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The tropical liver fluke Fasciola gigantica affects livestock and humans in many Asian countries, large parts of Africa, and parts of Europe. Despite the public health and economic impacts of F. gigantica, understanding of F. gigantica biology and how the complex lifecycle of this liver fluke is transcriptionally regulated remain unknown. Here, we tested the hypothesis that the regulatory small non-coding RNAs (sncRNAs), microRNAs (miRNAs) and tRNA-derived fragments (tRFs) play roles in the adaptation of F. gigantica to its intermediate and definitive hosts. We sequenced sncRNAs of eight lifecycle stages of F. gigantica. In total, 56 miRNAs from 33 conserved families and four Fasciola-specific miRNAs were identified. Expression analysis of miRNAs suggested clear stage-related patterns. By leveraging the existing transcriptomic data, we predicted a miRNA-based regulation of metabolism, transport, growth and developmental processes. Also, by comparing miRNA complement of F. gigantica with that of Fasciola hepatica, we detected a high level of conservation and identified differences in some miRNAs, which can be used to distinguish the two species. Moreover, we found that tRFs at each lifecycle stage were predominantly derived by tRNA-Lys and tRNA-Gly at 5' half sites, but relatively high expression was related to the buffalo-infecting stages. Taken together, we provided a comprehensive overview of the dynamic transcriptional changes of small RNAs that occur during the developmental stages of F. gigantica. This global analysis of F. gigantica lifecycle stages revealed new roles of miRNAs and tRFs in parasite development and will facilitate future research into understanding of fasciolosis pathobiology.

DNA sequencing is the current key technology for historic or ancient biological samples and has led to many exciting discoveries in the field of paleogenomics. However, functional insights into tissue identity, cellular composition, or gene regulation cannot be gained from DNA. Recent analyses have shown that, under favorable conditions, RNA can also be sequenced from ancient samples, enabling studies at the transcriptomic and regulatory level. Analyzing ancient RNA data from a Pleistocene canid, we find hundreds of intact microRNAs that are taxonomically informative, show tissue specificity and have functionally predictive characteristics. With an extraordinary age of 14,300 yr, these microRNA sequences are by far the oldest ever reported. The authenticity of the sequences is further supported by (i) the presence of canid/Caniformia-specific sequences that never evolved outside of this clade, (ii) tissue-specific expression patterns (cartilage, liver, and muscle) that resemble those of modern dogs, and (iii) RNA damage patterns that are clearly distinct from those of fresh samples. By performing computational microRNA-target enrichment analyses on the ancient sequences, we predict microRNA functions consistent with their tissue pattern of expression. For instance, we find a liver-specific microRNA that regulates carbohydrate metabolism and starvation responses in canids. In summary, we show that straightforward paleotranscriptomic microRNA analyses can give functional glimpses into tissue identity, cellular composition, and gene regulatory activity of ancient samples and biological processes that took place in the Pleistocene, thus holding great promise for deeper insights into gene regulation in extinct animals based on ancient RNA sequencing.

Whole genome duplications (WGDs) have long been considered the causal mechanism underlying the dramatic increase in vertebrate morphological complexity relative to invertebrates. This is due to the retention and neo-functionalization of paralogues generated during these events, evolving new regulatory circuits, and ultimately morphological novelty. Nonetheless, an alternative hypothesis suggests that behind the retention of most paralogues is not neo-functionalization, but instead the degree of the inter-connectivity of the intended gene product, as well as the mode of the WGD itself. Here, we explore both the causes and consequences of WGD by examining the distribution, expression, and molecular evolution of microRNAs (miRNAs) in both gnathostome vertebrates as well as chelicerate arthropods. We find that although the number of miRNA paralogues tracks the number of WGDs experienced within the lineage, few of these paralogues experienced changes to the seed sequence, and thus are functionally equivalent relative to their mRNA targets. Nonetheless, the paralogues generated by the gnathostome 2R allotetraploidization event are retained in higher numbers on one sub-genome relative the second, with the miRNAs found on the preferred set of paralogons showing both higher expression of mature miRNA transcripts and slower molecular evolution of the precursor miRNA sequences. Importantly, WGDs do not result in the creation of miRNA novelty, nor do WGDs correlate to increases in complexity. Instead, it is the number of miRNA seed sequences in the genome itself that not only better correlate to instances in complexification, but also mechanistically explain why complexity increases when new miRNA families are established.

Since the original publication of sRNAtoolbox in 2015, small RNA research experienced notable advances in different directions. New protocols for small RNA sequencing have become available to address important issues such as adapter ligation bias, PCR amplification artefacts or to include internal controls such as spike-in sequences. New microRNA reference databases were developed with different foci, either prioritizing accuracy (low number of false positives) or completeness (low number of false negatives). Additionally, other small RNA molecules as well asmicroRNA sequence and length variants (isomiRs) have continued to gain importance. Finally, the number of microRNA sequencing studies deposited in GEO nearly triplicated from 2014 (280) to 2018 (764). These developments imply that fast and easy-to-use tools for expression profiling and subsequent downstream analysis of miRNAseq data are essential to many researchers. Key features in this sRNAtoolbox release include addition of all major RNA library preparation protocols to sRNAbench and improvements in sRNAde, a tool that summarizes several aspects of small RNA sequencing studies including the detection of consensus differential expression. A special emphasis was put on the user-friendliness of the tools, for instance sRNAbench now supports parallel launching of several jobs to improve reproducibility and user time efficiency., European Union [765492 to M.H.]; Spanish Government [AGL2017-88702-C2-2-R to M.H., J.L.O.]; Instituto de Salud Carlos III, FEDER funds [PIE16/00045 to J.A.M.]; Chair ‘Doctors Galera-Requena in cancer stem cell research’ to JMA and by the Ministry of Education of Spain [FPU13/05662 to R.L., IFI16/00041 to E.A.]; Strategic Research Area (SFO) program of the Swedish Research Council (to V.R.) through Stockholm University (to B.F.). Funding for open access charge: SpanishGovernment [AGL2017-88702-C2-2-R].

