Trypanosoma brucei. While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3′ end of the coding region, through the 3′ tail, to the 5′ start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence, to our knowledge, of circular mRNA in the mitochondrion of the eukaryotic parasite,  The presence of organelles is a distinguishing feature of all eukaryotes. Of the organelles evolutionarily acquired by symbiosis and thus retaining independent genomes, the mitochondrion exists in nearly all extant eukaryotes. The pared-down mitochondrial genome generally retains a remarkably similar core subset of a few coding loci and rRNAs across eukaryotic species. However, the overall organization of these mitochondrial genomes, and the mechanisms for expression and processing of their RNA products vary widely.1 but soon after discovered in the cytoplasm of HeLa cells2 and yeast mitochondria3. Once thought rare, circRNAs’ ubiquity in nature is being increasingly recognized6. The precise mechanism of ligation remains an active area of research, and different mechanisms may operate in different cellular contexts. Ribozymes, cellular ligases, and spliceosomes have all been shown to mediate RNA circularization7. Most identified circRNAs are formed from eukaryotic mRNA introns or exons spliced at normal splice sites and subsequently ligated6. However, a 2018 report revealed the presence of circRNAs formed from full-length mitochondrial mRNAs in the unicellular alga Chlamydomonas reinhardtii8. The mitochondrial mRNA of this alga, like that of a mammalian cell, lacks a 5′ UTR and thus a ribosome binding site. The authors speculated that circularization of Chlamydomonas mitochondrial mRNAs provided a mechanism for ribosome binding upstream of the translational start site. The same group subsequently explored whether such a mechanism might also exist in the mammalian mitochondrion9. This was determined to be unlikely, because while circRNAs derived from Chlamydomonas mitochondrial mRNAs were full length with a termination codon, those from mammalian cells were truncated and thus non-translatable. Very recently, widespread mitochondrial circRNAs originating from mRNAs were discovered in plants, and some were shown to be ribosome associated10. The possibility that diverse regulatory roles for mitochondrial circRNAs exist has recently been confirmed with studies demonstrating that mitochondrial circRNAs regulate protein import into the mitochondria of metazoans and the output of mitochondrial reactive oxygen species in human cells12. Despite these few discoveries of functional roles for mitochondrial circRNAs, their functions and their associated mechanisms remain largely elusive. Further, the landscape of mitochondrial species of circRNAs among eukaryotic species across the tree of life has been barely scratched. The possibility of functional mitochondrial circRNAs in less-studied eukaryotic species is intriguing.Circular RNAs (circRNAs) are a class of RNAs in which the 5′ and 3′ ends of a single RNA molecule are covalently ligated. They were first reported as viroidsTrypanosoma brucei is an insect-transmitted parasitic protozoan of mammals that is part of a larger group called the kinetoplastids. These flagellates possess one unique mitochondrion per cell with a concatenated network of DNA and associated proteins called the kinetoplast. The kinetoplast (k)DNA contains 20 genes: 2 rRNAs and 18 mRNAs that mostly code for proteins of mitochondrial electron transport chain complexes. The process of kDNA transcription into RNA and subsequent RNA maturation steps have been extensively studied and a working model has emerged13. Evidence suggests that transcription is monocistronic14, contrary to previously held hypotheses of polycistronic transcription15, but that there may be a degree of both cannot be ruled out. After transcription, T. brucei RNAs may possess a 5′ triphosphate, which on mRNAs is converted to a 5′ monophosphate by the 5′ pyrophosphohydrolase complex (PPsome)14. All RNA is processed by the mitochondrial 3′ processome (MPsome), which trims the 3′ end and can add oligouridine (U) tails16. mRNAs generally have short adenine (A)-rich A/U heterogenous tails added by kinetoplast poly(A) polymerase I (KPAP1) as part of the kinetoplast polyadenylation complex (KPAC) in combination with KRET1 of the MPsome. Interactions between the 5′ and 3′ bound protein complexes are hypothesized to create a molecule with protected 5′ and 3′ ends in close proximity and are a key regulatory step against mis-tailing and extensive 3′ trimming13. These interactions are thought to be mediated and possibly coordinated by the RNA editing substrate-binding complex (RESC)13. The RESC is one of the main protein complexes that is involved in a complicated editing process that takes place for 12 of the 18 mRNAs. This unique uridine insertion/deletion RNA editing requires a multitude of proteins, many of which are found in the RESC and the RNA editing catalytic complex (RECC). After editing is complete, the short A-rich mRNA tail is elongated to a more heterogenous A/U tail before translation takes place. A multitude of RNA binding proteins control this process, including 3′ trimming, tailing, and editing.T. brucei. Using circular RT-PCR and deep sequencing, we confirmed that the 3′ and 5′ ends of these mRNAs are covalently linked together. We have investigated trends in the UTRs and tails that these transcript populations contain and show that for each transcript examined, its circRNAs are a distinct subpopulation of total mRNA that possess different tail and 5′ UTR characteristics. We hypothesize that the differences between a transcript’s linear and circular mRNA populations are an indication of an alternative pathway in the mRNA lifecycle differing from its normal maturation and turnover pathways.Here, we present evidence of mitochondrial mRNA-derived circRNAs in the kinetoplastids that are very evolutionarily distant from both plants and metazoans. Mitochondrial circRNA of multiple mRNA transcripts are present in both the insect and mammalian bloodstream life stage forms of 17. Library preparation first requires circularizing total RNA with T4 RNA ligase prior to the generation of cDNA, after which outward facing primers  Lister 427 life stage parasites both with and without performing in vitro RNA circularization Table , Fig. 1DCO1 unless it was first circularized in vitro, indicating that whether a transcript undergoes editing may be related to its likelihood of being circularized. To confirm that some transcripts in the untreated samples did not produce a product with circular primers, we increased the amount of PCR cycles further , pre-edited cytochrome b (CYbp), and pre-edited ATP synthase subunit 6 (A6p) from insect life stage parasites. Sequencing confirmed that these products were indeed circRNA: 3′ ends of mRNAs, sometimes including a short tail sequence, covalently ligated to 5′ ends of the same transcript. We attempted to clone and sequence products from edited CYb (CYbe) that appeared when PCR cycles were increased, but were unsuccessful.Notably, for transcripts requiring editing, reliable products were detected for the pre-edited (p) but not the edited (e) versions of the tested mRNA. This suggests that if edited transcripts are circularized in vivo, their abundance is very low. Further, an amplicon was not generated for never-edited transcript her Fig. C. For inND1; CYb, which is edited in only a small region; and A6, an extensively edited transcript .With increased confidence that the amplicons generated were from native mitochondrial circRNA molecules, we further characterized them to distinguish how they may differ from linear mRNAs of the same transcript. We examined ligation point junction sequences between high-throughput sequencing reads from insect-stage parasite libraries obtained with and without in vitro circularization of RNA. We extracted the ligation point junction sequences from Illumina reads of libraries obtained from 29-13 strain parasites untreated RNA (sequences only from circRNAs) and in vitro circularized parasite RNA from control cells described above to identify any termini characteristics unique to circRNA Table . The RNAA6p tails are between 10 and 15 nucleotides in length when the library derived from total RNA was examined, whereas