray-based assays came in the late 1990s, when researchers were first able to utilize high-quality and reproducible results using DNA chips ,3. ProteThe breakthrough in the fabrication of microarrays came through the development of two parallel key approaches . The firThe soft lithography method developed by Whitesides’ group  is a comin situ by actually producing the biological molecules directly on the substrate with extremely high density using photochemistry and solid-phase synthesis [® [i.e., increasing the current low capacity  of microarrays to and ultradense (mega-giga) capacity. As shown in The second major approach to microarray fabrication was the development of photolithography by Stephen Fodor and colleagues at Affymax Research Institute, which has made an important contribution to the fabrication of “synthesis-on-chip”-type microarrays . In thisynthesis ,18. Affyhesis [® . Parallehesis [® . Howeverin situ synthesis and patterning of microarrays.This method is based on the principle of the gravure printing (roll-to-roll printing) process using a dense array of microengraved plates (micromold plates). 2, were employed.A microchamber-array chip is a basic requirement for soft lithography-based µIP. Micromold plates with desired features can be designed by computer software and prepared by photolithography or ordered from commercial suppliers. These plates are then used to fabricate arrays of microchambers using soft lithography by the replica molding of monolithic slabs of PDMS. To demonstrate the concept of µIP, microchambers of 1.5–100 µm in diameter and depth, giving a density of 40,000–20,000 uniformly-distributed independent microchambers per mmBiomolecular libraries, such as proteomes in a cell or tissue lysate, which are anticipated to be composed of over 100,000 proteins, exist as mixtures in a solution and must first be individualized and then organized into a one-to-one format prior to analysis using a microarray approach. This can be carried out by several techniques, including photolithography and contact printing. However, these are not high-throughput and robust ways of dealing with a full-length library. µIP uses a BEAMing  approach  to firstThis step enables the co-synthesis and immobilization of biomolecular ink from individual microchambers arrays to corresponding positions on a glass substrate. A droplet of a cell-free-coupled transcription/translation system is sandwiched between a Ni-NTA-modified glass surface and a bead-incorporated microchamber array chip by applying pressure. To ensure perfect contact between the microchamber array chip and the glass substrate, the chip is vertically pressed onto the glass using a bench-mounted hand press on ice. Then, the coupled transcription/translation reaction is initiated by increasing the temperature from 4 °C to room temperature and incubating the assembly at 30 °C for 60 min. Multiple copies of a messenger RNA are transcribed from the bead-bound DNAs in the individual compartments of the microchamber-array chip, which is followed by the immediate translation of the double-histidine-tagged protein, which diffuses to the glass surface and is captured by the Ni-NTA-modified glass surface.in situ co-synthesis and immobilization of biological molecules, the need for chemical modification and the risk of degrading labile RNA can be reduced.RNA microarrays, such as RNA aptamer microarrays for the detection and quantization of protein biomarkers in biological samples, are a powerful tool for analyzing RNA-related phenomena. DNA microarrays are commonly used and fabricated by well-developed methodologies. However, a similar methodology may not be applicable to the fabrication of RNA microarrays, because of the extremely labile nature of RNA molecules. The previously reported methods of fabricating RNA microarrays were performed by the direct attachment of a messenger RNA onto a substrate after chemically-modifying the RNA using thiol-gold chemistry  or by me2 [i.e., low contrast between the foreground (spots) and background) is observed, which is approximately 250 RFU (relative fluorescence units), i.e., nearly one-third of the actual signal intensity. This suggests that the printing of ready-to-print ink molecules  must be improved for high-resolution patterning using the µIP approach. Note that there is a risk of part of the ink molecules  remaining in the non-array region (outside the grooves on the printing mold) while achieving a tight seal between the microchamber-array chip and the substrate. Thus, the ink molecules remaining in contact with the non-array region before being forced out by the sealing step may also be undesirably printed