Toxoplasma gondii infection. We have previously demonstrated that the recombinant T. gondii nucleoside triphosphate hydrolase-II (NTPase-II) protein is able to provide protective Th1 cell-mediated immunity against T. gondii. Herein, we evaluated the immunogenic potential of a self-amplifying RNA vaccine-encoding T. gondii NTPase-II gene, RREP-NTPase-II, delivered by a synthetic lipid nanoparticle (LNP). Immunization of mice with naked RREP-NTPase-II induced a strong cellular and humoral immune response with high-IgG antibody titers and IFN-γ production. The immunized mice displayed significantly prolonged survival time and reduction in brain parasite load (46.4%) compared with control group. Furthermore, mice vaccinated with RREP-NTPase-II-encapsulated LNP displayed significantly enhanced protection against acute infection as well as chronic infection with PRU cyst, which shows 62.1% reduction in brain cyst burden in comparison to control group. These results suggest that the combination of self-amplifying RNA and LNP ion would be beneficial to the development of a safe and long-acting vaccine against toxoplasmosis.RNA-based vaccine represents an irresistible and safe immunization strategy with decreasing theoretical risks of genomic integration and malignant cell transformation. To our knowledge, however, there is no report about development of RNA vaccine against  Toxoplasma gondii, the pathogen of toxoplasmosis, is an obligate intracellular parasitic protozoan, which has a wide range of hosts including humans and warm-blooded animals  genome has been utilized to improve the weak immune responses induced by mRNA vaccines . The skein vitro . Moreove(in vivo .Toxoplasma gondii nucleoside triphosphate hydrolase (NTPase), accounting for 2–8% of the total protein of tachyzoites, has a potent apyrase activity and is released from dense granules into parasitophorous vacuole for successively degrading ATP to ADP and finally AMP  were purchased from Laboratory Animal Center, Wenzhou Medical University, Zhejiang, China. They were bred and handled in strict accordance with the Good Animal Practice requirements of the Animal ethics Procedures and Guidelines of China. This research was approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University (Permit number: wydw2015-0073). Humane endpoints are considered to avoid the mice pain or suffering via euthanasia. Mice were monitored daily for signs of toxoplasmosis, such as fatigue, difficulty in feeding and severe ascites. Mice shown above signs would be sacrificed immediately with COThe cyst-forming type II strain PRU was maintained in female ICR mice. Forty-five days after infection, cysts were harvested from infected mouse brains for challenge of immunized mice. The virulent type I strain RH was routinely propagated in human foreskin fibroblast (HFF) as previously described  and emplXma I and Spe I site of pRREP-eGFP  and stored at -20°C. Prior to immunization, the plasmids were linearized and RNAs in vitro transcription were made by using MEGAScriptTM Kit  as described previously .The DNA sequence of NTPase-II gene was amplified from the construct pBAD-HisB-NTPase-II  using a REP-eGFP  to swap eviously . Finally® 2000 Reagent . Twenty-four hours after transfection, expression of NTPase-II was confirmed by indirect immunofluorescence assay (IFA) with the mouse monoclonal antibody anti-NTPase-II (1:2000 dilution) that has been raised in our laboratory , cholesterol (Sigma–Aldrich), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]  (PEG DMG 2000) , and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) synthesized as reported previously  according to the manufacturer’s instructions. Briefly, RNA/LNP samples were diluted in TE buffer prior to addition of the dye, and the RNA concentrations outside the LNP were determined by detecting the fluorescence with a microplate reader . Afterward, the total RNA concentrations, including the RNA outside and inside LNPs, were obtained after the lipid membranes were breached using Triton X-100. To evaluate RNase protection of the encapsulated RNA, the RNA/LNP samples were treated with RNase A  for 30 min. After RNase inactivation with proteinase K , RNA was extracted and denaturing agarose gel electrophoresis was performed.Ribonucleic acid/lipid nanoparticle formulations were characterized for encapsulation efficiency and the ability to protect the encapsulated RNA from RNase as previously described . To deteOne hundred 6- to 8-week-old female BALB/c mice were divided into 5 groups with 20 mice per group. They were injected intramuscularly into bilateral quadriceps with 10 μg of vaccines diluted in PBS on day 0 and 21. Mice that received PBS were used as non-immunized control. For the determination of specific antibodies, serum samples from all groups were obtained 14 days after the last immunization. For the analysis of cellular immune responses, 5 spleen samples from each group were aseptically collected on day 35.2SO4 after 10 min, and the optical density at 450 nm was detected with a microplate reader. Endpoint titers were calculated or at the dilution when the absorbance fell below 0.2.Specific anti-NTPase-II IgG antibody levels were measured by ELISA according to our previous description  with min5 cells/well) in presence of 10 μg/ml recombinant NTPase-II proteins or in plain complete medium as negative control or 5 μg/ml ConA as positive control. Plates were incubated for 72 or 96 h at 37°C and 5% CO2.Two weeks after the last booster immunization, five mice per group were sacrificed to obtain single splenocyte suspensions as described previously . After e450 value from recombinant NTPase-II-stimulated cultures/the average OD450 value from cells with medium cultures.For lymphocyte proliferation assay, the stimulated splenocytes were cultured for 72 h, and the spleen lymphocyte proliferative activity was detected with CCK-8 reagent  in accordance to the manufacturer’s instruction. The absorbance at 450 nm was read, and then, the stimulation index (SI) was calculated as the ratio of the average OD® , their IFN-γ concentrations were determined by reference to a standard curve constructed with known amounts of mouse IFN-γ and the sensitivity limit was 15 pg/ml.For IFN-γ analysis, cell-free supernatants from the stimulated splenocytes were collected and the IFN-γ activity was analyzed at 96 h after stimulation. According to the manufacturer’s instructions of Mouse IFN-γ ELISA Ready-SET-Go!3 tachyzoites of RH strain and observed for an additional 30 days. The survival time and the percentages of mice survived were recorded daily.Two weeks after the last booster immunization, five mice per group were infected intraperitoneally with 10In addition, 10 mice of each group were orally challenged with 20 tissue cysts of PRU strain on day 35. Mortality was observed daily for 45 days, and the surviving mice were sacrificed for quantification of cysts in brain as described previously .p < 0.05.Statistical analysis was carried out with one-way ANOVA and multiple comparison procedures using SPSS 13.0 software for each assay, and Kaplan–Meier test for survival analysis. Data were represented as the mean ± SEM. Differences were considered statistically significant when in vitro, transfection of self-amplifying RNA into 293T