the A6p derived from naturally circularized mRNAs generally incorporated tails of less than five nucleotides. Tail length trends for CYbp and ND1 also indicate shorter tails in circRNA than total RNA  or about 20 nucleotides, whereas total RNA tails are either less than 15 nucleotides or about 40 nucleotides. Nucleotide compositional differences were also observed. For each transcript’s tail population, Fig. CYbp and ND1 show similar patterns in tail length between the circularized mRNA in the two cell lines, A6p has a peak of tail lengths that are under 10 nucleotides in the EATRO164 cells, unlike that of 29-13 cells  and/or Us (state 2) rarely interrupted by a different nucleotide. For never-edited transcripts such as ND1, these shorter tails are sometimes elongated with a sequence characterized by frequent switching between As and Us presumably involving both KPAP1 and KRET1 combined activities (state 3). Thus, although it is appropriate to model pre-edited transcripts CYbp and A6p with a 2-state model, a 3-state model is required for never-edited ND1 Us state  rarely iUs state  rarely iA6p, 95–96% to 88–89% for CYbp, 93% to 85–88% for ND1). For the pre-edited transcripts (A6p and CYbp), we also see an increase in the percentage of tails that stay in the U-adding state , indicating that the U-adding KRET1 may be more processive. The HMM for ND1 indicates a different enzymatic activity pattern, with the predominant tail percentage starting in an atypical U-rich A/U-adding state, which indicates a less processive KRET1 that is often interrupted by KPAP1.In addition to confirming data seen in Fig. ND1 and A6p, circRNAs indeed had a higher percentage of reads that lacked evidence of 3′ mRNA tail addition prior to circularization  oligo(U) sequences   Table . The seqs) Table  and founs) Table , these pIn summary, when compared to a transcript’s total mRNA population, its circRNA population exhibits shortened 5′ UTRs and altered non-templated tail characteristics, but consistent 3′ UTR termini. Together, these data support an mRNA processing model in which (1) modification of the 3′ and 5′ ends of mRNA are physically linked, and (2) circularization happens after the MPsome has trimmed the 3′ UTR and added an initial oligo(U) tail, but before the oligo(U) tails acquire A-rich extensions.T. brucei mitochondria. The presence of circRNAs suggest a previously unidentified pathway in the T. brucei mitochondrial mRNA lifecycle.We have described, for the first time, kinetoplastid mitochondrial-encoded circular mRNAs. These circRNAs comprise part of the total mRNA population of at least 4 of the 5 tested transcripts in both insect- and bloodstream-stage parasites. However, for transcripts requiring editing, only the pre-edited form showed convincing evidence of circularization Fig. D. After 13. 5′ and 3′ end proximity is theoretically advantageous for subsequent mRNA covalent circularization.Since a primary mechanism of trypanosome mitochondrial mRNA processing has been proposed and proteins involved identified, we can derive models for how and when circRNAs are formed. Figure 13. However, while sequenced circRNAs have similar 3′ UTR lengths as those of the total transcript population, they exhibit fewer non-encoded 3′ nucleotide additions , and EATRO164 Istar1 (EATRO164) insect-stage cells were grown in SDM-79 at 27 °C in 5% CO2. G418 and hygromycin were added to 29-13 cultures. Bloodstream-stage ‘single marker’ (SM) Lister 427 T. brucei was grown at 37 °C in 5% CO2 in G418-supplemented HMI-9. All cell lines were a gift from Dr. Laurie Read, University at Buffalo in 2013. Most experiments of mitochondrial circRNAs, including all deep sequencing of circRNAs, were performed on 29-13, EATRO164, and SM parasite RNA. However, mitochondrial circRNAs were first identified with gel electrophoresis and Sanger sequencing on a 29-13 strain with an additional nonphenotypic genetic modification. Currently unpublished and termed “control”, this cell line under tetracycline induction will express the protein KPAP1 from an exogenous locus and simultaneously silence endogenous KPAP1. Lacking tetracycline (uninduced), “control” cells are phenotypically identical to 29-13 in growth and other parameters. CircTAIL-seq (including an in vitro ligation step) of total transcript mRNA (likely majority non-circular) was performed on this cell line. These same data from “control” serve as wild-type in a different study to be published elsewhere. “Control” cell line was maintained with G418, hygromycin, puromycin, and phleomycin.28, circularized using T4 RNA ligase 1 (Epicentre or New England BioLabs) and reverse transcribed using Superscript III (Invitrogen) and gene specific primers as in17. Table RNA was collected at approximately the same cell densities for each cell line as described inA LiCor Odyssey Fc Imager was used to image agarose gels stained with ethidium bromide using Image Studio Amalysis software, version 5.2.5. Gels were imaged for 30 s on the 600 channel. Contrast and brightness were adjusted for the entire gel until bands were visible while minimizing background color. Original gel images are presented in Fig. ND1, CYbp, and A6p  performed quality control on all libraries by assessing quality and quantity on an Agilent BioAnalyzer and performing KapaQC. Amplicon libraries were then sequenced at UMGC on an Illumina MiSeq analyzer using the MiSeq V2 Chemistry 150 PE kit, acquiring 150 bp paired-end reads. One exception, CYbp control replicate 1, was sequenced using MiSeq V2 Chemistry 250 PE kit. Eight libraries were run in one lane and sorted using barcodes when possible and unique gene primer sequences otherwise. Table ND1 libraries, in which only 51–57% of reads were used for the control libraries and between 8 and 19% were used in the circularized libraries. Initial downstream read processing was performed as in17. Briefly, read quality was assessed with FastQC, paired reads were merged using PEAR, Illumina barcodes were trimmed with Trimmomatic, reads were reverse complemented and converted to fasta files using the FastX toolkit, and merged read quality was checked using FastQC. An updated tail identification protocol was used to identify which nucleotides constituted tail sequence for each read and which were part of the 3′ and 5′ UTRs surrounding any tail. The Needleman Wunch algorithm29, coded in C, was modified for the alignments, while all downstream analysis was done by Python scripts . Reads within files were aligned to the DNA sequence corresponding to both the 3′ sequence immediately downstream of the primer binding site to identify the last 3′ templated nucleotide for each read and to the 5′ region just upstream of the 5′ primer binding site to identify the 5′ last untemplated nucleotide. These alignments returned files of 3′ ends, 5′ ends, and non-encoded tails. Because the aligner predicts the longest possible UTR, tails were occasionally mis-aligned to A/U rich UTRs. To address this, we implemented a pruning step in circTAIL-seq alignment. Pruning starts from the end of the alignment and, based on a transcript-specific scoring matrix, terminates the 3′ and 5′ UTR alignment at the point where an A/U rich region follows a gap or mismatch that cannot be attributed to RNA insertion/deletion editing. For EATRO164 A6p, the DNA template used for alignment was modified to accommodate differences from the 29-13 A6p DNA sequence in the 5′ terminus. All graphs were made using R, version 4.2.1.CircTAIL-seq was performed as described inet Table  to decre30 and19.Hidden Markov modelling (HMM) was performed as described inSupplementary Figures.Supplementary Table S1.Supplementary Table S2.


Correction to: Clinical rheumatologyhttps://doi.org/10.1007/s10067-023-06659-9The affiliations in the published version of the attached above article was incorrectly designated to the authors. The correct presentation are as follows:Fangfang Chen1, Yitian Lang3, Shikai Geng1, Xiaodong Wang1, Liangjing Lu1, Shuang Ye1, Le Zhang1,2, Ting Li11 Department of Rheumatology, Ren Ji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China 2 Department of Pharmacy, Ren Ji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 3 Department of Pharmacy, Huangpu Branch, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaThe original article has been corrected.