Made available in DSpace on 2021-06-25T10:55:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: During vertebrate evolution, the heart has undergone remarkable changes that lead to morphophysiological differences in the fully formed heart of these species, such as chamber septation, heart rate frequency, blood pressure, and cardiac output volume. Despite these differences, the heart developmental process is guided by a core gene set conserved across vertebrates. Nonetheless, the regulatory mechanisms controlling the expression of genes involved in heart development and maintenance are largely uncharted. MicroRNAs (miRNAs) have been described as important regulatory elements in several biological processes, including heart biology. These small RNA molecules are broadly conserved in sequence and genomic context in metazoans. Mutations may occur in miRNAs and/or genes that contribute to the establishment of distinct repertoires of miRNA-target interactions, thereby favoring the differential control of gene expression and, consequently, the origin of novel phenotypes. In fact, several studies showed that miRNAs are integrated into genetic regulatory networks (GRNs) governing specific developmental programs and diseases. However, studies integrating miRNAs in vertebrate heart GRNs under an evolutionary perspective are still scarce. Results: We comprehensively examined and compared the heart miRNome of 20 species representatives of the five major vertebrate groups. We found 54 miRNA families with conserved expression and a variable number of miRNA families with group-specific expression in fishes, amphibians, reptiles, birds, and mammals. We also detected that conserved miRNAs present higher expression levels and a higher number of targets, whereas the group-specific miRNAs present lower expression levels and few targets. Conclusions: Both the conserved and group-specific miRNAs can be considered modulators orchestrating the core and peripheral genes of heart GRNs of vertebrates, which can be related to the morphophysiological differences and similarities existing in the heart of distinct vertebrate groups. We propose a hypothesis to explain evolutionary differences in the putative functional roles of miRNAs in the heart GRNs analyzed. Furthermore, we present new insights into the molecular mechanisms that could be helping modulate the diversity of morphophysiology in the heart organ of vertebrate species. Laboratório Especial de Toxinologia Aplicada (LETA) CeTICS Instituto Butantan Department of Chemical and Biological Sciences Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biophysics and Pharmacology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biological Sciences Vanderbilt University Department of Molecular Biosciences The Wenner-Gren Institute (MBW) Stockholm University Department of Chemical and Biological Sciences Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biophysics and Pharmacology Institute of Biosciences of Botucatu São Paulo State University (UNESP) FAPESP: 2012/15589-7 FAPESP: 2013/06864-7

High-throughput sequencing for micro-RNAs (miRNAs) to obtain expression estimates is a central method of molecular biology. Surprisingly, there are a number of different approaches to converting sequencing output into micro-RNA counts. Each has their own strengths and biases that impact on the final data that can be obtained from a sequencing run. This chapter serves to make the reader aware of the trade-offs one must consider in analyzing small RNA sequencing data. It then compares two methods, miRge2.0 and the sRNAbench and the steps utilized to output data from their tools.

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data resource for the RNA community. RNAcentral is freely available at https://rnacentral.org.

Aging is a key risk factor for chronic diseases of the elderly. MicroRNAs regulate post-transcriptional gene silencing through base-pair binding on their target mRNAs. We identified nonlinear changes in age-related microRNAs by analyzing whole blood from 1334 healthy individuals. We observed a larger influence of the age as compared to the sex and provide evidence for a shift to the 5’ mature form of miRNAs in healthy aging. The addition of 3059 diseased patients uncovered pan-disease and disease-specific alterations in aging profiles. Disease biomarker sets for all diseases were different between young and old patients. Computational deconvolution of whole-blood miRNAs into blood cell types suggests that cell intrinsic gene expression changes may impart greater significance than cell abundance changes to the whole blood miRNA profile. Altogether, these data provide a foundation for understanding the relationship between healthy aging and disease, and for the development of age-specific disease biomarkers., Aging is a key risk factor for chronic diseases of the elderly. Here the authors perform large-scale miRNA profiling of blood from individuals of a range of ages and show that common diseases alter the physiological age-related blood microRNA profile.

Trematodes are widespread parasitic flatworms that significantly affect mankind either directly as human parasites, or indirectly via the infection of livestock and the related economic damage. The two most important trematode taxa are the blood flukes Schistosoma and the liver flukes Fasciola, but detection and differentiation of these parasites remains a challenge. Recently, microRNAs (miRNAs) were described from extracellular vesicles (EV) for both parasites secreted into respective hosts. These molecules have been proposed as mediators of parasite-host communication, and potential biomarkers for the detection of parasitic infections from host blood. Our aim here was to study similarities and differences in the miRNA complements of Schistosoma mansoni and Fasciola hepatica, EV-load in particular, to predict their targets and potential functions in the parasite-host interaction. We reanalyzed the known miRNA complements of S. mansoni and F. hepatica and found 16 and 4 previously overlooked, but deeply conserved miRNAs, respectively, further moving their complements closer together. We found distinct miRNA enrichment patterns in EVs both showing high levels of flatworm miRNAs with potential for the detection of an infection from blood. Two miRNAs of the protostome specific MIR-71 and MIR-277 families were highly expressed in EVs and could, therefore, have potential as biomarkers for trematode infection. Curiously, we identified nucleotide differences in the sequence of Mir-277-P2 between S. mansoni and F. hepatica that hold great promise for the distinction of both parasites. To test whether the EV-miRNAs of S. mansoni and F. hepatica could be modulating the expression of host genes, we predicted miRNA targets in 321 human and cattle messenger RNAs that overlapped between both hosts. Of several predicted targets, wnt signaling pathway genes stood out and their suppression likely leads to changes in the glucose concentration in host blood and the reduction of inflammatory and immune responses.