in the non-array region on the substrate, leading to high background noise or low-contrast printing.The first demonstration of µIP was the successful patterning of a 5-µm-diameter spot of presynthesized messenger RNAs at a density of 10,000 per mm2 . Figure 2 with high contrast  screening purposes, which deal with huge aptamer libraries with up to 1015 variants. Conventional microarrays fabricated by a robotic spotter or a photolithographic approach have a number of immobilized spots on a single array substrate that is limited to the 105 order. However, µIP can be considered as an ultrahigh-throughput printing tool for the instrument-free and inexpensive printing of arrays of biomolecules. Owing to the fundamental nature of µIP, this technology would allow the printing of spotter-free ultra-small microarrays while maintaining a high resolution and, thus, can overcome the low-medium-density limitations of current methods. Using µIP, we demonstrated the printing of in situ-transcribed messenger RNAs onto part of a 30 mm × 30 mm glass substrate with a density of 40,000 spots per mm2, which to our knowledge, is the highest ever density reported for microarrays. To demonstrate the high-resolution characteristic of µIP, the printing of an arbitrary shape, a pattern comprising the letters “RNA” with a line width of 7.5 µm, was also achieved [in situ-synthesized biomolecular (messenger RNA/protein) microarrays with an ultrahigh density  and a high resolution (sub-micrometer).The printing of higher density protein microarrays is highly desirable for global proteome analysis. Similarly, ultrahigh density DNA or RNA microarrays are a prerequisite for on-chip novel DNA or RNA probe  the self-assembly of microarrays with an increased density of up to 25 million per chip, which is 3–4 orders of magnitude higher than that of existing approaches; and (4) high resolution with sub-micrometer features. Despite the success of µIP, several aspects of printing must be improved. First, the current protocol of µIP requires the use of a microchamber-array chip fabricated by top-down microfabrication using soft lithography, which relies on the use of photolithography to generate the master, which is not a universally-affordable technique. In an advanced form of µIP, it may be possible to produce the microchamber-array chip by solution processing, i.e., a solution-based deposition technique, which will help significantly to reduce the cost and increase the availability of µIP. Second, a PDMS-based microchamber-array chip is used for µIP; the limitations of the use of PDMS in cell-free protein synthesis-based microarrays are its propensity to adsorb analytes and its porosity, which allows small molecular components of the cell-free system, such as amino acids, to diffuse into the bulk of PDMS [in situ synthesis of GFP inside PDMS microchambers with different sizes, and thus, different surface area-to-volume (S/V) ratios, and observed that the yield in the cell-free synthesis of GFP in PDMS microchambers decreased monotonically with decreasing microchamber diameter from 100 down to 25 µm [The development of µIP has provided fundamental insight into: (1) the spotter-free arraying of ready-to-print biological molecules; (2) the  of PDMS . Minimizto 25 µm . Therefo


Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of  This study uses computational metagenomics and molecular experimentation to massively expand the known genomic and ecological diversity of RNA bacteriophages, identifying novel tropisms and genes. Bacteriophages (viruses that infect bacteria) can alter biological processes in numerous ecosystems. While there are numerous studies describing the role of bacteriophages with DNA genomes in these processes, the role of bacteriophages with RNA genomes (RNA bacteriophages) is poorly understood. This gap in knowledge is in part because of the limited diversity of known RNA bacteriophages. Here, we begin to address the question by identifying 122 novel RNA bacteriophage partial genome sequences present in metagenomic datasets that are highly divergent from each other and previously described RNA bacteriophages. Additionally, many of these sequences contained novel properties, including novel genes, segmentation, and host range, expanding the frontiers of RNA bacteriophage genomics, evolution, and tropism. These novel RNA bacteriophage sequences were globally distributed from numerous ecological niches, including animal-associated and environmental habitats. These findings will facilitate our understanding of the role of the RNA bacteriophage in microbial communities. Furthermore, there are likely many more unrecognized RNA