Drosophila and honeybee is already known to benefit the host by increasing proteomic diversity in a spatial-temporal manner (namely “diversifying hypothesis”).C-to-U RNA editing in plants is believed to confer its evolutionary adaptiveness by reversing unfavorable DNA mutations. This “restorative hypothesis” has not yet been tested genome-wide. In contrast, A-to-I RNA editing in insects like Arabidopsis thaliana, Drosophila melanogaster, and Apis melifera. We unprecedentedly defined the haplotype diversity (HD) of RNA molecules based on nonsynonymous editing events (recoding sites).We profiled the RNA editomes of multiple tissues of Arabidopsis by observing higher frequencies and levels at nonsynonymous editing sites over synonymous sites. Compared to A-to-I recoding sites in Drosophila, the C-to-U recoding sites in Arabidopsis show significantly lower HD, presumably due to the stronger linkage between C-to-U events.Signals of adaptation is confirmed in Arabidopsis is adaptive but it is not designed for diversifying the proteome like A-to-I editing in Drosophila. Instead, C-to-U recoding sites resemble DNA mutations. Our observation supports the restorative hypothesis of plant C-to-U editing which claims that editing is used for fixing unfavorable genomic sequences.C-to-U RNA editing in The online version contains supplementary material available at 10.1186/s13062-023-00404-7. RNA editing is prevalent in all kingdoms of lives –3. TradiRNA editing has various biological functions. Apart from the non-coding RNA editing sites that are dedicated to immune and anti-viral responses , the manIn plants, C-to-U RNA editing events take place in chloroplast and mitochondrial genes. These RNA editing events are mediated by various factors. These factors include the first discovered site-specific editing/splicing factors PPRs that recognize and bind to target RNA regions , the secLines of evidences show that the C-to-U RNA editing events (mainly in non-nuclear genome) conform to the restorative hypothesis . This me(1) Bear high editing levels. Since T allele is optimal and C allele is non-optimal under the restorative hypothesis, the C-to-U editing at a particular site should achieve as high level as possible. For DNA mutations (homozygote), the alternative allele level is 100%. Similarly, natural selection would force the RNA editing level to increase. Note that this rule is only for nonsynonymous sites because we presume that synonymous sites are neutral. Moreover, we should also explain that no strict criteria could define what is the value of being high. The restorative hypothesis is a new theory which was first proposed in 2019  and was Nicotiana tabacum) ];df2 <- list_candi[[k]].df3 <- cbind],df2)df3$p <- apply{.x[1] <- x[1]-x[2];x[3] <- x[3]-x[4].fisher.test)$p.value.})df3$fdr <- p.adjust.df_des_nonsyn <- sum(df3$fdr < 0.05).}}The same goes for the code for synonymous editing sites.P value of the difference in editing level = alt/. Then the P values were adjusted for multiple testing correction to get a false discovery rate (FDR) [D. melanogaster and A. mellifera samples.In brief, for each editing site, the reads count numbers  of two samples were extracted. Fisher’s exact tests were used to calculate the te (FDR) . EditingA. thaliana , including 402 nonsynonymous and 105 synonymous sites. The Nonsyn/Syn ratio is 402/105 = 3.8, which is significantly higher than random expectation  under neutral evolution  .Although signals of adaptation were observed in the C-to-U editome of pothesis , 10 and pothesis  require es) Fig. B. Here, es) Fig. C, suggeses) Fig. . Among tes) Fig. D. The avues Fig. D and theThe evidences demonstrated in this section would support the predictions made by restorative hypothesis. Note that although the restorative purpose of specific C-to-U editing sites has already been proposed by early studies , 25–29, D. melanogaster and 3 tissues of honeybee A. mellifera  seems much lower than the faction in honeybee, but we should notice that Drosophila has 10 samples so the pairwise DES number is diluted by the combination number We calculated the pairwise DES among the 10 fruitfly samples and 3 honeybee samples. Remarkably, in both insect species, head samples had many DES compared with other samples Fig. B. We alsA. thaliana has less power to increase proteomic diversity compared to the A-to-I editing in Drosophila (which is well-known for its diversifying role). By showing this comparison, it will be more confident to claim that C-to-U editing in plants is used for restorative purpose rather than diversifying purpose.Philosophically, if group A and group B are mutually exclusive, then “X does not belong to group A” would increase the probability that “X belongs to group B”. Given that the diversifying hypothesis and restorative hypothesis are complementary, we intend to prove that C-to-U editing in A. thaliana compared to Drosophila brains  parameter for each pair of nonsynonymous editing sites Fig. . When meins Fig. A. This scoverage . This reely Fig. A. All thA. thaliana than in D. melanogaster. According to the HD formula as well as the intuitive definition of diversity, the “evenly distributed” haplotypes will produce a higher HD than the case where all individuals are skewed to a particular “dominant” haplotype. Therefore, we calculated the haplotype frequency for each pair of nonsynonymous editing sites. In pooled samples of A. thaliana, the CT and TC haplotypes are strongly suppressed while TT haplotype is dominant  the frequencies of AG, GA, and GG haplotypes are comparable, and (2) the frequency of AA is only slightly higher than the frequencies of other three haplotypes  of synaptic gene hig (hikaru genki) produce a combination of four protein isoforms, amplifying the neural proteomic diversity (HD = 0.63) in synapses  only have the all edited TT haplotype and the none edited CC haplotype in all tissues . These observations and implications all support the restorative hypothesis but not diversifying hypothesis of C-to-U RNA editing in plants.Intriguingly, strong linkage is also a representative feature for DNA mutations: for instance, two DNA mutations will always be linked in the RNA molecules; compared to the random RNA editing events on two nearby editing sites (which are weakly linked), DNA mutations have 100% linkage. Thus, the fact that C-to-U editing in Drosophila, it was reported that alternative splicing is prevalent in the neuronal genes to create various versions of transcripts [Drosophila nerve systems, it is not surprising to observe that edited genes on average bear more isoforms than unedited genes  or for reversing unfavorable DNA mutations (restorative hypothesis). We observed signals of adaptation in the C-to-U RNA editomes of No matter nonsynonymous editing is designed for diversifying or restorative purpose, it has to be positively selected compared to synonymous editing. Therefore, signal of adaptation is expected under both hypotheses. Although the original restorative hypothesis states that the edited allele is no better than the ancestral DNA allele, it is intuitive to think that the edited allele is better than the unedited allele in extant species . Then, tNotably, both diversifying and restorative hypotheses only focus on nonsynonymous editing. There are many non-coding editing sites in repeat regions that function as anti-virus defense in mammals . These eX. Obviously, m will drastically increase with X. When the sequencing coverage is not deep enough, the observed number of haplotypes (n) might be small and n/m will be even smaller. This will significantly reduce the HD value. HD values under different X (number of mutation sites) are not comparable. Therefore, this current HD formula needs to be refined to adapt to a broader range of cases. Nevertheless, the HD values are comparable when given the same number of mutation sites. The specific cases shown in our study prove that our HD parameter accurately reflects the proteomic diversity.HD is strongly affected by the number of mutation sites. In this study, we only considered pairs of nonsynonymous C-to-U editing sites . If we extend the HD formula to X editing sites, then m = 2Arabidopsis is adaptive but it is not designed for diversifying the proteome like A-to-I editing in Drosophila. Instead, C-to-U recoding sites resemble DNA mutations. Our observation supports the restorative hypothesis of plant C-to-U editing which claims that editing is used for fixing unfavorable genomic sequences.C-to-U RNA editing in Below is the link to the electronic supplementary material.Supplementary Material 1