cells was performed at once post-transcription and capping. At 24 h post-transfection, expression of the NTPase-II protein was verified by IFA with specific monoclonal antibody. The fluorescence was observed in the cells transfected with self-amplifying RNA, whereas no fluorescent signal was observed in the negative control , demonstrating that NTPase-II protein could be expressed in mammalian cells. The identity of the expressed NTPase-II protein was further confirmed by Western blot. A single protein band was detected at the size of about 70 kDa corresponding to the predicted size of NTPase-II protein .Plasmid pRREP-NTPase-II as templates for transcription of RNA was constructed successfully by verification with sequencing (data not shown). For the assessment of protein expression Figure 1D). The result showed that the encapsulated RNA was maintained, while the naked RNA was degraded by RNase A, demonstrating that the LNP has the ability to protect the encapsulated RNA from degradation.After RNA was encapsulated in LNP, characteristics of RREP-NTPase-II/LNP formulations were analyzed. For the assessment of encapsulation efficiency, the concentrations of both free RNA and total RNA in solution post-encapsulation were measured. The result showed that ∼80% of the RNA was encapsulated. In addition, the integrity of encapsulated RNA was validated by denaturing agarose gel electrophoresis after treatment with RNase A  in contrast to three control groups, PBS (0.495 ± 0.040), RREP (0.60 ± 0.07), and RREP/LNP . In an additional experiment, the distribution of both IgG1 and IgG2a was also analyzed . The levels of both IgG subtypes were significantly increased in mice immunized with RREP-NTPase-II (p < 0.001). Noteworthy, RREP-NTPase-II vaccine elicited slightly higher specific IgG2a (2.625 ± 0.174) levels when compared to IgG1 (1.59 ± 0.019), consistent with a Th1 response.In order to evaluate if the immunization with self-amplifying RNA vaccine encoding NTPase-II antigen induced specific humoral immune response, the antibody titers in serum samples from immunized mice were detected by indirect ELISA 2 weeks after last vaccination. Results were presented as the mean of logp < 0.001) as well as both IgG subtypes titers, IgG1 (3.095 ± 0.106) and IgG2a (3.970 ± 0.094), than naked RNA (RREP-NTPase-II). Similarly, a Th1 response was elicited in immunized mice. These results indicate that the presence of LNP enhances the specific humoral immune response.Furthermore, to investigate if LNP improved the humoral immune responses, mice were immunized with 10 μg of encapsulated RNA (RREP-NTPase-II/LNP). The encapsulated RNA elicited significantly higher total IgG  and RREP-NTPase-II/LNP  elicited a significantly higher SI values to the NTPase-II antigen stimulation. On the contrary, administration with PBS (1.098 ± 0.100), RREP (1.240 ± 0.129), or RREP/LNP (1.482 ± 0.143) induced little lymphocyte proliferation to NTPase-II stimulation .Two weeks after last vaccination, splenocytes were harvested from immunized mice and an p < 0.001). For RREP-NTPase-II/LNP (451.34 ± 16.26), the positive effect was even more pronounced and increased twofold than those from RREP-NTPase-II (p < 0.01). In contrast, neither the PBS control group (18.05 ± 1.98) nor the groups that immunized with RREP (16.59 ± 2.75) or RREP/LNP (23.64 ± 2.29) developed any detectable amount of IFN-γ . These results suggest that RNA/LNP was able to elicit the antigen-specific IFN-γ-mediated responses.To further determine if immunization with self-amplifying RNA vaccine induced a cellular immune response, the concentration of IFN-γ in supernatants of splenocytes stimulated with NTPase-II antigen was measured. Upon stimulation, RREP-NTPase-II (214.10 ± 46.01) produced significantly higher levels of IFN-γ . All mice immunized with PBS, RREP, or RREP/LNP died within 9 days after challenge. However, a significantly prolonged survival time was observed in mice immunized with both RREP-NTPase-II and RREP-NTPase-II/LNP (p < 0.01). Especially in the RREP-NTPase-II/LNP group, one mouse still survived after 30 days of challenge, which showed the highest survival rate and was significantly higher than that of the RREP-NTPase-II group (p < 0.01).To evaluate if self-amplifying RNA vaccine could confer effective protection against T. gondii chronic infection was also evaluated. The immunized mice were challenged with 20 PRU strain cysts 2 weeks after last immunization . Forty-five days after the challenge, direct cyst counting was performed by optical microscopy in the brains of survived mice. Similar to the results of acute challenge, the number of brain cysts showed a significant reduction in both RREP-NTPase-II by 46.4%  and RREP-NTPase-II/LNP by 62.1%  compared with PBS (2370.17 ± 310.36), RREP (2245.86 ± 319.89), or RREP/LNP (2231.67 ± 306.02). Therefore, immunization with self-amplifying NTPase-II RNA encapsulated with LNP enhanced a substantial resistance to the acute and chronic challenges of T. gondii.Furthermore, the resistance to T. gondii challenge. Significant information obtained demonstrates that a potent immune response could be induced by self-amplifying RNA, and this response can be further enhanced by LNP delivery system.This study applies a RNA/LNP vaccination strategy, explored for inducing protective immunity against viral infection, to induce effective immune responses against Currently, there is a popular view that the nuclear membrane of transfected cells is attributed to the major barrier for restriction of delivering DNA vaccine into nuclear and consequently reduction of the expression level of AOI . Particuin vitro transcription were completed using standard molecular cloning techniques. The self-amplifying RNA was then transcribed in vitro using a commercial kit, which can produce up to milligram quantities of RNA. Compared with other mRNA vaccine, the production of the self-amplifying RNA not only eliminates the potential risks that produce infectious virus via recombination, but circumvents the immunity against viral vectors because the viral replicon is intrinsically able to induce apoptosis of the transfected cell, resulting in transient and self-eliminating expression of the self-amplifying RNA vaccine (In addition, in our study, the production and amplification of DNA templates for  vaccine . ImportaT. gondii in a good deal of studies. However, to our knowledge, the LNP delivery system is not so far utilized as an adjuvant in the development of vaccines against T. gondii, even though it has been developed to encapsulate self-amplifying mRNA for eliciting specific immune responses against antigens from HIV (Currently, many kinds of adjuvants have been applied and reported to enhance immune responses and protection against from HIV  or influfrom HIV . In thesT. gondii (Toxoplasma in vivo (Toxoplasma by induction of lymphocyte proliferation, high IFN-γ production, and activation of CD8+ T cells, the major T cell subset associated with acquired immune protection against T. gondii (Toxoplasma NTPase-II protein in mice immunized with naked self-amplifying RNA. More importantly, the enhanced lymphocyte proliferation response and higher IFN-γ production were promoted by LNP encapsulation. These findings indicate that immunization with naked