Although microRNAs (miRNA) are involved in all hallmarks of cancer, miRNA dysregulation in metastasis remains poorly understood and contradictory results have been published. The aim of this work was to identify miRNAs associated with metastatic progression of colorectal cancer (CRC). Novel and previously published next generation sequencing (NGS) datasets generated from 268 samples with primary (pCRC) and metastatic CRC (mCRC; liver, lung and peritoneal metastases) and tumor adjacent tissues were analyzed. Differential expression analysis was performed using a meticulous bioinformatics pipeline, including only bona fide miRNAs, utilizing miRNA-tailored quality control and processing, and applying a physiologically meaningful cut-off value (100 reads per million). The results were adjusted for host tissue background expression and samples from the different metastatic sites were independently analyzed. A metastatic signature containing five miRNAs up-regulated at multiple metastatic sites was identified (Mir-210_3p, Mir-191_5p, Mir-8-P1b_3p (mir-141-3p), Mir-1307_5p, and Mir-155_5p) along with a number of miRNAs that were differentially expressed at individual metastatic sites. Several of these have previously been implicated in metastasis through involvement in epithelial-to-mesenchymal transition and hypoxia, while other identified miRNAs represent novel findings. The identified differentially expressed miRNAs confirm known associations and contribute novel insights into miRNA involvement in the metastatic process. The use of open science practices facilitates reproducibility, and new datasets may easily be added to the publicly available pipeline to continuously improve the knowledge in the field. The identified set of miRNAs provides a reliable starting-point for further research into the role of miRNAs in metastatic progression.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

Background To date, mitochondrial genomes of more than one hundred flatworms (Platyhelminthes) have been sequenced. They show a high degree of similarity and a strong taxonomic bias towards parasitic lineages. The mitochondrial gene atp8 has not been confidently annotated in any flatworm sequenced to date. However, sampling of free-living flatworm lineages is incomplete. We addressed this by sequencing the mitochondrial genomes of the two small-bodied (about 1 mm in length) free-living flatworms Stenostomum sthenum and Macrostomum lignano as the first representatives of the earliest branching flatworm taxa Catenulida and Macrostomorpha respectively. Results We have used high-throughput DNA and RNA sequence data and PCR to establish the mitochondrial genome sequences and gene orders of S. sthenum and M. lignano. The mitochondrial genome of S. sthenum is 16,944 bp long and includes a 1,884 bp long inverted repeat region containing the complete sequences of nad3, rrnS, and nine tRNA genes. The model flatworm M. lignano has the smallest known mitochondrial genome among free-living flatworms, with a length of 14,193 bp. The mitochondrial genome of M. lignano lacks duplicated genes, however, tandem repeats were detected in a non-coding region. Mitochondrial gene order is poorly conserved in flatworms, only a single pair of adjacent ribosomal or protein-coding genes – nad4l-nad4 – was found in S. sthenum and M. lignano that also occurs in other published flatworm mitochondrial genomes. Unexpectedly, we unambiguously identified the full metazoan mitochondrial protein-coding gene complement including atp8 in S. sthenum and M. lignano. A subsequent search detected atp8 in all mitochondrial genomes of polyclad flatworms published to date, although the gene wasn’t previously annotated in these species. Conclusions Manual, but not automated genome annotation revealed the presence of atp8 in basally branching free-living flatworms, signifying both the importance of manual data curation and of diverse taxon sampling. We conclude that the loss of atp8 within flatworms is restricted to the parasitic taxon Neodermata. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3807-2) contains supplementary material, which is available to authorized users.

A lack of knowledge of the cellular origin of microRNAs (miRNAs) has greatly confounded functional and biomarkers studies. Recently, three studies characterized miRNA expression patterns across >78 human cell types. These combined data expand our knowledge of miRNA expression localization and confirm that many miRNAs show cell-type specific expression patterns.

Ancient DNA sequencing is the key technology for paleogenomic studies and today a routine method in many laboratories. Recent analyses have shown that, under favoring conditions, also RNA can be sequenced from historical and even ancient samples. We have re-analyzed ancient RNA data from a Pleistocene canid and find - in addition to the previously described messenger RNA fragments - intact microRNAs, which are short transcripts with important gene regulatory functions. With an extraordinary age of 14,300 years, the canid microRNA profiles are the oldest ever reported. Despite their age, we show that the microRNA profiles are conclusive of taxonomic origin, tissue identity with organ- and cell-type specific signatures, and that they yield glimpses into gene regulatory activity and biological processes from the Pleistocene. In summary, we here show that straightforward microRNA analyses hold great promise for deeper insights into gene regulation in extinct animals.