The relative Ct values were normalized using abundance of cyclophilin or the geometric mean of specific miRNA for the ISG and miRNA genes of interest, respectively. The DA of the ISG were increased due to pregnancy (p < 0.05); however, there was no expected day of gestation by pregnancy interaction. Relative abundance of Bta-miR-16 increased on d18 regardless of pregnancy status (p < 0.05). None of the miRNA evaluated in this study were associated with pregnancy status. These data indicate that certain ISG may serve as early indicators of pregnancy in cattle, but abundance of the miRNA does not.Interferon-stimulated genes (ISG) and microRNA (miRNA) present in maternal circulation have been reported to be diagnostic of pregnancy in cattle prior to day (d)30 of gestation. The objective of this study was to assess specific ISG and miRNA abundance on d 18 of gestation. Cattle were subjected to estrous synchronization and artificially inseminated to a single Angus sire. At time of insemination (d 0) and d 18 post-insemination, blood was collected and total RNA isolated. Differential abundance (DA) in specific ISG and miRNA between d 0 and d 18 samples in pregnant ( High rates of pregnancy loss have been observed when using assisted reproductive techniques such as IVF ,2,3 and It has also been argued that determination of pregnancy prior to the day 28 to day 30 of gestation would be beneficial to producers enabling quick financial decisions. Pregnancy recognition in cattle occurs around day 16 of gestion . This prOther biomolecules are also being evaluated as determinants of pregnancy and as markers for competence of early embryonic development. MicroRNA (miRNA) are small non-coding RNA produced within the cell and can inhibit transcription of mRNA by association with the 3′ UTR, suppressing translation and eventually targeting the mRNA for degradation within the cell ,20. miRNg for 10 min. The serum was collected and stored at −80 °C until used in analysis. Prior to analysis, serum was thawed, subjected to centrifugation and subjected to spectrophotometry to determine the degree of hemolysis [All animal procedures were approved by Clemson University Animal Care and Use Committee (AUP #2018-048 and #2019-023), and all animals were housed at a single location, and subjected to all treatments within the time in the same year. Primi- and multiparous Angus and Angus crossbred cows (n = 80) greater than 45 days postpartum were synchronized using the C0-synch plus CIDR protocol . All aniemolysis ,34, as hemolysis . Blood sAll animals were subjected to pregnancy detection. Pregnancy was determined by an animal being diagnosed as pregnant or open using trans-rectal ultrasonography and the BioPryn ELISA , which detects pregnancy specific protein b (PSPb) . BrieflyRNA was isolated either from whole blood collected into PAX blood collection tubes or from serum. Total RNA was isolated from blood samples using the PAXgene Blood RNA Kit  as described by the manufacturer. Quantity and quality of total RNA isolated were assessed using spectrophotometry  and the Agilent 6000 Nano Chip kit on the Agilent 2100 . Only samples with a RIN of greater than or equal to 7 were used in further analysis. Total RNA was isolated from serum using the Qiagen miRNeasy kit  per manufacturer directions. Concentration and purity of RNA was determined using the Nanodrop 1000  and quality assessed using the Agilent 2100 small RNA kit.1/slope) × 100. For RT-qPCR, an equal mass of each RNA sample (100 ng) was subjected to reverse transcription using qScript cDNA synthesis kit . qPCR was conducted using 5 ng of resulting cDNA with PerfeCTa SYBR Green FastMix  in duplicate for each sample for the mRNA for MX2, Oas1 and Isg11. Data were normalized using cyclophilin [gene of interest) ΔCt gene of interest/(E cyclophilin) ΔCt cyclophilin where E is the efficiency of each primer set [RNA isolation from samples using the PAXgene blood RNA kit would contain blood cells, including white blood cells, in the lysate producing total RNA. These samples were utilized in RT-qPCR to determine the relative abundance of 3 INF stimulated genes . Genes, lophilin . Relativimer set . Receiveimer set .gene of interest/Geometric mean (RQ of reference genes). RQ is defined as the primer efficiency ΔCt [To determine the abundance of specific miRNA in maternal serum at d0 or d18 of gestation, Taqman  PCR was conducted on samples extracted using the PAXgene method (contained cells) or the Qiagen miRNeasy method (no cells present). The assay ID is given in the table and checked against Bos taurus miRNA sequences. The miRNA evaluated were reported to be present in maternal serum ,8,29,39.ency ΔCt . Receiveency ΔCt .p < 0.05.ISG RT-qPCR data were subjected to analysis using ANOVA . Model fixed effects were pregnancy status determined at day 30 of gestation (Open or Pregnant), day of gestation (d0 or d18) or their interaction. RNA isolation and miRNA relative differences in abundance data were evaluated where model fixed effects were pregnancy status determined at day 30 of gestation (Open or Pregnant), day of gestation (d0 or d18), RNA isolation method or their interactions. Student’s t-test among LS means for RNA quality, INF stimulated gene abundance and miRNA abundance was used to determine significance at the RNA was isolated from whole blood using PAXgene blood RNA kit that isolated RNA from all components of blood including red and white blood cells or serum (no cellular RNA) using the Qiagen miRNeasy procedure. The RNA profiles for RNA isolated using the PAXgene or RNeasy procedures are shown in p < 0.05); however, only ISG15 exhibited a trend for a pregnancy by day interaction (p < 0.1).End-point PCR analysis was conducted on all 20 samples with a representative gel shown in p < 0.05); however, only Bta-miR-25 tended to have increased abundance for isolation methods by day interaction (p < 0.1) (p < 0.05) and tended to be elevated in the d 0 samples of cows that were open at d 30 of gestation (p < 0.1). No miRNA evaluated exhibited increased abundance or tended to exhibit an elevated abundance in d 0 or d 18 samples from cows verified as pregnant at d 30 of gestation. The relative abundance for Bta-let-7a, miR-15b, miR-25 and miR-26 were highest in the PAXgene RNA samples (p < 0.1) . Method Receiver operator characteristic curves (ROC) were generated for each target ISG and miRNA evaluated to determine their usefulness as a diagnostic tool for accurately predicting pregnancy. ROC curves were generated for the ddCT of each ISG are shown in The reproductive efficiency when using assisted reproductive technologies, such as AI or ET, could be increased if reliable methods to detect developmentally incompetent embryos or at risk pregnancies versus developmentally normal pregnancies early in gestation were available. These detection methods would allow producers to make management decisions earlier in the process saving time and money. Circulating miRNA are present in biological fluids ,26 and hOur long-term goal is to utilize maternal miRNA as markers for pregnancy health in ruminants when using either traditional or assisted reproductive technologies such as ET or SCNT. Others investigators have similar interests. Some focused on early embryonic loss and identified miRNA associated with pregnancy or pregnancy loss ,29,30,31The PAXgene blood RNA kit gave high-quality samples with distinct 28 s and 18 s bands, 260/280 ratios of over 2.0 and RIN values > 7.5. All these parameters indicate quality total RNA for the subsequent use in RT-qPCR for ISG and specific miRNA. Previous reports determining the relative abundance of ISG were performed by the isolation of white blood cells . The metThe relative abundance of ISG15, MX2 and OAS1 mRNA was assessed by RT-qPCR from RNA isolated using the PAXgene blood RNA isolation kit, and our data would tend to support previous observations ,16,17. WRelative abundance of maternal circulating miRNA was evaluated using two separate RNA isolation procedures. The miRNA chosen were previously reported to be present ,8,29 and


Fungi with a striking expansion in members of the phylum Basidiomycota representing the most complex fungal organisms. Our study unveils a large diversity of unknown ambiviruses with as little as 27% protein sequence identity to known members and sheds new light on the evolution of this distinct class of infectious agents with RNA genomes. No evidence for the presence of ambiviruses in human microbiomes was obtained from a comprehensive screen of respective metatranscriptomes available in the SRA.Ambiviruses are hybrid infectious elements encoding the hallmark gene of RNA viruses, the RNA-dependent RNA polymerase, and self-cleaving RNA ribozymes found in many viroids. Ambiviruses are thought to be pathogens of fungi, although the majority of reported genomes have been identified in metatranscriptomes. Here, we present a comprehensive screen for ambiviruses in more than 46,500 fungal transcriptomes from the Sequence Read Archive (SRA). Our data-driven virus discovery approach identified more than 2,500 ambiviral sequences across the kingdom  Infectious genetic elements with RNA genomes (IGERs) encompass viroids and viroid-like RNAs as well as certain viruses. These agents have been demonstrated to cause a multitude of economically and medically important diseases despite major differences in genome size, genetic complexity, and life cycle between the various IGER classes and subclasses. The vast diversity of known and potentially unknown IGERs makes them ideal systems to study micro- and macro-evolutionary processes, the emergence of hybrid elements with features from difference IGER classes, and the origin(s) of life.Viroids are the simplest form among known infectious pathogens, consisting of a single-stranded, covalently closed circular RNA genome of a few hundred nucleotides in length . AlthougFungi is lacking. This paucity includes a description of the presence or absence of ambiviruses in the human mycobiome formed by fungal components of the microbiome which interact with both the bacterial microbiome and host immunity, and can influence pathophysiological processes in humans . We discovered more than 2,500 viral sequences from 345 distinct ambiviruses and demonstrate an expansion of ambiviruses in the most complex fungal organisms from the phylum We have applied a data-driven virus discovery (DDVD) approach  to screeAscomycota  as well as Mucoromycota (p = 4.1e–28) and Chytridiomycota (p = 3.6e–19) was statistically highly significant, while no significant differences were observed between the other pairs of phyla  reproduce asexually and are characterized by internal spore production in a sac-like structure called the ascus. Members from the Basidiomycota form spores externally by specialized cells called basidia, and sexual reproduction is considered to be more common among Basidiomycota species. It is tempting to speculate that the mode of reproduction may play a role in the spread of ambiviruses, a hypothesis that warrants further investigation, for instance via comparative infection experiments. Another factor of susceptibility to ambivirus infection might be linked to the higher complexity, in terms of cell cycle and multicellularity, of Basidiomycota species compared to those of other orders  and constitute two