self-amplifying RNA or LNP-encapsulated RNA indeed induces the IFN-γ-related Th1-type immunity, requiring for prevention of Toxoplasma infection, although more experiments should be designed to further evaluate the Th1-type immunity. Nevertheless, it is worth noticing that the immunity induced by RNA/LNP elicits a considerably higher protection rate than the naked self-amplifying RNA in experimental mice against Toxoplasma RH strain, significantly prolonging their surviving time up to 30 days after challenge. As a result, the higher survival rate and prolonged survival days further support that LNP encapsulation is better in inducing strong NTPase-II-specific IFN-γ production to sustain a protective immune response against T. gondii.As an obligate intracellular parasitic protozoan, Th1 cell-mediated immunity is considered to play a critical role in protective immunity to . gondii , 2010. I in vivo . Our pre in vivo  have dem. gondii . TherefoToxoplasma NTPase-II could be a potential vaccine candidate (In addition, one advantage of self-amplifying RNA is the ability to elicit a significantly cellular memory immune response . Althougandidate .Toxoplasma infection, a widespread theory is that the specific IgG antibodies play a partial role in the resistance to T. gondii during secondary infection (T. gondii infection. More importantly, the IgG titers after vaccination with the RNA/LNP were superior to those induced by vaccination of naked RNA, which is consistent with previous findings that LNP encapsulation promotes a stronger Th1 cell response (Although now it is not entirely clear to what extent the function of IgG antibodies is in anti-nfection . In thisresponse .T. gondii NTPase-II RNA with LNP to produce RNA/LNP that can prevent the RNA degradation from RNase A. The naked self-amplifying RNA is capable to induce the protective NTPase-II-specific humoral and cellular immune responses to protect mice from T. gondii challenge. Additionally, RNA/LNP administered in the mouse enhances further the protective immunity. Noteworthily, although the partial protection was provided by this vaccine candidate, other effective immunization strategies and multi-antigens constructs should be included to produce better protective immunity in the future. In any case, the capability of the combination of self-amplifying RNA and LNP encapsulation to successfully elicit and extend protective immune response would be beneficial to the development of a safe and long-acting vaccine against T. gondii for future use in animals and humans.In summary, we have successfully encapsulated self-amplifying FT conceived and designed the study. LZ and YH carried out plasmid construction and RNA synthesis. ZX prepared the lipid nanoparticle and encapsulated self-amplifying RNA. FL, SL, and YW performed immunization of mice and evaluation of protective immunity. FL and LZ analyzed the data and drafted the manuscript. XH and FT critically revised the manuscript. All authors read and approved the final manuscript.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.


PAGs) in the spermatozoa suggest a possible influence of sperm transcripts beyond early embryonic development. The specific regions  of the particular spermatozoal transcripts might help regulate fertilization. This study demonstrates that the use of two different RNA-seq platforms provides a comprehensive profile of bovine spermatozoal RNA. Spermatozoal RNA profiling may be useful as a non-invasive method to delineate possible causes of male infertility and to predict fertility in a manner that is more effective than the conventional methods.Mammalian spermatozoa deliver various classes of RNAs to the oocyte during fertilization, and many of them may regulate fertility. The objective of the present study was to determine the composition and abundance of spermatozoal transcripts in fresh bull semen. The entire transcriptome of the spermatozoa from bulls (n = 3) was sequenced using two different platforms (Ion Proton and Illumina) to identify the maximum number of genes present in the spermatozoa. The bovine spermatozoa contained transcripts for 13,833 genes . Both intact and fragmented transcripts were found. These spermatozoal transcripts were associated with various stages of spermatogenesis, spermatozoal function, fertilization, and embryo development. The presence of intact transcripts of pregnancy-associated glycoproteins ( The mammalian spermatozoon transfers coding as well as non-coding transcripts to the oocyte during fertilization465The spermatozoa contain both intact and biologically degraded RNAs in such low quantities that conventional techniques such as qPCR10Whole-transcriptome sequencing has been employed for assessing male fertility in humans5The spermatozoon contained 20–30 fg of RNA and these RNAs were fragmented, with a peak fragmentation size of 75 bp. RNA from the testis had intact 18 S and 28 S ribosomal RNAs (rRNAs), whereas the spermatozoa lacked any of rRNA peaks in the Bioanalyzer profile . The total RNA isolated from the spermatozoa was ensured to be free of gDNA and other RNA contaminants from somatic cells, leucocytes and germ cells. Testicular RNA and spermatozoal gDNA were used as positive controls .The isolated RNA was subjected to two different library preparation procedures. A single-end RNA sequencing library without size selection was prepared for Ion Proton  analysis, and a paired-end RNA sequencing library with size selection was prepared for Illumina platform  analysis. The constructed libraries were subjected to sequencing on the respective platforms and generated approximately 20 million reads/sample. The 95% of the reads generated from the Ion Proton and Illumina platform were between 10–130 bp , and 74–The spermatozoa contained 12,307 and 12,470 genes (>10 TPM) based on the Ion Proton and Illumina platform analyses, respectively , and of PRM1, CHMP5 and YWHAZ were among the most abundant , which are essential for sperm nuclear formation. The spermatozoa also contained transcripts associated with cytoskeletal organization . Transcripts associated with biological processes such as the regulation of amide (p = 2.35E-06) and peptide (p = 3.69E-06) biosynthesis, translation (p = 4.61E-06), energy homeostasis and the generation of precursor metabolites , motility  and acrosomal reactions  were significantly enriched in the spermatozoa.The abundant (TPM > 100) spermatozoal transcripts were significantly associated with germ cell development (p = 4.04E-03)  A. The tPRSS37, TPM = 137), the binding of spermatozoa to the zona pellucida  and egg activation  , whereas the less abundant transcripts are associated with ion transporter activity (The spermatozoa contained transcripts (p = 2.2E-02) associated with reproductive processes (p = 3.87E-0.02) such as sperm-egg recognition (M = 114) . In addiactivity , and theactivity .THSD4, PAG5, PAG7 and PAG10, were abundantly transcribed in spermatozoa, suggesting that these spermatozoal transcripts might play a role in implantation and placental development  with previously reported human spermatozoal transcriptsGAPDHS and ENO1), capacitation , and events associated with fertilization   . The maj SPADH2) .Existing spermiogram tests are insufficient to accurately assess male fertility; thus, research is