The prevalent porcine helminth, Ascaris suum, compromises pig health and reduces farm productivity worldwide. The closely related human parasite, A. lumbricoides, infects more than 800 million people representing a disease burden of 1.31 million disability-adjusted life years. The infections are often chronic in nature, and the parasites have a profound ability to modulate their hosts' immune responses. This study provides the first in-depth characterisation of extracellular vesicles (EVs) from different developmental stages and body parts of A. suum and proposes the role of these vesicles in the host-parasite interplay. The release of EVs from the third- (L3) and fourth-stage (L4) larvae and adults was demonstrated by transmission electron microscopy (TEM), and sequencing of EV-derived RNA identified a number of microRNAs (miRNAs) and transcripts of potential host immune targets, such as IL-13, IL-25 and IL-33, were identified. Furthermore, proteomics of EVs identified several proteins with immunomodulatory properties and other proteins previously shown to be associated with parasite EVs. Taken together, these results suggest that A. suum EVs and their cargo may play a role in host-parasite interactions. This knowledge may pave the way to novel strategies for helminth infection control and knowledge of their immune modulatory potential.

Peritoneal malignant mesothelioma is a rare disease with a generally poor prognosis and poor response to chemotherapy. To improve survival there is a need for increased molecular understanding of the disease, including chemotherapy sensitivity and resistance. We here present an unusual case concerning a young woman with extensive peritoneal mesothelioma who had a remarkable response to palliative chemotherapy (platinum/pemetrexed). Tumor samples collected at surgery before and after treatment were analyzed on the genomic and transcriptional levels (exome sequencing, RNA-seq, and smallRNA-seq). Integrative analysis of single nucleotide and copy-number variants, mutational signatures, and gene expression was performed to provide a comprehensive picture of the disease. LATS1/2 were identified as the main mutational drivers together with homozygous loss of BAP1 and PBRM1, which also may have contributed to the extraordinary chemotherapy response. The presence of the S3 mutational signature is consistent with homologous recombination DNA repair defects due to BAP1 loss. Up-regulation of the PI3K/AKT/mTOR pathway after treatment, supported by deactivated PTEN through miRNA regulation, is associated with cancer progression and could explain chemotherapy resistance. The molecular profile suggests potential benefit from experimental targeting of PARP, EZH2, the PI3K/AKT/mTOR pathway and possibly also from immune checkpoint inhibition. In addition to providing the molecular background for this unusual case of peritoneal mesothelioma, the results show the potential value of integrative genomic analysis in precision medicine.

Gastrotrichs&mdash, 'hairy bellies&rsquo, &mdash, are microscopic free-living animals inhabiting marine and freshwater habitats. Based on morphological and early molecular analyses, gastrotrichs were placed close to nematodes, but recent phylogenomic analyses have suggested their close relationship to flatworms (Platyhelminthes) within Spiralia. Small non-coding RNA data on e.g., microRNAs (miRNAs) and PIWI-interacting RNAs (piRNA) may help to resolve this long-standing question. MiRNAs are short post-transcriptional gene regulators that together with piRNAs play key roles in development. In a &lsquo, multi-omics&rsquo, approach we here used small-RNA sequencing, available transcriptome and genomic data to unravel the miRNA- and piRNA complements along with the RNAi (RNA interference) protein machinery of Lepidodermella squamata (Gastrotricha, Chaetonotida). We identified 52 miRNA genes representing 35 highly conserved miRNA families specific to Eumetazoa, Bilateria, Protostomia, and Spiralia, respectively, with overall high similarities to platyhelminth miRNA complements. In addition, we found four large piRNA clusters that also resemble flatworm piRNAs but not those earlier described for nematodes. Congruently, transcriptomic annotation revealed that the Lepidodermella protein machinery is highly similar to flatworms, too. Taken together, miRNA, piRNA, and protein data support a close relationship of gastrotrichs and flatworms.

Since their discovery in 1993, microRNAs (miRNAs) have been identified as important gene regulators in many biological processes and as key molecular players in human disease, including cancer where they show specific pathogenic deregulation. Their remarkable chemical stability, the availability of very sensitive miRNA detection methods and the fact that miRNAs can be extracted from and detected in various kinds of clinically relevant samples, such as solid tissues, body fluids and secretions make them excellent candidate biomarkers. However, no miRNA has yet entered the level of practical clinical relevance. We present a brief background and some key aspects and challenges of miRNAs as cancer biomarkers, we discuss shortfalls and identify possible routes towards the use of miRNAs as reliable biomarkers for cancer.

Robust markers of invasiveness may help reduce the overtreatment of in situ carcinomas. Breast cancer is a heterogeneous disease and biological mechanisms for carcinogenesis vary between subtypes. Stratification by subtype is therefore necessary to identify relevant and robust signatures of invasive disease. We have identified microRNA (miRNA) alterations during breast cancer progression in two separate datasets and used stratification and external validation to strengthen the findings. We analyzed two separate datasets (METABRIC and AHUS) consisting of a total of 186 normal breast tissue samples, 18 ductal carcinoma in situ (DCIS) and 1,338 invasive breast carcinomas. Validation in a separate dataset and stratification by molecular subtypes based on immunohistochemistry, PAM50 and integrated cluster classifications were performed. We propose subtype-specific miRNA signatures of invasive carcinoma and a validated signature of DCIS. miRNAs included in the invasive signatures include downregulation of miR-139-5p in aggressive subtypes and upregulation of miR-29c-5p expression in the luminal subtypes. No miRNAs were differentially expressed in the transition from DCIS to invasive carcinomas on the whole, indicating the need for subtype stratification. A total of 27 miRNAs were included in our proposed DCIS signature. Significant alterations of expression included upregulation of miR-21-5p and the miR-200 family and downregulation of let-7 family members in DCIS samples. The signatures proposed here can form the basis for studies exploring DCIS samples with increased invasive potential and serum biomarkers for in situ and invasive breast cancer.