relatively young lineages compared to the other fungal orders , followed by Hairpin ribozyme  and Hairpin-meta1 ribozyme . Similar to other viroid-like elements, such as HDV, many of the ambivirus genomes are predicted to adopt a rod-shaped RNA secondary structure conformation (The novel ambiviruses with full-length or near full-length genome sequences showed the expected genomic organization involving two open reading frames (ORFs) encoded in opposite reading directions . Two of them were from the same study analyzing lung metatranscriptomes of patients with pneumonia and acute respiratory infections (SRA run accessions: SRR13677688 and SRR13677804). The third ambivirus-positive experiment (SRR5963935) was from a stool sample of a patient with Crohn’s disease. The very low number of identified ambivirus makes it very challenging to discriminate between origin of the ambiviruses sequences by infection of fungi from the human microbiome and origin by any source of contamination  distinct class(es) of sub-viral elements with ancient and extant RNA viruses may offer unprecedented insights into the emergence of RNA viruses and their hallmark RdRp protein.Fungi (except those of the model organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe) in the NCBI SRA database was compiled as of October 2022. The following search query was performed to obtain the SRA run identifiers: ‘txid4751[Organism:exp] NOT txid4932[Organism:exp] NOT txid4896[Organism:exp] AND (cluster_public[prop] AND “biomol rna”[Properties])’. A list of all 12,694 human metatranscriptome experiments were compiled from the SRA database using the following search query: ‘txid1131769[Organism:exp] OR txid1504969[Organism:exp] OR txid1632839[Organism:exp] OR txid1633571[Organism:exp] OR txid1679718[Organism:exp] OR txid1712573[Organism:exp] OR txid1837932[Organism:exp] OR txid1842734[Organism:exp] OR txid2489051[Organism:exp] OR txid2705415[Organism:exp] OR txid408170[Organism:exp] OR txid433733[Organism:exp] OR txid447426[Organism:exp] OR txid539655[Organism:exp] OR txid646099[Organism:exp] AND (cluster_public[prop] AND “biomol rna”[Properties])’. SRA data were downloaded using the SRA Toolkit  in Python .The original contributions presented in the study are included in the article/CL and LC performed experiments, analyzed the data and prepared figures. CL designed the study, supervised the project, and wrote the manuscript with contributions from LC. All authors contributed to the article and approved the submitted version.LC and CL are supported by the Project “Virological and immunological determinants of COVID-19 pathogenesis—lessons to get prepared for future pandemics (KA1-Co-02 ‘COVIPA’),” a grant from the Helmholtz Association‘s Initiative and Networking Fund. CL was supported by the Deutsche Forschungsgemeinschaft  under Germany’s Excellence Strategy—EXC 2155—project number 390874280. This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the “Open Access Publikationskosten” program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time.Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods  for poliovirus molecular detection using stool samples (IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool. Poliovirus (PV) surveillance is vital to the Global Polio Eradication Initiative (GPEI) effort to eradicate and contain all wild, vaccine-related, and Sabin polioviruses. Surveillance operates through both the detection of the virus in the stools of children suffering from acute flaccid paralysis (AFP)  and from5–Nucleic acid extraction marks a starting point in any molecular method and serves as a key step to recover viral nucleic acid and remove unidentified constituents that inhibit downstream molecular applications , 12. DirTherefore, different RNA extraction methods were compared with a view to identify if specific methods produced better results for different poliovirus molecular detection assays, such as quantitative PCR (qPCR), long-range reverse transcription-PCR (RT-PCR), and suitability for poliovirus direct detection by the DDNS method .The aim of the study was to compare 11 extraction methods  for their ability to efficiently recover poliovirus RNA from stool for subsequent poliovirus direct detection. Performance of the 11 extraction methods was evaluated in two different laboratories in terms of their ability to detect polioviruses in stool suspensions, as determined by poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a near-whole-genome amplification pan-poliovirus (PanPV) RT-PCR, pan-enterovirus (PanEV) RT-PCR amplifying the entire capsid, and nested VP1 PCR amplifying the full VP1 region. The QIAamp viral RNA minikit is a commonly used and accepted RNA extraction method in the GPLN and has been used on all the samples included in this study; therefore, it was used as a comparator for all results.We examined the effect of the extraction methods on these PCR amplification assays and how they differ in their ability to efficiently recover poliovirus genome copies present in stool suspensions. The viral RNA recovered by each of the 11 extraction methods was also assessed for quality based on RNA integrity number equivalent (RINe) and quantity (concentration).P = 0.251, P = 0.199, and P = 0.472, respectively). However, RINe was found to be statistically significant in determining RT-qPCR PV results (P < 0.001).The mean RNA concentration of the 11 extraction methods ranged from 1.54 ± 3.60 ng/μL to 3.30 ± 4.51 ng/μL. The mean RINe  valTC) value (30.0 ± 5.38) and highest mean copies/μL in log10 (2.29 ± 1.52). Samples extracted using the AllPrep PowerFecal DNA/RNA kit had the highest average TC value (37.8 ± 2.69) and lowest mean copies/μL in log10 (0.634 ± 0.68) (P < 0.001). For both TC values and log viral copies, statistically significant and positive differences were observed for most methods except for the KingFisher MagMAX viral RNA isolation kit, MagMAX viral RNA isolation kit , KingFisher MagMAX pathogen RNA/DNA kit (medium volume [MV]), and High Pure viral RNA kit , which were statistically nonsignificant and positive compared to the QIAamp viral RNA minikit extraction method  had the lowest average cycling threshold ( ± 0.68)  and B. Tn method .The proportion of PV-positive samples in the RT-qPCR, nested VP1, PanEV RT-PCR, and PanPV RT-PCR assays extracted by 11 extraction methods ranged from 0.50 to 0.94, from 0.46 to 0.90, from 0.46 to 0.97, and from 0.29 to 0.90, respectively. The highest proportion of PV positive stool samples in the RT-qPCR was observed in stool samples extracted using the Zymo quick RNA viral kit (protocol B), High Pure viral RNA minikit (protocol B), MagMAX viral RNA isolation kit , and KingFisher MagMAX viral RNA minikit, with results comparable to those obtained with the QIAamp viral RNA minikit . The lowThe highest proportion of PV positive stool samples for the nested VP1 assay was observed in samples extracted using the Zymo quick RNA viral kit (protocol B), High Pure viral RNA minikit (protocol B), KingFisher MagMAX pathogen RNA/DNA kit (LV), KingFisher MagMAX viral RNA minikit, and MagMAX viral RNA isolation kit , with results comparable to those obtained with the QIAamp viral RNA minikit. The lowest proportion was observed in samples extracted using the Zymo quick RNA viral kit (protocol A), AllPrep PowerFecal DNA/RNA kit, and KingFisher MagMAX pathogen RNA/DNA kit (SV) . For PV The highest proportion of PV-positive stool samples for the whole-genome PanPV RT-PCR assay was observed in samples extracted using the High Pure viral RNA kit (protocol A), High Pure viral RNA minikit (protocol B), Zymo Quick RNA viral kit (protocol B), KingFisher MagMAX viral RNA isolation kit, and MagMAX viral RNA isolation kit , with results comparable to those obtained with the QIAamp viral RNA minikit. The lowest proportion was observed in samples extracted using the Zymo quick viral RNA kit (protocol A) and KingFisher MagMAX pathogen RNA/DNA kit (LV) .P < 0.001). Overall, most methods were not inferior to the QIAamp viral RNA minikit except for the AllPrep PowerFecal DNA/RNA kit, KingFisher MagMAX pathogen RNA/DNA kit (SV), and Zymo quick RNA viral kit (protocol A) . Additionally, results indicated that some extraction methods proved to be successful at yielding PV results for a given sample, as detected using PV-specific RT-qPCR, nested VP1 PCR, and PanEV and PanPV RT-PCR, while other methods proved to be unsuccessful .All the samples extracted using the High Pure viral RNA kit (protocol A), High Pure viral RNA minikit (protocol B), Zymo quick RNA viral kit (protocol B), and QIAamp viral RNA minikit were successful at yielding PV results in a combination of the different PCR assays . There wP < 0.001).In the logistic regression analysis that accounted for RNA extraction methods, the different molecular detection assays did not differ significantly in the proportion of samples that were positive (Table S2). In general, performance of the RNA extraction methods was consistent across the different detection assays, although there was a significant negative interaction between the PanPV RT-PCR and the KingFisher MagMAX pathogen RNA/DNA kit (LV), indicating worse performance of this combination of assay and extraction method (To assess if increasing the input sample volume would increase PV molecular detection particularly for the nested VP1 (a PCR assay that forms part of the DDNS method), three sample volumes were extracted using the MagMAX pathogen DNA/RNA kit  on the KingFisher Duo. Comparison of the percentage of all positive samples by the nested VP1 indicated that using a large volume led to a higher percentage of samples that were PV positive than the medium- and small-input sample volume .The approximate amount of time required to process samples by the 11 extraction methods was determined, and the cost per sample was calculated for the kits and additional reagents required (excluding plasticware) . The autEfficient direct detection