being focused on identifying novel molecular markers that can be incorporated into the existing tests for the best possible accuracy. In this regard, spermatozoal transcriptomes have been investigated to assess male fertilityRPL23, RPL27A and RPS18) or degraded  and might be packaged into the cells during spermatogenesis12Each bovine spermatozoon contained 20–30 fg of fragmented RNA, with intact or biologically degraded ribosomal RNA21Also, because the library preparation methods and sequencing platforms used influence gene profiles and expression levelsBull spermatozoa contained transcripts for 13,838 genes, a number that is comparable with the findings of earlier reports in bovines121412YWHAZ, observed in the present study has been reported to regulate spermatogenesis and acrosomal reactionsYWHAZ gene-knockout males have been found to be infertileKIF5C and KCNJ6, found to be abundant in spermatozoa are involved in microtubule function. Such transcripts are also abundant in the cerebral cortex27The majority of the abundant spermatozoal transcripts were intact, and these transcripts might play a role in sperm function, fertilization events and early zygote development upon their transfer to the oocytes11314miR-365-2, is known to have BCL2 as one of its targetsBCL2 is essential for the progression of a fertilized oocyte into the cleavage stage; therefore sperm-derived factors are thought to be essential for the development of preimplantation embryosApart from these findings, studies have suggested that sperm cells provide noncoding RNA (ncRNA)589TNP1, RAD21, UBE2B and RAN transcripts were highly abundant (TPM > 100) in spermatozoa and are involved in the crucial stage of spermatid development, wherein high-order chromosomal organization takes place. RAD21 is expressed in spermatogonia3637RAD21 had been found to have gonadal atrophy and infertilityRAN, in association with calcineurin, regulates spermatogenic meiosisRNABP1) might result in male infertilitySpermatozoal transcripts are significantly over represented in spermatids, indicating that many of the spermatozoal transcripts are related to sperm function12TEKT1 is important for spermatozoal cytoskeleton development, and this protein is localized in the apical region of the acrosome and on flagella and could play a key role during fertilization44MAP7, PTK2, PLK1S1, MYH9 and PRKCZ) involved in centrosome organization were observed in the ejaculated spermatozoa, suggesting that the presence of these transcripts might reflect events associated with spermatogenesis and sperm function. The presence of motility-associated transcripts, such as CATSPER and SPAG, indicate the fertility of the male47SPAG genes are also essential for the structural integrity of spermatozoaThe presence of abundant nuclear-encoded transcripts/proteins that are essential for the condensation of nuclear material in spermatozoa  was observed in the study. These transcripts help determine the ability of spermatozoa to fertilize the egg, and any alterations in the expressions of these nuclear-encoded transcripts might lead to male infertilityPLCZ1 and PLCB1 are involved in calcium signaling and the spermatozoon-induced activation of an oocyte leading to its final maturation49PLCZ1 interacts with phosphatidylinositol and thus activates the process of oocyte maturation via Ca2+oscillations51Genes such as ADAM1B, ADAM2 and ADAM32) along with the corresponding proteins. These proteins regulate sperm motility in the reproductive tract and sperm-zona pellucida binding and penetration5253COX5 A and COXI1 transcripts, which are associated with mitochondrial function, were observed in the spermatozoa along with their respective proteins. The altered expression of these genes might cause mitochondrial dysfunction and impaired fertilitySpermatozoa carry fertilin transcripts  in spermatozoa. The expression of BCL2L11 by cumulus cells can be used to assess the status of the embryo quality in humansThis study confirms the existence of embryogenesis-associated transcripts, such as PAG5, PAG7 and PAG10, were observed to be full length and intact in spermatozoa, suggesting that sperm-borne transcripts have an influence beyond early embryonic development, such as during the processes of implantation and placentation.Studies conducted so far suggest that the spermatozoal transcripts have a role in fertilization and early embryonic development. However, the placental abnormalities reported from somatic cell nuclear transfer studies60YBX2 regulates male fertilityYBX1 is essential for survival of embryosThe functional annotation of the common transcripts observed in animals revealed that these transcripts are involved in metabolism, motility and translation regulation. In mouse, cold shock protein with nucleic acids binding properties, Enriched spermatozoal transcripts and protein pathways associated with axon guidance and oocyte meiosis were identified, further suggesting that these transcripts might have a potential role in sperm function and fertilization. Similarly, the spermatozoal proteins produced from their respective transcripts might play a role in sperm metabolism, oocyte meiosis and fertilization. The occurrence of functional transcripts in the spermatozoa indicate their significant role in regulating spermatogenesis and embryogenesis.Studies have confirmed the presence of many RNAs in mature spermatozoa from different species31415124Bull spermatozoa contain many functional genes, and several of these genes have functions associated with different stages of spermatogenesis, spermatozoa kinetics, fertilization and embryonic development. As the mRNAs are short lived, many of the proposed functions of the spermatozoal transcripts beyond early embryonic development are speculative and need to be proved experimentally. The findings from the present study provide two different insights. The first is that spermatozoal carry remnant mRNAs along with the corresponding proteins, which are retrospectively indicative of successful spermatogenic events. Second, spermatozoal contain the transcripts that might play vital roles in fertilization and embryogenesis. Spermatozoa transcriptome profiling could be a non-invasive approach for the identification of transcripts that are indicative of sperm fertility.Semen samples from bulls (n = 3) that were known to be fertile were obtained from Nandini Spermatozoa Station . All experiments were performed in accordance with the relevant guidelines and regulations. The experimental protocols were approved by the Institute Research Committee, ICAR-NIANP, Bengaluru and Task Force , Department of Biotechnology, Government of India. From each bull, four ejaculates were pooled to minimize transcript expression profile biases. The pooled semen samples of individual bulls were subjected to RNA isolation and to whole-transcriptome sequencing using Ion Proton and Illumina (NextSeq-500) platforms.g at room temperature (28 °C) for 20 min. After centrifugation, the gradient solutions were removed without disturbing the spermatozoal pellet. The spermatozoal pellet was re-suspended in 10 mL of PBS and centrifuged at 2500 g for 5 min at 4 °C. It was then re-suspended in 1 mL of PBS, and spermatozoon concentrations were measured using a hemocytometer.The semen samples were purified