SUMMARY We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated miRNAs, we utilized antagomirs to inhibit their expression and successfully rescue simulated deep-space-radiation-mediated damage in human 3D vascular constructs., Graphical Abstract, In Brief Malkani et al. uncover the role of circulating microRNAs as both a potential biomarker for health risks associated with spaceflight and a countermeasure to mitigate the damage caused to the body by the space environment.

microRNAs are conserved noncoding regulatory factors implicated in diverse physiological and developmental processes in multicellular organisms, as causal macroevolutionary agents and for phylogeny inference. However, the conservation and phylogenetic utility of microRNAs has been questioned on evidence of pervasive loss. Here, we show that apparent widespread losses are, largely, an artefact of poorly sampled and annotated microRNAomes. Using a curated data set of animal microRNAomes, we reject the view that miRNA families are never lost, but they are rarely lost (92% are never lost). A small number of families account for a majority of losses (1.7% of families account for >45% losses), and losses are associated with lineages exhibiting phenotypic simplification. Phylogenetic analyses based on the presence/absence of microRNA families among animal lineages, and based on microRNA sequences among Osteichthyes, demonstrate the power of these small data sets in phylogenetic inference. Perceptions of widespread evolutionary loss of microRNA families are due to the uncritical use of public archives corrupted by spurious microRNA annotations, and failure to discriminate false absences that occur because of incomplete microRNAome annotation.

microRNAs (miRNAs) are often highly conserved across species, but species-specific sequences are known. In addition, miRNA “isomiRs” arise from the same precursor molecule but differ in post-processing length and modification, usually at the 3’ end. A recently published feeding study reported the intriguing result that two bovine milk-specific miRNAs were taken up into human circulation after ingestion of bovine milk. Unfortunately, this interpretation is based on annotation errors in a public microRNA database. Reanalysis using databses including the MirGeneDB database reveals that the miRNAs in question, miR-21-5p and miR-30a-5p, arise from 100% identical 5’ precursor sequences in human and bovine, and the putative bovine-specific isomiRs appear to be depleted, not enriched, in bovine milk. Thus, enrichment of these isomiRs in human blood is inconsistent with uptake of xenomiRs and likely betrays endogenous miRNA regulation in response to diet or technical artifact.

The prediction of novel miRNA genes generally requires the availability of genome sequences in order to assess important properties such as the characteristic hairpin-shaped secondary structure. However, although the sequencing costs have decreased over the last years, still many important species lack an assembled genome of certain quality. We implemented an algorithm which for the first time exploits characteristic biogenesis features like the 5’ homogeneity that can be assessed without genome sequences. We used a phylogenetically broad spectrum of well annotated animal genomes for benchmarking. We found that between 90-100% of the most expressed miRNA candidates (top quartile) corresponded to known miRNA sequences.

Non-coding RNA (ncRNA) molecules have fundamental roles in cells and many are also stable in body fluids as extracellular RNAs. In this study, we used RNA sequencing (RNA-seq) to investigate the profile of small non-coding RNA (sncRNA) in human serum. We analyzed 10 billion lllumina reads from 477 serum samples, included in the Norwegian population-based Janus Serum Bank (JSB). We found that the core serum RNA repertoire includes 258 micro RNAs (miRNA), 441 piwi-interacting RNAs (piRNA), 411 transfer RNAs (tRNA), 24 small nucleolar RNAs (snoRNA), 125 small nuclear RNAs (snRNA) and 123 miscellaneous RNAs (misc-RNA). We also investigated biological and technical variation in expression, and the results suggest that many RNA molecules identified in serum contain signs of biological variation. They are therefore unlikely to be random degradation by-products. In addition, the presence of specific fragments of tRNA, snoRNA, Vault RNA and Y_RNA indicates protection from degradation. Our results suggest that many circulating RNAs in serum can be potential biomarkers.

Earthworms show a wide spectrum of regenerative potential with certain species likeEisenia fetidacapable of regenerating more than two-thirds of their body while other closely related species, such asParanais litoralisseem to have lost this ability. Earthworms belong to the phylum annelida, in which the genomes of the marine oligochaeteCapitella telata, and the freshwater leechHelobdella robustahave been sequenced and studied. The terrestrial annelids, in spite of their ecological relevance and unique biochemical repertoire, are represented by a single rough genome draft ofEisenia fetida(North American isolate), which suggested that extensive duplications have led to a large number of HOX genes in this annelid. Herein, we report the draft genome sequence ofEisenia fetida(Indian isolate), a terrestrial redworm widely used for vermicomposting assembled using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm, showed that many miRNA gene families have also undergone extensive duplications. Genes for several important proteins such as sialidases and neurotrophins were identified by RNA sequencing of tissue samples. We also usedde novoassembled RNA-Seq data to identify genes that are differentially expressed during regeneration, both in the newly regenerating cells and in the adjacent tissue. Sox4, a master regulator of TGF-beta induced epithelial-mesenchymal transition was induced in the newly regenerated tissue. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. The metagenome of the worm, characterized using 16S rRNA sequencing, revealed the identity of several bacterial species that reside in the nephridia of the worm. Comparison of the bodywall and cocoon metagenomes showed exclusion of hereditary symbionts in the regenerated tissue. In summary, we present extensive genome, transcriptome and metagenome data to establish the transcriptome and metagenome dynamics during regeneration.