methods for the testing of stool samples from individuals with AFP will be crucial to future poliovirus surveillance. In this study, a comparison of 11 extraction methods  was conducted to assess their ability to recover poliovirus RNA from stool suspensions that had been stored for over 6 months following previous identification as containing poliovirus through virus isolation. All 11 extraction methods assessed use a lysis buffer containing guanidine thiocyanate or guanidine hydrochloride, a component that has been shown to lyse cells and inactivate nucleases –20. The –In terms of RNA quality (RINe) and quantity, the automated extraction methods and MagMAX viral RNA isolation kit  recovered RNA with higher RINe values and higher RNA concentration, respectively, than all the other methods tested in this study. This indicates that magnetic bead-based methods likely recover better-quality RNA than column-based methods, owing to the lack of centrifugation steps through purification columns that can cause nucleic acids to break. Most of the variations in the mean RNA concentration and RINe value observed for the 11 extraction methods can be attributed to the effects of the extraction method used because the matrix effect was controlled for by using the same stool suspension. Although the High Pure viral RNA kit without proteinase K (protocol A) and the Zymo viral RNA kit (protocol B) had the lowest average RINe, indicating low quality RNA, a high proportion of near-whole-genome PV amplicons was observed for RNA extracts recovered with these methods. Moreover, RNA extracts from the Zymo quick RNA minikit (protocol B), High Pure viral RNA minikit with proteinase K (protocol B), QIAamp viral RNA minikit, and MagMAX viral RNA isolation kit that failed to generate a RINe value still showed successful PCR amplification. RINe was found to be statistically significant for only RT-qPCR due to the assay using a much shorter target sequence than other PCR assays performed in this study. This finding suggests that RINe value that reflects total RNA may not necessarily be an indicator of RT-PCR amplification performance.The results demonstrated that the AllPrep PowerFecal DNA/RNA kit was the worst performer for the molecular detection of enteroviruses, including PV, as the lowest proportion of PV-positive results observed in the RT-qPCR, PanEV RT-PCR, and nested VP1 were found in RNA extracted with this method. No viral contamination was observed in the negative controls used during RNA extraction for all 11 extraction methods. Extraction method was found to have a statistically significant effect on the PanPV RT-PCR, PanEV RT-PCR, and nested VP1 PCR assays. All samples (59/59) in this study extracted by a combination of the different extraction methods and tested by different PCR assays were identified as PV positive by one or more of the PCR assays used. Performing only the PV-specific RT-qPCR and nested VP1 PCR assays would have been sufficient to allow PV detection in all the tested samples. This is not surprising because the RT-qPCR and nested VP1 PCR assays have increased sensitivity due to requiring a very short target to amplify and additional amplification cycles, respectively. Interestingly, we observed that if one extraction method did not yield a PV-positive result for a given sample, often another method proved to be successful. This is vital for PV surveillance, as missed or undetected PV in any given stool sample would increase the risk of undetected circulation, hence delaying the global polio endgame. Overall, results showed that the High Pure viral RNA minikit with proteinase K (protocol B), MagMAX viral RNA isolation kit , Zymo quick RNA minikit (protocol B), and QIAamp viral RNA minikit are better suited for PV molecular direct detection using amplicon-based PCR methods. Although the QIAamp viral RNA minikit performed well, using a 560 μL sample input (four times the manufacturer’s recommended starting input) proved to be labor intensive and time-consuming due to multiple loading of the column. Therefore, it would probably not be ideal for processing medium to large sample sets.Direct detection by molecular methods for any pathogen is dependent on efficient recovery of high-quality extracted nucleic acid from the sample. Selecting the right extraction method for the research question at hand that also meets the particular laboratory’s needs requires planning and evaluation. Therefore, the following factors should be considered when selecting an extraction method. (i) Sample type used to recover the nucleic acid should be considered. Different sample matrices require different approaches to lysis and extraction, making certain extraction methods more suitable for a given sample type than others . (ii) TaTC values and the assay cutoff point or the way for determining if a sample is positive or negative should be based on the sensitivity and specificity of the given qPCR assay. These need to be established by testing an appropriate sample panel consisting of negative and positive samples, negative and positive controls, and dilution series of the target. Additionally, a reference method used for comparison should be one that is well established in the literature and widely used for targeting the particular pathogen.The range of While considering performance, simplicity of the procedure, cost factor, and flexibility to use manually or automatically, we currently consider the MagMAX viral RNA isolation kit to be the preferred method for PV detection in stool samples among the 11 methods compared in this study. Future work could investigate the application of the MagMAX viral RNA isolation kit for PV detection in wastewater, where the sample matrix will differ and where the possibility of processing large-volume sample aliquots could maximize direct detection of the low PV copies present in wastewater. For direct detection of PV by DDNS (using the PanEV RT-PCR followed by the nested VP1 PCRs for nanopore sequencing) to be accepted instead of virus isolation in the polio eradication program, the method would need to be noninferior to virus isolation in terms of sensitivity and specificity and be rapid, robust, easy to use, less expensive, and accessible.There are limitations to this study. There were insufficient volumes for some of the stool suspensions; hence, not all samples could be extracted by the 11 extraction methods. Relatively old stool samples were used in this study (over 6 months old). Therefore, although all the samples used in this study had been previously identified as PV positive via virus isolation, some were identified as negative by one or more of the PV detection PCR assays used in this study. This suggests that some of the stool samples had lost their integrity. Harrington et al. found PV direct detection following RNA extraction to be significantly more sensitive than virus isolation by cell culture for PV detection in retrospective stool samples . In thisIn conclusion, this study provides useful information for the selection of RNA extraction methods for stool samples that maximizes their application for PV detection using qPCR or amplicon sequencing, such as the DDNS method . The finIn this study, a sample panel of 59 supernatants from chloroform-treated stool suspensions processed according to the WHO guidelines  that hadGGGTTTGTGTCAGCCTGTAATGA-3′) and Q8 (5′-AAGAGGTCTCTRTTCCACAT-3′) (http://cels.uri.edu/gsc/cndna.html). A substock standard at 107 copies/μL was made from the purified DNA amplicon and used for the preparation of a 10-fold dilution series in the range 2 × 105 to 2 × 101 copies/μL. The dilutions were aliquoted in 0.2-mL tubes for single use to avoid nucleic acid degradation.The RNA from 140 μL of Sabin 1 virus cell culture supernatant was extracted using the QIAamp viral RNA minikit (Qiagen), as per the manufacturer’s instructions. Five microliters of the RNA extract was amplified by a nested RT-PCR, first targeting the entire capsid region using pan-enterovirus primers  followedACAT-3′)  and was The extraction methods included seven manual methods  and two semiautomated extraction methods  on the KingFisher Duo instrument . The 28 The RNA quality  and concentration was determined using RNA ScreenTape and the high-sensitivity RNA ScreenTape assay with 2 μL of extracted RNA performed on the Agilent 2200 or 4200 TapeStation system . According to the high-sensitivity RNA TapeStation technical bulletin, RINe values range from 1 to 10, with 1 being the lowest, and a RINe of over 7 expected as an indicator of intact RNA.For samples processed at ICL, the extracted RNA for each method was subjected to RT-qPCR with the same primer/probe sets as those provided in the poliovirus intratypic differentiation (ITD) diagnostic real-time reverse transcription PCR (rRT-PCR) version 5.0 kit (US CDC) . The reaFor samples processed at NIBSC, the extracted RNA for each method was subjected to RT-qPCR with poliovirus-specific primers and probe from the Poliovirus rRT-PCR ITD version 5.1 kit using the qScript XLT one-step RT-qPCR Quanta tough mix kit  on the Qiagen Rotor Gene Q platform .The qPCR was performed in duplicate on material from single RNA extractions. The samples were run in parallel with the Sabin 1 DNA standards to estimate the viral genome copies present in the samples. A AGAGGCCCACGTGGCGGCTAG-3′) and PCR 3′ (5′-CCGAATTAAAGAAAATTTACCCCTACA-3′)  A nested RT-PCR that amplifies the entire capsid region using pan-enterovirus primers (PanEV RT-PCR), followed by full VP1 amplification using degenerate pan-poliovirus primers (nested VP1 PCR used by the DDNS method), as previously described , 8. BrieTACA-3′) . The PCRTC threshold of 40 for the RT-qPCR) were analyzed using a logistic regression model (estimated using maximum likelihood).The model included sample ID as a random effect on the intercept, and QIAamp viral RNA minikit was used as the reference group, as it showed good overall performance in all assays, and it included all samples that were extracted by the methods tested in this study. To compare the sensitivity of the 11 extraction methods, the proportion of PV-positive samples as identified by each PV direct detection PCR assay was determined along with the 95% confidence intervals. We also compared log viral copy numbers estimated using RT-qPCR using a linear mixed effects regression with sample ID as a random effect. P values of ≤0.05 were considered statistically significant. All analyses were performed in R environment, version 4.1.3.The RT-qPCR and conventional PCR results scored as positive and negative (using a