using a 50% Bovipure gradient solution  in phosphate-buffered saline . Then, 1 ml of each semen sample was slowly layered on top of 4 mL of 50% Bovipure solution in 15 mL conical-bottom centrifuge tubes. The samples were centrifuged at 200 6 cells) were homogenized and lysed with a lysis buffer cocktail (0.5 mL of lysis buffer from the kit + 0.5 mL of TRIzol), followed by phase separation with 0.2 mL of chloroform. The remaining protocol was performed following the manufacturer’s instructions . The isolated RNA was treated with DNase  to eliminate traces of gDNA. Total RNA was quantified using a fluorometer  with an RNA high-sensitivity assay kit , and RNA quality was analyzed using a spectrophotometer to check for contamination from proteins and organic solvents. The Bioanalyzer system was used to assess the fragment size distribution of spermatozoal RNA. After quality and quantity analyses, total RNA samples were converted into cDNA using Superscript II . qPCR experiments were carried out to check for contamination with an input RNA concentration of 10 ng/reaction using cell-specific primers for RNA from other cell types and intron-spanning primers to detect spermatozoal gDNA contamination  according to the manufacturer’s instructions, with slight modifications. During the library preparation, the fragmentation step was omitted due to the fragmented nature of spermatozoal RNA. Spermatozoal RNA was not enriched, and size selection was avoided. The template was prepared for sequencing using an Ion Express Template Kit . The constructed libraries were subjected to sequencing using the Ion Proton platform , generating 20 million reads/sample.Total RNA was converted to double-stranded cDNA (ds cDNA) using a SMARTer Ultra-Low Input RNA Kit for the Illumina platform  followed by covaris shearing and end-repair of the overhangs resulting from the shearing. The paired-end RNA sequencing library was prepared using an Illumina TruSeq Total RNA Library Preparation Kit as described in the manufacturer’s protocol. The libraries were quantified using a DNA high-sensitivity assay kit and were sequenced using 2 × 75 paired-end (PE) chemistry using the NextSeq-500 platform, and approximately 20 million PE reads were generated for each sample.The initial quality of the raw reads was analyzed using FastQC . After analyzing read quality , the adapter sequence, primers and low quality reads were trimmed to improve read quality and to reduce transcript expression profile biases. The reads (before and after trimming) were mapped to the bovine genome (Btau_4.6.1/Btau7) using the CLC Genomics Workbench . Transcript expression levels were calculated based on TPM.To determine the number of genes present in bull spermatozoa, the transcripts from both platforms were combined based on TPM. The highly abundant transcripts expressed in the spermatozoa were identified based on average TPM along with unique gene reads (>50 reads/transcript). The unique gene read parameter was included to make the selection process more stringent to ensure that the true abundance of the transcripts was identified. To assess the possible functions of spermatozoal RNAs, the common transcripts obtained from both RNA-seq platforms with TPM > 10 and >100 underwent gene ontology and functional annotation analyses.The transcripts were selected based on the RNA-seq data analysis to validate the sequence data  of spermatozoal RNA. The primers were designed for the selected transcripts  and vali4HCO3 buffer. Protein concentrations were determined using a BCA assay , and the pH of samples was adjusted to 8.5. For in-solution digestions, 20 μg of protein was dissolved in 20% ACN in a 100 mM NH4HCO3 buffer, followed by the reduction of the cysteine residues in the sample with the addition of 10 mM dithiothreitol , and samples were then incubated at 56 °C for 30 min. Then, the samples were alkylated in the presence of 56 mM iodoacetamide  and incubated in the dark for 30 min. Ice-cold trypsin (13 ng/μL) was added to the samples at a 1:20 ratio and mixed well. The tubes were incubated at 37 °C for 16 hours, and the tubes were then chilled on ice and very briefly centrifuged. Formic acid (5%) was then added to the tubes to stop the reaction, and the pH was adjusted to ~3.0. The digested peptides were vacuum dried, reconstituted in 15 μL of 2% ACN in 0.1% formic acid, and incubated with occasional vortexing for 30 min. The contents in the tubes were spun at 5000 rpm for 1 min, and the digested peptides were subjected to mass spectrometry analysis.To identify translated transcripts in spermatozoa, 22,948 genes (TPM > 1) from both platforms were compared with the sperm proteome obtained via LC-MS/MS. Protein samples from randomly selected spermatozoa from two bulls were precipitated using a mixture of acetone (50%), ethanol (50%) and acetic acid (0.1%) and incubated overnight at −20 °C. The obtained protein pellet was resuspended in 20% acetonitrile (ACN) in a 100 mM NHBos taurus database (NCBI), and a minimum of two high-confidence peptide matches were considered a prerequisite for identifying the proteins. The proteome data obtained from two bulls were consolidated based on the criteria of a minimum of one peptide per protein to obtain significant protein identities for matching with sperm transcripts with >1 TPM.The samples  were injected into an LC-MS/MS column and allowed to separate for 180 min in a RPLC gradient, followed by data acquisition with a Thermo LTQ Orbitrap Discovery system. The mass precision for the experiment was set to 4 ppm, with a S/N threshold of one. The minimum/maximum precursor mass range was 350–5000 Da, and the maximum collision energy was set to 1000 in an ESI-TRAP. The precursor mass tolerance was set to 10 ppm and fragment mass tolerance to 0.8 Da. The oxidation of methionine (M), carbamidomethylation of cysteine (C), and carboxymethyl (C), acetyl (N-term), Gln- > pyro-Glu (N-term Q) and Glu- > pyro-Glu (N-term E) alterations were considered as dynamic modifications. The target Decoy PSM validator was selected as the processing node with a target false discovery rate of not more than 0.01. The data were analyzed via a standard approach using MASCOT 2.4 as the search engine in Proteome Discoverer 1.4 . The data were searched against the Total read counts/transcript were measured for 30,304 genes for each sample obtained from both RNA-seq methods. The TPM values were calculated, and comparisons were made between and within the RNA-seq platform results. The intactness of transcripts was analyzed by manually visualizing the read coverage of the top 100 transcripts selected based on their TPM. The intactness was assessed based on their 5′ and 3′ read coverage with unique gene reads (more than 5). The proportion-based Kal’s test (Z-test) was performed to identify the transcripts that differed significantly between the RNA-seq methods.Bos taurus as the background. Tissue enrichment associated with spermatozoal transcripts was analyzed using Gene Ranker  using Homo sapiens as the background. The statistical test for GO enrichment from the DAVID analysis was the Benjamini p value corrected for the false discovery rate, and for Gene Ranker, it was Fisher’s exact test. For all experiments, p values less than 0.05 were considered statistically