bacteriophages that remain to be discovered. Bacteria play key roles in metabolic and immunological processes; however, at this time many of the factors that define the composition of a given microbial population are still unknown –4. BacteLeviviridae that includes four recognized species  and the segmented, double-stranded RNA (dsRNA) family Cystoviridae that contains a single recognized species (Pseudomonas phage ϕ6) [DNA bacteriophages are currently classified by the International Committee for the Taxonomy of Viruses (ICTV) into eight separate families with a total of 494 species, 55 single-stranded DNA (ssDNA), and 439 double-stranded DNA (dsDNA) bacteriophage species. These species derive from a diverse group of host bacteria; additionally, there are over 1,000 genomic sequences of DNA bacteriophage species in GenBank. By contrast, according to the latest (2014) report of the ICTV, only two official families of RNA bacteriophages are recognized: the single-stranded RNA (ssRNA) bacteriophage family hage ϕ6) ,12. Therhage ϕ6) . For theBacteriophage identification has historically relied on culture-based methods –18. GiveHere, by mining multiple metagenomic datasets that were generated such that RNA could be evaluated, we identify partial genomes of over 120 highly diverse RNA bacteriophage phylotypes that are highly divergent from each other and all of the known RNA bacteriophage genomes. This expansive diversity enabled us to identify new dimensions of RNA bacteriophage biology, including bacteriophages with novel genome organizations, numerous open reading frames (ORFs) that contain novel genes with no detectable homology to known bacteriophage genes, presence in novel ecological niches, and the first data in support of a RNA bacteriophage infection of a gram-positive bacterium. We additionally assess the prevalence of two novel RNA bacteriophages in a cohort of macaques, presenting the first description of the ecological dynamics of these novel RNA bacteriophages. Our results critically illuminate an unexamined dimension of molecular and ecological bacteriophage diversity and fundamentally establish a necessary framework that enables a more accurate dissection of RNA bacteriophage modulation of microbial populations.−4). In contrast, multiple nucleotide sequences in datasets from stool-associated and sewage specimens were identified that, following translation to amino acid sequences, aligned to leviviral proteins. The four studies of relevance, which were previously generated by our laboratory, included a study of raw sewage [−4) was selected for assembly. Using a National Center for Biotechnology Information (NCBI) conserved-domain search  or Phyre2 (confidence > 90%), partial genomes of RNA bacteriophage phylotypes were defined as any assembled sequence greater than 750 nucleotides in length that contained a translated frame with a recognizable RNA bacteriophage-specific domain, such as a bacteriophage-specific RNA-dependent RNA polymerase (RdRp), capsid, maturation protein, or packaging nucleoside triphosphatase (NTPase) [To detect RNA bacteriophages, we initially queried multiple metagenomic nucleotide sequence datasets with protein sequences from the known leviviruses and cystoviruses. We focused on datasets generated by our laboratory that contained cDNA sequences derived from RNA in the original material and that represented ecological niches known to support DNA bacteriophages, such as the vertebrate gastrointestinal tract and sewage. Cystovirus protein queries yielded no significant alignments  ,34.In order to focus our analyses on truly unique RNA bacteriophage phylotypes, any partial genomes that shared > 70% nucleotide identity in either the RdRp or the maturation gene were defined as belonging to a single phylotype. The longest partial genome for a given phylotype was selected as the representative sequence for that phylotype in all downstream analyses. By these criteria, partial genomes of 20 unique RNA bacteriophage phylotypes were identified in 17 distinct specimens. Five partial genomes were assembled from metagenomic data from sewage specimens, 14 were from rhesus macaque stool data, and one was from mouse stool data. Additional partial genomes that shared 85%–97% nucleotide identity to these 20 unique partial genomes were also identified in multiple other specimens in these studies, but they did not represent novel phylotypes by our criteria and therefore were not analyzed further. Based on the sequence diversity of each of these assembled partial genomes within a single phylotype, we believe it is unlikely that these RNA bacteriophages originate from laboratory contamination. The 20 unique