Small RNAs  are defined as polymeric ribonucleic acid molecules that are less than 200 nucleotides in length and serve a variety of essential functions within cells. Small RNA species include microRNA (miRNA), PIWI-interacting RNA (piRNA), small interfering RNA (siRNA), tRNA-derived small RNA (tsRNA), etc. Current evidence suggest that small RNAs can also have diverse modifications to their nucleotide composition that affect their stability as well as their capacity for nuclear export, and these modifications are relevant to their capacity to drive molecular signaling processes relevant to biogenesis, cell proliferation and differentiation. In this review, we highlight the molecular characteristics and cellular functions of small RNA and their modifications, as well as current techniques for their reliable detection. We also discuss how small RNA modifications may be relevant to the clinical applications for the diagnosis and treatment of human health conditions such as cancer. RNA molecules play essential and diverse roles in numerous biological functions, as studied in organisms ranging from prokaryotes to eukaryotes –3. From N6-methyladenosine (m6A), first discovered in the 1970s , 41, is In 2015, Alarcon and colleagues reported that alterations to levels of the methyltransferase METTL3 affected the expression of mature miRNA as well as unprocessed primary miRNA (pri-miRNA), in addition to its known effect on mRNAs . This reIn addition to its functions with methylation readers, writers and erasers, m6A modification may also facilitate miRNA maturation by promoting Dicer splicing of precursor miRNAs Fig. C. In theGab2) gene [Interestingly, in the absence of changes to its primary transcript, it has been reported that levels of several mature miRNAs are decreased within cells after downregulation of the m6A demethylase known as FTO, and this suggests that m6A can negatively regulate miRNA biogenesis . Consistb2) gene . Contrasb2) gene . Thus, wb2) gene , 68.Furthermore, m6A can be found in mature miRNAs . HoweverTaken together, m6A modification is essential to miRNA processing, and our understanding of the functional impact of m6A modification of miRNAs remains to be better clarified.Another RNA modification is 2′-O-methylation (Nm), which is an abundant and highly conserved modification that replaces hydrogen (-H) atom on the ribose fraction 2′-hydroxyl (-OH) with a methyl group -CH3) . The Nm  . The NmArabidopsis as a methylase for miRNAs and siRNAs [Drosophila [HEN1 knockout or mutation in Arabidopsis leads to elevated levels of heterogeneous 3′-ends and poly-U, both features of which are known to disrupt RNA stability [Drosophila, loss of Pimet (HEN1 homolog) activity leads to deletion of Nm in piRNA and siRNA [HEN1 gene accelerate neurodegeneration and shorten lifespan, suggesting that Nm of small RNAs may affect age-related signaling events within cells [The first identified 2′-O-methyltransferase responsible for Nm modification of small RNAs was the HUA ENHANCER 1 (HEN1) protein, discovered in d siRNAs , 121. Suosophila , 122–127tability , 118, antability , 117 andtability . For exand siRNA . In addiin cells . In zebrin cells . CollectIn mammals, the HEN1 homolog, HENMT1, plays an essential role in fertility. For example, in mice, loss of HENMT1 expression leads to piRNA instability, observed as reductions to piRNA volume and length, as well as developmental arrest of germ cells during the process of spermatogenesis. Particularly, loss of HENMT1 and the associated loss of piRNA collectively lead to defective meiosis and precocious and selective expression of haploid transcripts during meiosis . This fiDrosophila leads to small RNA pathway dysfunction and increased susceptibility to RNA virus infection. This phenomenon is also associated with small RNA-induced gene silencing pathways [In addition to HEN1, other 2′-O-methyltransferases, such as FTSJ1 and FBL, are also crucial for Nm modification on small RNAs and their functions. For instance, studies have shown that the reduction of human FTSJ1 orthologs-mediated tRNA Nm modification in pathways . FTSJ1-mpathways . Yi et apathways . OverallAnother RNA modification is 5-Methylcytidine (m5C) in which the fifth carbon atom (C) of cytosine is methylated within RNAs . The higAll these methyltransferases can also methylate small RNAs. For example, Shobbir Hussain and coworkers found that vault RNAs, a class of small RNAs of approximately 88 to 100 nucleotides in length and transcribed by RNA polymerase III, contain 6 methylated cytosines by NSUN and formed riboprotein granules called Vault with proteins . The autIn addition, m5C modifications on tRNAs affects the generation of tsRNA species. The formation of tsRNAs is reported to be induced in response to stress, since unconventional 5′ initiation sites can be found in the 5′ UTR of stress response transcripts to inhibit canonical translation and favor ribosome assembly . Thus, mIn addition to the modifications mentioned above, various other small RNA modifications have also been reported, including pseudouridine (Ψ) modifications, m7G modifications and m1A modifications, and of which also seem to play important roles in cell biological processes, as described below. One caveat is that the underlying effects of such modifications on the structure and functions of RNAs remain to be better characterized.Pseudouridine (Ψ), an isomeric form of uridine also known as 5-ribosyluracil, was discovered in the 1950s and is described as the most abundant type of post-transcriptional RNA modification discovered in all kingdoms of life –153. PseRecent studies have found that pseudouridine catalyzed by PUS7 could activate tsRNAs that are involved in protein synthesis which are vital to the functions of mammalian stem cells. For example, inhibition of a pseudouridine-driven regulatory network can severely affect hematopoiesis and promote the incidence and pathogenesis of human myeloid malignancies . With reN7-methylguanosine (m7G) is a methylated modification of the seventh nitrogen atom of guanine in RNA, and the modification is documented in tRNAs, rRNAs, mRNAs and miRNAs –170. TheN1-methyladenosine (m1A) modification involves the methylation of the first nitrogen atom of adenine within RNA. This modification is mainly detected in tRNAs, rRNAs, mRNAs and small RNAs, and its presence has been found to influence the structure and functions of these RNA species –174. ParCurrent evidence in the literature has indicated that therapeutic manipulation of small RNA modification may be clinically beneficial in the treatment and prevention of human disease conditions. For example, Wang and colleagues investigated changes in methylation of 22 miRNAs in 57 cases of human neural tube defects (NTDs) and reported that methylation of the microRNA hsa-let-7 g directly effects its expression . Further22G) modifications on tsRNAs and rsRNAs in bladder cancer cells [22G modifications in 15–25 nt RNA from cells within the cerebral cortex of Alzheimer's disease patients were significantly increased, compared with normotypic control samples [In 2006, Seidel and colleagues applied a chromatography method to discover that the modified nucleosides in urine samples have high sensitivity and specificity as potential biomarkers to identify a variety of cancers in patients . Recentler cells . Yan ander cells . Zhang a samples . Konno a samples . Further samples .In addition to their potential value as diagnostic markers, the presence and relative abundance of small RNA modifications may also serve as prognostic biomarkers. For example, in patients diagnosed with glioma, low miRNA-181a-5p expression and cytosine methylation levels were associated with poor survival prognosis , while glioma patients with high levels of unmethylated miRNA-181a-5p were found to have a better survival prognosis (median 16.5 months) . Guzzi aTaken together, these findings suggest that small RNA modifications may be informative as markers that reflect the pathogenesis and progression of human disease.Given the examples of direct effects for small RNA modifications on human diseases such as bladder cancer and armed with the knowledge that evolutionarily conserved mechanisms drive small RNA modifications within cells, researchers are now exploiting these discoveries to design novel RNA-based treatments for human disorders . For exaRecently, as indicated above, Su and colleagues reported that m1A modifications are highly enriched in 22-nucleotide long 3′ tRNA fragments and is dependent on its methylase TRMT6/61A . In bladAs researchers discover the critical roles for small RNAs in physiological and pathological processes, an increasing number of sequencing techniques to detect small RNAs with high sensitivity and specificity have been reported. However, the complex landscape of small RNA modification presents as a challenge for high-throughput analysis of small RNAs because such modifications interfere with the preparation of RNA-seq libraries and can limit their detection. Table Several RNA-seq methods involve sequencing of the cDNA intermediate of RNA, and the conversion of RNA to cDNA can lead to loss of detection of small RNA modification. Table As a recently discovered class of regulatory RNAs, small RNAS  can negatively or positively regulate the expression of tumor markers through different molecular pathways and intracellular signaling mechanisms. These functions for small RNAs depend on their sequence, their three-dimensional structure and their extent of RNA modification. As highlighted in this review, emerging evidence suggests that RNA modifications are essential not only in their capacity to influence the biogenesis and function of small RNAs, but their potential value as biomarkers for human diseased states is also noted . Novel d