Recent studies on microRNA (miRNA) evolution focused mainly on the comparison of miRNA complements between animal clades. However, evolution of miRNAs within such groups is poorly explored despite the availability of comparable data that in some cases lack only a few key taxa. For flatworms (Platyhelminthes), miRNA complements are available for some free-living flatworms and all major parasitic lineages, except for the Monogenea. We present the miRNA complement of the monogenean flatworm Gyrodactylus salaris that facilitates a comprehensive analysis of miRNA evolution in Platyhelminthes. Using the newly designed bioinformatics pipeline miRCandRef, the miRNA complement was disentangled from next-generation sequencing of small RNAs and genomic DNA without a priori genome assembly. It consists of 39 miRNA hairpin loci of conserved miRNA families, and 22 novel miRNAs. A comparison with the miRNA complements of Schmidtea mediterranea (Turbellaria), Schistosoma japonicum (Trematoda), and Echinococcus granulosus (Cestoda) reveals a substantial loss of conserved bilaterian, protostomian, and lophotrochozoan miRNAs. Eight of the 46 expected conserved miRNAs were lost in all flatworms, 16 in Neodermata and 24 conserved miRNAs could not be detected in the cestode and the trematode. Such a gradual loss of miRNAs has not been reported before for other animal phyla. Currently, little is known about miRNAs in Platyhelminthes, and for the majority of the lost miRNAs there is no prediction of function. As suggested earlier they might be related to morphological simplifications. The presence and absence of 153 conserved miRNAs was compared for platyhelminths and 32 other metazoan taxa. Phylogenetic analyses support the monophyly of Platyhelminthes (Turbellaria + Neodermata [Monogenea {Trematoda + Cestoda}]).

Is there a correlation between miRNA diversity and levels of organismic complexity? Exhibiting extraordinary levels of morphological and developmental complexity, insects are the most diverse animal class on earth. Their evolutionary success was in particular shaped by the innovation of holometabolan metamorphosis in endopterygotes. Previously, miRNA evolution had been linked to morphological complexity, but astonishing variation in the currently available miRNA complements of insects made this link unclear. To address this issue, we sequenced the miRNA complement of the hemimetabolan Blattella germanica and reannotated that of two other hemimetabolan species, Locusta migratoria and Acyrthosiphon pisum, and of four holometabolan species, Apis mellifera, Tribolium castaneum, Bombyx mori and Drosophila melanogaster. Our analyses show that the variation of insect miRNAs is an artefact mainly resulting from poor sampling and inaccurate miRNA annotation, and that insects share a conserved microRNA toolkit of 65 families exhibiting very low variation. For example, the evolutionary shift toward a complete metamorphosis was accompanied only by the acquisition of three and the loss of one miRNA families., This work was supported by the Spanish Ministry of Economy and Competitiveness (grants CGL2012-36251 and CGL2015-64727-P to X.B.), Spanish Ministry of Science and Innovation (grant BFU2011-22404 to M.D.P.) and Catalan Government (2014 SGR 619). The research has also benefited from European Fund for Economic and Regional Development (FEDER) funds. B.F. was supported by the South-Eastern Norway Regional Health Authority grant #2014041.

microRNAs (miRNAs) are integrated parts of the developmental toolkit in animals. The evolutionary history and origins of animal miRNAs is however unclear, and it is not known when they evolved and on how many occasions. We have therefore investigated the presence of miRNAs and the necessary miRNA biogenesis machinery in a large group of unicellular relatives of animals, Ichthyosporea. By small RNA sequencing we find evidence for at least four genes in the genus Sphaeroforma that satisfy the criteria for the annotation of animal miRNA genes. Three of these miRNAs are conserved and expressed across sphaeroformid species. Furthermore, we identify homologues of the animal miRNA biogenesis genes across a wide range of ichthyosporeans, including Drosha and Pasha which make up the animal specific Microprocessor complex. Taken together we report the first evidence for bona fide miRNA genes and the presence of the miRNA-processing pathway in unicellular Choanozoa, implying that the origin of animal miRNAs and the Microprocessor complex predates multicellular animals.

Summary Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune-system and live inside a hostile environment such as the mammalian liver and bile-ducts. Recently, microRNAs, a class of short non-coding gene-regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction and recent data illustrates that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe), and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions, that we review here. This article is protected by copyright. All rights reserved.

Genome-wide, cell-type-specific profiles are being systematically generated for numerous genomic and epigenomic features. There is, however, no universally applicable analytical methodology for such data. We present GSuite HyperBrowser, the first comprehensive solution for integrative analysis of dataset collections across the genome and epigenome. The GSuite HyperBrowser is an open-source system for streamlined acquisition and customizable statistical analysis of large collections of genome-wide datasets. The system is based on new computational and statistical methodologies that permit comparative and confirmatory analyses across multiple disparate data sources. Expert guidance and reproducibility are facilitated via a Galaxy-based web-interface. The software is available athttps://hyperbrowser.uio.no/gsuite

The advent of next-generation sequencing methods is fuelling the discovery of multiple non-coding RNA transcripts with direct implication in cell biology and homeostasis. This new layer of biological regulation seems to be of particular importance in human pathogenesis, including cancer. The aberrant expression of ncRNAs is a feature of prostate cancer, as they promote tumor-suppressive or oncogenic activities, controlling multicellular events leading to carcinogenesis and tumor progression. From the small RNAs involved in the RNAi pathway to the long non-coding RNAs controlling chromatin remodeling, alternative splicing, and DNA repair, the non-coding transcriptome represents the significant majority of transcriptional output. As such, ncRNAs appear as exciting new diagnostic, prognostic, and therapeutic tools. However, additional work is required to characterize the RNA species, their functions, and their applicability to clinical practice in oncology. In this review, we summarize the most important features of ncRNA biology, emphasizing its relevance in prostate carcinogenesis and its potential for clinical applications.