significant.Gene ontology was analyzed with 20,689 annotations available in the bovine gene ontology consortium. The commonly expressed transcripts among the animals were identified irrespective of the platforms and the transcripts with non-significant difference between the platforms were selected and the subgroup of RNAs were identified. The ontological analysis  for the transcripts with >10 TPM, commonly expressed subgroup of RNAs and the translated transcripts was performed using the online Database for Annotation, Visualization and Integrated Discovery , PANTHER (v10.0) and Gene Ranker  using How to cite this article: Selvaraju, S. et al. Occurrence and functional significance of the transcriptome in bovine (Bos taurus) spermatozoa. Sci. Rep.7, 42392; doi: 10.1038/srep42392 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently.The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp “snatches” capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide IMPORTANCE Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis. RNA viruses use an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe their viral RNA (vRNA) genome. One of the best-studied negative-strand RNA viruses is the influenza A virus (IAV). The IAV genome is replicated and transcribed in the nucleus of the host cell by the IAV RdRp, an enzyme that consists of the viral proteins PB2, PB1, and PA –5, while–de novo  that are complementary to the second, third, and fourth nucleotides of the template , 15. Thiin vitro polymerase activity assays to provide in vitro evidence for the existence of low-processive transcription elongation events that result in a duplication of the first 3 nucleotides of the vRNA 3′ terminus. Moreover, we show that the synthesis of these duplications is dependent on the sequence of the capped primer, the sequence of the template, and the interaction of the body of the RNA primer with the body of the priming loop. These observations thus provide mechanistic insight into IAV RNA synthesis and redefine the function of the priming loop as a platform for both efficient replication IAV transcription uses a capped RNA primer that is snatched from host cell mRNAs and subsequently hybridized to the 3′ 1U and/or 2C of the vRNA promoter for extension from 3′ 2C or 3G, respectively . Althoug14–20–To characterize the three products in more detail, we cut the part of the gel containing the three bands into three fragments and extracted the RNA contained in the three gel fragments. Subsequent Sanger sequencing analysis revealed that the gel fragment with the bottom band contained an RNA species that had initiated at 3′ 3G of the template , whereasTo verify that the GP species had indeed been synthesized after initiation at 3′ 3G in our assay, we first mutated 3′ 2C of the vRNA promoter to A (2C→A) in order to disrupt G-C base pairing between the AG primer and template. As shown in In the above-described experiments, RP formation was independent of 3′ 2C but dependent on 3′ 1U . This isThe above-described results imply that transcription initiation relies on base pairing when the IAV RdRp uses primers ending in AG or CA. However, the IAV RdRp can also use primers that are not complementary to the template , 16, 18.To investigate if we could make the AU primer initiate from G3, we next used the 2C→A mutant promoter in the reaction  and founTo investigate this further, we performed a transcription reaction with a primer ending in 3′ UG (UG primer). Up to the point of realignment, this primer is extended into the same e formed . Moreovee formed , withoutpol elongation complex shows that the PB1 priming loop, which is located between the active site and the entrance of the nascent strand exit channel, is ideally positioned to interact with the incoming capped primer  when the RdRp is extending a primer that can form a double-Watson-Crick base pair with the template . However, 15 other nucleotide combinations exist for the last 2 bases of the primer, which implies that realignment may be more frequent in viral infections. Indeed, in viral infections, up to 30% of transcription elongation events involve realignment, as shown by deep sequencing . WithoutIn summary, here we propose that the priming loop of the IAV RdRp interacts with the primer that it snatched from the host cell to improve transcription elongation, which, as we argue above, may be crucial for IAV mRNA and protein synthesis. This thus provides the IAV RdRp with two mechanisms to optimize viral transcription and help the enzyme elongate efficiently after a cap-snatching event. These observations provide a deeper insight into IAV RNA synthesis, and they may be relevant for other negative-strand RNA viruses that have been observed to use realignment mechanisms as well.Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% fetal calf serum (FCS). Sf9 cells were grown in Xpress medium (Lonza) supplemented with penicillin and streptomycin. Plasmids pPolI-NA, pcDNA-NP, pcDNA-PB1, pcDNA-PA, pcDNA-PB2-TAP, and pcDNA-PB1a were described previously , 21, 26.pol elongation complex (PDB accession number 3OL7) and the influenza B virus RdRp crystal structure (PDB accession number 5MSG), we aligned active-site residues 324 to 332 of the poliovirus enzyme with residues 442 to 449 of the bat influenza virus PB1 subunit in PyMOL 1.3.Amino acid sequences of the PB1 subunits of influenza A/WSN/33 (H1N1), influenza B/Michigan/22687/09, and influenza C/JJ/50 viruses were aligned by using ClustalX . The ali32P]GTP , 2.5 U/μl 2′-O-methyltransferase (NEB), and a vaccinia virus capping kit (NEB). The products were denatured in formamide and purified as described previously , 1,500 cpm capped RNA primer, 0.7 μM vRNA promoter (Sigma), 5% glycerol, 0.05% NP-40, 75 mM NaCl, 10 mM HEPES (pH 7.5), and ∼1 μM RdRp. The reaction mixtures were preincubated for 20 min at 30°C, and the reactions were then started by adding 500 μM UTP, 500 μM ATP, 500 μM CTP, and 500 μM GTP to the mixtures. Aliquots were taken at the time points indicated, and reactions were stopped with 4 μl formamide loading buffer. Samples were subsequently denatured and analyzed by 20% denaturing PAGE. The extended capped primers were visualized by phosphorimaging. P values were determined by using an unpaired t test. To sequence the extended capped primers, products were excised from the 20% denaturing PAGE gel, eluted overnight in water and desalted over NAP-10 columns . The isolated RNA was next polyadenylated with poly(A) polymerase (NEB), reverse transcribed with the dTGG primer (5′-CACGACGCTCTTCCGATCTTTTTTTTTTTTTTTTTTGG-3′), and turned into double-stranded DNA (dsDNA) by using second-strand primer 2ND_GA (5′-GTTCAGACGTGTGCTCTTCCGATCTGA+AT+A+CTCAAG-3′ [here “+” indicates a locked nucleic acid (LNA) base]). To remove the excess primer, the DNA was treated with exonuclease VII (NEB) for 1 h and subsequently heated to 95°C for 10 min to denature the exonuclease. Finally, the DNA was amplified with GoTaq (Promega) by using primers P5 (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) and i7003 (5′-CAAGCAGAAGACGGCATACGAGATACTGGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′) and TOPO cloned (Invitrogen) for Sanger sequencing. The β-globin mRNA transcription assay was performed as described previously , 5 mM MgCleviously .