Heterotrophic microbes in the Southern Ocean are challenged by the double constraint of low concentrations of organic carbon (C) and iron (Fe). These essential elements are tightly coupled in cellular processes; however, the prokaryotic requirements of C and Fe under varying environmental settings remain poorly studied. Here, we used a combination of metatranscriptomics and metaproteomics to identify prokaryotic membrane transporters for organic substrates and Fe in naturally iron-fertilized and high-nutrient, low-chlorophyll waters of the Southern Ocean during spring and late summer.Nitrincolaceae, and groups with a more restricted repertoire of expressed transporters, such as Alphaproteobacteria and Flavobacteriaceae.Pronounced differences in membrane transporter profiles between seasons were observed at both sites, both at the transcript and protein level. When specific compound classes were considered, the two approaches revealed different patterns. At the transcript level, seasonal patterns were only observed for subsets of genes belonging to each transporter category. At the protein level, membrane transporters of organic compounds were relatively more abundant in spring as compared to summer, while the opposite pattern was observed for Fe transporters. These observations suggest an enhanced requirement for organic C in early spring and for Fe in late summer. Mapping transcripts and proteins to 50 metagenomic-assembled genomes revealed distinct taxon-specific seasonal differences pointing to potentially opportunistic clades, such as Pseudomonadales and The combined investigations of C and Fe membrane transporters suggest seasonal changes in the microbial requirements of these elements under different productivity regimes. The taxon-specific acquisition strategies of different forms of C and Fe illustrate how diverse microbes could shape transcript and protein expression profiles at the community level at different seasons. Our results on the C- and Fe-related metabolic capabilities of microbial taxa provide new insights into their potential role in the cycling of C and Fe under varying nutrient regimes in the Southern Ocean.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01600-3. Heterotrophic prokaryotes consume roughly half of primary production and thereby influence the flux of carbon through the marine food web. The transformation of phytoplankton-derived organic matter by prokaryotes shapes the amount and quality of dissolved organic matter (DOM) that can accumulate in surface waters on a seasonal time scale and eventually be exported to depth via overturning circulation –3.The concentration and composition of a variety of individual compounds that make up the pool of DOM determine its overall bioavailability and thus the quantity of carbon (C) that can be transformed over different time scales . This prMarine DOM is composed of diverse substrates of varying bioavailability. The complexity of organic compounds contained in DOM has become more accessible through advances in analytical methods , 7. ConcThe objective of the present study was to identify seasonal differences in compound-specific transporters for C and Fe of microbial communities at contrasting sites in the Southern Ocean. Our study was carried out in the Kerguelen region where natural Fe fertilization leads to annually recurring phytoplankton blooms in otherwise high-nutrient-low-chlorophyll (HNLC) waters . These bSurface seawater samples 10 m) were collected during the Southern Ocean and Climate (SOCLIM) cruise in early spring (ES)  and during the Marine Ecosystem Biodiversity and Dynamics of Carbon around Kerguelen (MOBYDICK) cruise in late summer (LS) . Two stations were chosen for the present study: one station was located in high-nutrient low-chlorophyll (HNLC) waters , and one station was located in naturally iron-fertilized waters above the central plateau of Kerguelen  , are described elsewhere  supplem, 3 and 4n = 12) were sequenced on one lane of HiSeq 4000 with 150-bp paired-end reads yielding between 26 and 36 million reads per metatranscriptome . Additionally, ALDEx2 [For RNA extractions, 10-L seawater were pre-filtered through 0.8-µm membrane filters , and cells were collected on 0.2-µm membranes  using a 142-mm filtration system (Geotech Equipment Inc.) and a peristaltic pump. RNA was extracted using the NucleoSpin® RNA Midi kit  was divided by the protein length (L) and then divided by the sum of PSM/L for all N proteins.For protein extractions, 20-L seawater were pre-filtered through 0.8-µm filter membranes , and cells were collected on 0.2-µm filter membranes  using a 142-mm filtration system (Geotech Equipment Inc.) and a peristaltic pump. Filtrations were performed in duplicate for both seasons and frozen immediately at −80 °C. Whole protein extractions from filters were performed using a modified protocol from Bayer et al. , and extLC–MS/MS . The norhttp://www.cazy.org/) and Fe-specific transport  were ≈ 5-fold higher at the on-plateau station A3 (1.54 μg L−1) as compared to station KERFIX (0.32 μg L−1) in HNLC waters in early spring (Table −1 at A3 and ≈ 0.17 μg L−1 at KERFIX). An opposite seasonal pattern was observed for prokaryotic abundances at both sites with about 2-fold higher cell abundances in late summer (8.94 × 108 cells L−1 at A3 and 5.71 × 108 cells L−1 at KERFIX) than in early spring (4.27 × 108 cells L−1 at A3 and 2.89 × 108 cells L−1 at KERFIX) [−1) as compared to spring (52 ± 1 µmol L−1). Despite the continuous input of Fe, dissolved Fe concentrations were not substantially different between sites, an observation that can be explained by the rapid biological utilization of Fe in surface waters [Natural fertilization supplies waters above the Kerguelen plateau continuously with low quantities of Fe, thereby stimulating seasonal primary production and associated food web processes within otherwise HNLC waters . For the KERFIX) , 27. Aboe waters . On the e waters  than in ion A3 1. μg L−1 aA total of 144 and 156 transporter transcripts and proteins were identified by metatranscriptomics (MT) and metaproteomics (MP), respectively, of which 98 transporters were shared between the two datasets. The proportion of transporters made up between 3.5 and 12.3% of the total normalized transcript counts in the metatranscriptomes, whereas their proportion in the metaproteomes ranged from 42.8 to 96.8%   Fig. .Fig. 1NoHowever, most protein families  contained transcripts that either increased or decreased in relative abundance. As a consequence, no seasonal trend in Fe-related transport proteins was observed in the MT data , glycine betaine/proline (3.6 to 1.1%) and branched-chain amino acids (2.7 to 1.1%).Transporters for taurine, urea, fatty acids and nitrate/sulfonate/bicarbonate were low in relative abundance (< 2%) during both seasons at both sites Fig. A. TranspThe marked seasonal pattern in the proportion of transporters of organic substrates and Fe in the metaproteomes could reflect a switch in prokaryotic requirements of these elements between early spring and late summer. This temporal shift was observed at both sites despite the differences in the seasonal productivity regimes between fertilized and HNLC waters  were constructed, and gene sequences encoding for transporter proteins recovered from metatranscriptome and metaproteome datasets were mapped to metagenomic bins  had higher relative abundances in early spring. The role of these taxa in the expression of transporters for Fe (MAGs 103 and 62) and organic C substrates  in spring and summer was confirmed in the metaproteome dataset  and A3 (527m). B. Climatology of Chlorophyll a over 10 years at station A3 and C. station KERFIX. The green line indicates the 10-year mean; the blue line indicates the year 2018 (MOBYDICK cruise). Note different scales on y-axis.Additional file 2: Supplementary Figure 2. Bioinformatic pipeline for the analysis of all three ‘omics’ levels. A. Metagenomic assembly  and binning B. Construction of Ocean protein database from metagenomic assemblies C. Metatranscriptomic mapping to metagenomes after extraction of mRNA with SortMeRNA D. Metaproteomic analysis with metagenomic database and annotation in eggNOG and KEGG.Additional file 3: Supplementary Figure 3. Relative proportions of transporter families. Bar-plot showing the relative proportions of different transporter families according to the transporter database (TBDB) for A. Metaproteomes from NSAF and B. Metatranscriptomes based on the total normalized transcripts. All duplicates and triplicates are shown, note difference in scale. ES -early spring, LS –late summer, Transporter types are indicated by different colors: ABC – ATP-binding cassette transporter complex, AMT/NPP – Ammonium Channel Transporters/Nitrate, Nitrate Porters, CUT – Carbohydrate Uptake Transporters, OMP – Outer Membrane Proteins, OMR – Outer Membrane Receptors, SSS – Solute Sodium Symporters, TRAP-T - Tripartite ATP-independent periplasmic transporters, TTT – Tripartite Tricarboxylate Transporters.Additional file 4: Supplementary Figure 4. Compound-specific transporter transcripts. ES: Early spring; LS: Late summer. Relative proportion of compound-specific transporter transcripts of tranporters present in metatranscriptomes  from MAG contigs. Supplementary Table 12. Results for Biosynthetic gene clusters from MAG contigs - siderophore related. Supplementary Table 13. DESeq2 results for spring vs. summer for iron-related proteins. Supplementary Table 14. Aldex2 results for spring vs. summer comparison.