Background The rather species-poor oviparous gyrodactylids are restricted to South America. It was suggested that they have a basal position within the otherwise viviparous Gyrodactylidae. Accordingly, it was proposed that the species-rich viviparous gyrodactylids diversified and dispersed from there. Methods The mitochondrial genome of Aglaiogyrodactylus forficulatus was bioinformatically assembled from next-generation illumina MiSeq sequencing reads, annotated, and compared to previously published mitochondrial genomes of other monogenoidean flatworm species. Results The mitochondrial genome of A. forficulatus consists of 14,371 bp with an average A + T content of 75.12 %. All expected 12 protein coding, 22 tRNA, and 2 rRNA genes were identified. Furthermore, there were two repetitive non-coding regions essentially consisting of 88 bp and 233 bp repeats, respectively. Maximum Likelihood analyses placed the mitochondrial genome of A. forficulatus in a well-supported clade together with the viviparous Gyrodactylidae species. The gene order differs in comparison to that of other monogenoidean species, with rearrangements mainly affecting tRNA genes. In comparison to Paragyrodactylus variegatus, four gene order rearrangements, i.e. three transpositions and one complex tandem-duplication-random-loss event, were detected. Conclusion Mitochondrial genome sequence analyses support a basal position of the oviparous A. forficulatus within Gyrodactylidae, and a sister group relationship of the oviparous and viviparous forms.

After their discovery about 25 years ago, microRNAs (miRNAs) have today taken the center stage in many applied fields such as the search for new therapeutic and diagnostic biomarkers in cancer and as phylogenetic and developmental markers in biosystematics and evolutionary research. Indeed much is known about their highly conserved biogenesis and structural features, their distribution across the tree of life and dysregulation in many (human) diseases. However, this knowledge is hardly translated to the current miRNA prediction software, and recent work has shown that the majority of described miRNAs do not originate from bona fide miRNA genes, underlining the fact that the prediction and annotation of miRNAs is challenging and far from being a standard procedure—in particular when it comes to new organisms or novel miRNAs. Here I discuss the recent development in miRNA research and compare selected miRNA prediction and profiling pipelines including their shortcomings.

Summary The emergence of multicellular animals was associated with an increase in phenotypic complexity and with the acquisition of spatial cell differentiation and embryonic development. Paradoxically, this phenotypic transition was not paralleled by major changes in the underlying developmental toolkit and regulatory networks. In fact, most of these systems are ancient, established already in the unicellular ancestors of animals [1, 2, 3, 4, 5]. In contrast, the Microprocessor protein machinery, which is essential for microRNA (miRNA) biogenesis in animals, as well as the miRNA genes themselves produced by this Microprocessor, have not been identified outside of the animal kingdom [6]. Hence, the Microprocessor, with the key proteins Pasha and Drosha, is regarded as an animal innovation [7, 8, 9]. Here, we challenge this evolutionary scenario by investigating unicellular sister lineages of animals through genomic and transcriptomic analyses. We identify in Ichthyosporea both Drosha and Pasha (DGCR8 in vertebrates), indicating that the Microprocessor complex evolved long before the last common ancestor of animals, consistent with a pre-metazoan origin of most of the animal developmental gene elements. Through small RNA sequencing, we also discovered expressed bona fide miRNA genes in several species of the ichthyosporeans harboring the Microprocessor. A deep, pre-metazoan origin of the Microprocessor and miRNAs comply with a view that the origin of multicellular animals was not directly linked to the innovation of these key regulatory components., Highlights • The animal-specific miRNA Microprocessor is discovered in unicellular Ichthyosporea • The origin of the animal miRNA machinery was independent of animal multicellularity • The Microprocessor is lost in ctenophores and is not an ancestral animal trait • Several ichthyosporeans harboring the Microprocessor express bona fide miRNAs, In animals, microRNAs and the miRNA biogenesis machinery are essential for correct organismal development. Bråte et al. demonstrate that the core of this machinery, the Microprocessor, is not an animal innovation but originated among their unicellular relatives. Several unicellular species harboring the Microprocessor also express bona fide miRNAs.

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that fewer than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation, and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database -- MirGeneDB (http://mirgenedb.org) -- to catalog this set of robustly supported miRNAs, which complements the efforts of miRBase, but differs from it by annotating the mature versus star products, and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