Self-rated health (SRH) has been shown to be a good predictor of mortality. Data on SRH and its associated factors in the Chinese general population are limited. This study aims to assess the epidemiology of SRH in rural Anyang, China. SRH  was measured in a population-based study of 2,814 adults (including 697 couples) aged 25 to 69 who were recruited from rural Anyang in 2014. Of 2,814 subjects, 63.1% rated their health as “healthy”, whereas 28.1% and 8.8% rated their health as “fair” and “unhealthy”. Compared to males, females had a higher likelihood of reporting a better SRH. Health ratings declined with increasing age, unmarried status, lower education levels. Poor SRH was positively correlated with medical history as well as high levels of fasting plasma glucose and total cholesterol, but not with unhealthy lifestyle indicators including smoking, drinking, and obesity. High household income was predictive of better SRH in men but not in women. Among couples, a positive spousal SRH concordance was observed, although the strength of this concordance was low. These findings will be useful for formulation of appropriate strategies for improving risk perception and promoting general health in economically developing regions. Many international studies have consistently demonstrated that SRH is a good predictor of mortality and is closely associated with morbidity and disability4. Moreover, SRH has been found to have good test-retest reliability5 and has been recommended for health monitoring by the World Health Organization and the European Union Commission6.Self-rated health (SRH), which is a subjective perception of an individual’s health status, refers to a single-item health evaluation in which people are asked to assess their current health status on a five-point scale which ranges from very good to very poor8. However, the relationship between certain socioeconomic factors, health behaviors, clinical parameters and SRH varies depending on demographic and geographic factors14. In contrast with abundant data available from developed regions, studies in economically developing countries including China are not adequate and have generally been restricted to particular subpopulations, such as elderly people and floating population who refers to migrants without local household registration16. Furthermore, comprehensive assessments of the correlates of SRH among general Chinese populations are scarce. Most previous studies only evaluated the potential effects of interview data such as lifestyle characteristics on SRH ratings16. Other factors such as biochemical indicators, which may reflect subclinical physiologic conditions, have not been sufficiently investigated in existing studies carried out in China.The prevalence and determinants of SRH have been extensively investigated mainly in economically developed countries. Medical health status and its subsequent functional outcomes are widely recognized as major determinants when people rate their health17. In theory, environment and lifestyles shared by a couple could result in shared health risk18. However, factors which couples do not share such as genetic characteristics may cause spousal SRH discrepancy. The presence or absence of SRH correlation and the degree of SRH concordance in couples remains unclear.In addition, data concerning SRH spousal correlation is limited both in economically developed and developing regions. It has been suggested that good SRH is positively associated with spouses’ optimismTo overcome these limitations and fill important research gaps, this population-based cross-sectional study seeks to (1) investigate the distribution of SRH and its determinants including socio-demographic characteristics, behavioral factors, physical status and clinical measurements in rural China, and (2) assess the correlation and concordance of spousal SRH in this population.19. The current investigation utilized a subset including 5 of the 9 target villages which were cluster-sampled in the parent cohort study conducted in 2014. Eligibility criteria for enrolment were as follows: (1) permanent residency in the target villages; (2) age 25–69; (3) no prior diagnosis of cancer or mental illness; (4) no history of infection with HBV, HCV or HIV; (5) willingness to participate in the follow-up program and provide informed consent. All participants provided written informed consent, and the study was approved by the Institutional Review Board of the School of Oncology, Peking University. The methods were carried out in accordance with the approved guidelines.A population-based prospective cohort study of risk factors for esophageal cancer in rural Anyang, China, has been described elsewhere2 (kg/m2) and categorized into 4 groups: underweight (BMI < 18.5), normal weight (18.5 ≤ BMI < 24.0), overweight (24.0 ≤ BMI < 28.0), and obesity (BMI ≥ 28.0)20. History of disease was referred to as any disease(s) diagnosed by medical doctors.A one-on-one computer-aided interview was administered in a private room by a trained interviewer, and information on SRH, demographic characteristics , socioeconomic variables , indicators of healthy or unhealthy lifestyle , and disease status (history of disease) were collected during these interviews. SRH was initially recorded in 5 levels , and then categorized into 3 groups which included “healthy” (very good and good), “fair” and “unhealthy” (poor and very poor) for statistical analyses. Annual household income was categorized as low, moderate and high on the basis of annual income of ≤10,000, 10,001–30,000 or >30,000 RMB. Cigarette smoking was defined as consuming one cigarette or more per day for ≥12 months, and alcohol consumption was defined as drinking Chinese liquor at least twice per week for ≥12 months. A former smoker or drinker was characterized as an “ever” user who had not smoked or consumed alcohol in the preceding 12 months. Body mass index (BMI) was calculated as weight/height22. TC, TG, LDL and HDL were measured enzymatically on an automatic analyzer  with reagents purchased from Sichuan Xin Cheng Bio-Tech Co., Ltd. FPG was measured on the fasting blood glucose meter . All clinical measurements were performed by the clinical laboratory staff of Hua County Hospital, Anyang, China.Blood samples were obtained after overnight fasting. Clinical parameters were categorized as follows: fasting plasma glucose (FPG) , total cholesterol (TC) , triglycerides (TG) , high-density lipoprotein (HDL)  and low-density lipoprotein (LDL) The chi-square test was used to determine differences in self-rated health across model covariates. Due to the order of the outcome categories and satisfaction of the proportional odds assumption (measured by likelihood ratio tests and the Brant test), univariate and multivariate ordered logistic regressions adjusting for intracluster correlation (cluster variable: village) were conducted to estimate the relationships between explanatory variables and SRH. Potential risk factors that were statistically significant in univariate analyses, together with related variables which have been previously reported were included in multivariate models . The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated treating the group “healthy” as the reference group. Only a single odds ratio that described the odds of increasing by 1 category of the outcome for a 1-unit change in the explanatory variables was reported. For couple concordance analysis, Spearman’s rank test and weighted kappa statistics employing a linear set of weights  were used. The kappa statistics examine the level of agreement beyond what would happen by chance. The kappa value 0 indicates that the degree of agreement is no more than would be expected by chance, while the kappa value 1 indicates a perfect agreement.P values less than 0.05 (two-sided) were considered to be significant.Statistical analyses were performed using STATA version 12.0 software . Of 3,457 eligible residents, 2,814 (81.4%) were enrolled. The main reason why the other 643 individuals, who were generally more likely to be younger men, did not participate was loss of contact due to employment outside of Anyang. The median age of these 2,814 participants was 49 , with a female to male ratio of 1.6  , whereas 28.1% and 8.8% rated their health as “fair” or “unhealthy” , non-farming  and possession of higher levels of education  increased the likelihood of reporting a better SRH (Table vs. without current or past disease). The association with SRH increased significantly with the abnormal levels of FPG  and was markedly elevated among individuals with a high level of FPG . However, there was no such statistical association observed for the borderline high level of FPG and SRH  after adjustments. Similarly, elevated level of TC was statistically correlated with poorer SRH . Categorized by TC level, the high level of TC increased the likelihood of reporting a worse SRH , however, there was no such statistical association found for the borderline high level of TC and SRH . Abnormality of TG, LDL, and HDL showed no statistical