bacteriophage phylotypes were sequentially named based on whether it was identified from an environmental or animal specimen, followed by a two-letter descriptor of the ecological niche.To confirm the partial genome assemblies, the eight longest partial genomes (range 3.5–5.0 kb) out of the 20 identified were experimentally validated by generating multiple overlapping reverse transcription PCR (RT-PCR) amplicons followed by Sanger sequencing . The ave−4). The complete sequencing data from each of these 115 datasets were assembled, and RNA bacteriophage partial genomes were defined as above . We identified 138 unique partial genomes that contained ssRNA bacteriophage domains and five unique partial genomes that contained characteristic dsRNA bacteriophage motifs . These included transcriptomic and RNA-inclusive metagenomic studies. The metagenomic data analyzed were derived from environmental sources, such as oceans, sewage, and soil, and animal-associated sources, including stool. We aligned amino acid sequences from the 20 novel and 19 reference RNA bacteriophages against sequences in these datasets, following six-frame translation, using tBLASTn. Out of 2,765 RNA-inclusive metagenomes and 7,309 transcriptomic datasets examined, 115 contained at least ten sequences with significant alignments  . BrieflyFrom 36 primates that were a subset of a previously published SIV vaccination cohort, 71 stools were collected . Stools Stool pellets combined from two mouse cages infected with murine astrovirus were metagenomically analyzed as previously described ,46. BrieThe sewage sequencing reads have been previously described and are deposited in NCBI SRA (Accession SRA040148) . Briefly−3. We downloaded complete reference genomes of all 11 Leviviridae and five Cystoviridae species, as defined by NCBI Taxonomy, and isolated amino acid sequences of all annotated ORFs. These amino acid sequences were then queried against the described databases using tBLASTn .Nucleotide databases were constructed using either 454 FLX Titanium or Illumina MiSeq sequencing reads from specimens from the above studies. We constructed nucleotide databases from 454 sequencing-based reads that did not align to any known protein within the Genbank non-redundant (nr) database, as determined by VirusHunter . IlluminUsing the command line NCBI SRA tBLASTn, amino acid sequences were queried against deposited nucleotide databases that were annotated as having sequenced RNA . SRA was−4, all deposited reads from the specimen were then assembled with IDBA using default parameters [For every specimen that had more than ten reads that aligned with an e-value < 10rameters . SpecimeNovel RNA bacteriophages were named based on the ecology from which they were identified. The first letter designates whether the bacteriophage was identified from an animal (A) or an environmental (E) specimen. The second two letters designate a more specific ecological designation, where IN = invertebrate, VE = vertebrate, SE = sewage, SO = soil, MS = microbial sediment, MM = microbial mat, and OC = ocean. The three letters were followed by a three-digit number.Multiple PCR primers were designed to confirm eight  of the assembled partial genomes . RT-PCR −3 or structural alignments with greater than 90% accuracy using Phyre2. Global alignment of RNA bacteriophage proteins was performed with the MUSCLE algorithm using MEGA6, and pairwise percent identity was calculated based on the p-distance based on the alignment matrix.ORFs were predicted using ORF Finder from NCBI and subsequently analyzed for conserved domains using the NCBI Conserved-Domain Search. Domain annotation was defined using NCBI conserved domains that had e-values < 10Narnaviridae  were included as out-groups. Novel ssRNA bacteriophage that contained all five motifs of the RdRp palm domain as determined by multiple sequence alignment to the known ssRNA bacteriophage were selected for phylogenetic analysis. Amino acid sequences were aligned using 13 iterations of MUSCLE in MEGA6. The palm domain alignment was isolated, and noninformative sequences were trimmed using GBlocks  [ssRNA bacteriophage phylogeny was determined by acquiring RdRp protein sequences from NCBI GenBank for all reference levivirus genomes  as well as three additional known ssRNA bacteriophages: Marine bacteriophage MB: KF510034.2, Marine bacteriophage EC: KF616862.2, and Marine bacteriophage AC: KF616864. Additionally, the two type species of the family s = all) . The finPCR primers designed to detect AVE000 and AVE001  were desS1 Dataset(ZIP)Click here for additional data file.S2 Dataset(TXT)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.