MicroRNAs (miRNAs) are gene regulators that have recently been shown to down-regulate the immune response via extracellular vesicles in the mammalian host of helminthic parasites. Using the miRNA prediction pipeline miRCandRef, we expanded the current miRNA set of the liver fluke Fasciola hepatica (Platyhelminthes, Trematoda) from 16 to 54 miRNAs (42 conserved and 13 novel). Comparing the cellular expression levels with extracellular vesicles, we found all miRNAs expressed and enriched for miRNAs with immuno-regulatory function, tissue growth and cancer. Our findings support the hypothesis that miRNAs are the molecular mediators of the previously demonstrated immune modulatory function of extracellular vesicles. (C) 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The ectoparasitic Monogenea comprise a major part of the obligate parasitic flatworm diversity. Although genomic adaptations to parasitism have been studied in the endoparasitic tapeworms (Cestoda) and flukes (Trematoda), no representative of the Monogenea has been investigated yet. We present the high-quality draft genome of Gyrodactylus salaris, an economically important monogenean ectoparasite of wild Atlantic salmon (Salmo salar). A total of 15,488 gene models were identified, of which 7,102 were functionally annotated. The controversial phylogenetic relationships within the obligate parasitic Neodermata were resolved in a phylogenomic analysis using 1,719 gene models (alignment length of >500,000 amino acids) for a set of 16 metazoan taxa. The Monogenea were found basal to the Cestoda and Trematoda, which implies ectoparasitism being plesiomorphic within the Neodermata and strongly supports a common origin of complex life cycles. Comparative analysis of seven parasitic flatworm genomes identified shared genomic features for the ecto- and endoparasitic lineages, such as a substantial reduction of the core bilaterian gene complement, including the homeodomain-containing genes, and a loss of the piwi and vasa genes, which are considered essential for animal development. Furthermore, the shared loss of functional fatty acid biosynthesis pathways and the absence of peroxisomes, the latter organelles presumed ubiquitous in eukaryotes except for parasitic protozoans, were inferred. The draft genome of G. salaris opens for future in-depth analyses of pathogenicity and host specificity of poorly characterized G. salaris strains, and will enhance studies addressing the genomics of host-parasite interactions and speciation in the highly diverse monogenean flatworms.

The monogenean flatworm Gyrodactylus salaris is a serious threat to wild and farmed Atlantic salmon stocks in Norway. Morphologically, the closely related but harmless Gyrodactylus thymalli on grayling can hardly be distinguished from G. salaris. Until now, molecular approaches could not resolve unambiguously whether G. salaris and G. thymalli represent just one polytypic species, two polytypic species or a complex of more than two species. In the first known genome-wide analysis utilizing 37 conserved microRNA loci, the genetic differentiation of seven populations of G. salaris and G. thymalli was assessed. The concatenated alignment spanned 21,742bp including 62 variable positions. A neighbor-joining cluster analysis did not support any host-based or mitochondrial haplotype-based grouping of strains. We conclude that a two species concept for G. salaris and G. thymalli does not reflect meaningful biological entities. Instead, G. salaris and G. thymalli are just one species comprising several pathogenic and non-pathogenic strains on various primary hosts. Following the International Code for Zoological Nomenclature, G. salaris Malmberg, 1957 is the valid species name with G. thymalli Žitňan, 1960 becoming the junior synonym. Accordingly, the range of G. salaris is significantly increased, given that formerly G. salaris-free countries such as e.g., Great Britain are now within the species' natural range. The synonymization of G. salaris and G. thymalli implies severe challenges to current disease management routines, which assume that G. salaris and G. thymalli are readily distinguishable. Protocols for reliable identification of pathogenic and non-pathogenic strains of G. salaris need to be developed.

Background Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 μm in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). Findings We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen). Conclusions We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.

Background Neuropeptide ligands have to fit exactly into their respective receptors and thus the evolution of the coding regions of their genes is constrained and may be strongly conserved. As such, they may be suitable for the reconstruction of phylogenetic relationships within higher taxa. CAPA peptides of major lineages of cockroaches (Blaberidae, Blattellidae, Blattidae, Polyphagidae, Cryptocercidae) and of the termite Mastotermes darwiniensis were chosen to test the above hypothesis. The phylogenetic relationships within various groups of the taxon Dictyoptera (praying mantids, termites and cockroaches) are still highly disputed. Results Tandem mass spectrometry of neuropeptides from perisympathetic organs was used to obtain sequence data of CAPA peptides from single specimens; the data were analysed by Maximum Parsimony and Bayesian Interference. The resulting cladograms, taking 61 species into account, show a topology which is in general agreement with recent molecular and morphological phylogenetic analyses, including the recent phylogenetic arrangement placing termites within the cockroaches. When sequence data sets from other neuropeptides, viz. adipokinetic hormones and sulfakinins, were included, the general topology of the cladogram did not change but bootstrap values increased considerably. Conclusion This study represents the first comprehensive survey of neuropeptides of insects for solely phylogenetic purposes and concludes that sequences of short neuropeptides are suitable to complement molecular biological and morphological data for the reconstruction of phylogenetic relationships.

Extended FMRFamides are found throughout the central nervous system (CNS) of insects and exhibit diverse physiological effects on different target organs, such as muscles, intestine, and the nervous system. The genes encoding for extended FMRFamides are known from a number of flies, including Drosophila species, and the pest insects Lucilia cuprina, Calliphora vomitoria, and Musca domestica. No data, however, exist about the expression of the numerous paralogs of the latter three species, and studies on Drosophila melanogaster resulted in controversial findings. We could unambiguously verify, that all predictable products of the extended FMRFamide precursor are expressed in neurohemal tissues of the thoracic neuromers of these flies and can easily be identified and also sequenced by using single specimens. In addition to the confirmation of extended FMRFamides in species with known precursor sequences, the current knowledge about homologous peptides of Sarcophaga (=Neobellieria) bullata could be extended by de novo sequencing using tandem mass spectrometry. The most intriguing finding in this study was the detection of an internal gene duplication, followed by an amino acid substitution, in an insecticide-resistant strain of L. cuprina. To our knowledge, this is the first detection of such an intraspecific event and confirms the low conservation of the extended FMRFamide gene sequences. In insects, no other neuropeptide family is known that shows such sequence variability between related species.