correlation with SRH.In multivariate analyses, married status  vs. low ). However, there was no such independent association in females . Spearman’s rho test also showed that the SRH of one partner was mildly but significantly correlated with that of the other partner .As shown in Table To our knowledge, this is the first study assessing both the determinants and spousal concordance for SRH for general population in rural China. Of the male and female respondents, 9.5% and 8.4% reported “unhealthy” SRH in rural China. Poor SRH was positively correlated with disease status and abnormality of some clinical parameters but not with presence of unhealthy lifestyle indicators including smoking, drinking, and obesity. High household income was predictive of better SRH among males but not among females. Among couples, a statistically significant positive spousal concordance for SRH was observed, although the concordance strength was low. These findings will be useful to formulate appropriate strategies for health intervention.24. The variation found among populations may largely explain the heterogeneity of proportions across studies. It is well known that advancing age and low education levels are positively associated with poor SRH11. Our results further confirmed this correlation. With regard to gender, reports of its association with SRH have not been fully consistent27. In this study, SRH in women overall appeared to be better compared with SRH in men. Since poorer SRH reflects a higher burden of diseases28, gender disparity in SRH ratings could be partially explained by the fact that men in this population were more likely to report having a disease history .Our study showed that 8.8% of respondents rated their health status as “unhealthy”, while more than 60% reported “healthy” SRH. These proportions were in the middle of the wide range reported by prior studies from Asian countries9. Notably, in this study, an independent association between annual household income and SRH was found in males but not in females. This gender disparity in the relationship of family income and health has also been observed by other researchers24. It could be explained partially by specific sociological factors. For instance, household income is largely dependent on men’s income in rural China, which may affect their control over family resources, access to health care and decision-making power29. Accordingly, the impact of income inequality  on health is more likely to be observed in men (major controllers) than in women. However, the mechanism by which income inequality is linked to health and SRH yet to be convincingly established.It has been suggested previously that high household income is predictive of better SRH28. For clinical parameters, the degree of association increased with the abnormal levels of FPG and TC even after adjusting for disease status and other potential confounders. Additionally, high levels of FPG (≥7.0 mmol/L) and TC (≥6.2 mmol/L) were significantly associated with poorer SRH. The correlations between biochemical indexes and SRH observed by us and others groups may be the result of physical sensations associated with disease progression, such as fatigue and poor sleep patterns, indicating that SRH has a biologic basis and it may serve as a barometer of physiologic states14. For borderline high levels of FPG and TC as well as abnormality of TG, LDL and HDL, their association with SRH were not statistically significant after adjustments. This can be partially explained by the fact that rural residents rarely underwent biochemical examinations and most were unaware of the abnormality of clinical parameters. Thus, in contrast to high levels of clinical indexes which may influence health perception and relate to SRH ratings, marginally elevated levels of biochemical indicators without accompanying symptoms and signs would be unlikely to have an impact on SRH reporting14. For unhealthy lifestyle indicators , a null association was observed, in contrast with results from economically developed regions30. Taken together these findings may reflect the fact that adults in rural China often lack basic knowledge about disease prevention and are thus not aware that risk factor exposure may have adverse health effects31. In support of this assumption, other studies found that obesity is not regarded as unhealthy, but rather as a matter of good fortune in Chinese culture especially in rural area. This is especially true in rural areas, where it is believed that only rich people could afford to eat more and gain weight32. In view of the fact that physical health but not unhealthy lifestyles which are regarded as major causes of modern-day disease33, plays an important role in SRH in rural China, more measures for health promotion and intervention are recommended for rural individuals to improve lifestyle and risk perception. Further research is needed to confirm our findings.Consistent with most previous studies, current or past disease was strongly associated with poorer SRHOur study also augments knowledge regarding spousal SRH concordance in heterosexual couples based on a large sample. As expected, we found that the SRH of one partner was significantly associated with that of the other partner, although the degree of spousal concordance was low. This phenomenon may arise from the combined effect of shared and non-shared factors. The contribution of sharing a similar environment, social network, and financial resources  to similarity in spousal SRH could be attenuated to some extent by effects of non-shared factors  for respondents and their partners  may undermine the generalizability of our findings to a wider population. Second, bias potentially introduced by the individuals who did not participate must be noted, as such bias may have blurred the age/gender-related association. Third, with the exception of clinical parameters, all information was self-reported data. Recall bias or under-reporting of risk factors cannot be excluded, although interviews were administered by well-trained interviewers in a one-on-one private setting. Fourth, our findings may be subject to potential endogeneity bias. Additionally, information about depression, health service utilization and social networks was not collected, and their association with SRH could thus not be evaluated. Finally, owing to its cross-sectional nature, the temporal order of observed associations with SRH cannot be established.In conclusion, this study adds to current understanding of self-rated health and its determinants. SRH differed significantly with socio-demographic characteristics, disease status, and clinical parameters, but did not vary with healthy lifestyle indicators in rural China. Among couples, the shared lifestyle features may lead to a significant although low SRH concordance. These findings will be useful for future improvement in subjective well-being and objective health in economically developing regions.Supplementary Table S1


C. elegans TSN (cTSN) is a monomeric Ca2+-dependent ribonuclease, cleaving RNA chains at the 5′-side of the phosphodiester linkage to produce degraded fragments with 5′-hydroxyl and 3′-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca2+-binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay.Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1–SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the  Tudor staphylococcal nuclease  is a conserved multifunctional protein in eukaryotes that participates in various cellular events, from transcriptional to post-transcriptional regulation. TSN was first identified as a transcription coactivator interacting with transcription factors such as EBNA-2, cMyb, STAT6, and STAT5 to promote cell proliferation and differentiation in mammals . SubsequCaenorhabditis elegans, Drosophila, and mammals (X. laevis and human TSN degrades hyper-edited double-stranded RNA (dsRNA) containing multiple I•U and U•I pairs generated by ADAR (adenosine deaminase acting on RNA), which converts adenosines to inosines in dsRNA  decay. TSN was first identified as a component of the RNA-induced silencing complex (RISC) in  mammals ; althougin dsRNA . TSN thuin dsRNA . Recentlin dsRNA . TSN, thTSN is composed of four tandem repeats of staphylococcal nuclease-like domains (SN1-4), followed by an SN5 domain incorporating an inserted Tudor domain . The fouCaenorhabditis elegans TSN (cTSN) degrades both ssRNA and mismatched dsRNA, but not perfectly matched dsRNA. Therefore, TSN targets the unstructured regions of RNA, but not necessarily those with inosines or I•U/U•I pairs. We also identified multiple ribonuclease active sites in the SN1 and SN3 domains of cTSN and show by small-angle X-ray scattering (SAXS) that rice TSN (osTSN) has a flexible two-lobed conformation. Together, our results reveal how TSN selects its targets for degradation and explain how TSN degrades inosine-edited miRNA precursors and mature miRNA during miRNA decay.Compared to its bridging roles in protein–protein interactions, how TSN selects its RNA substrates for cleavage is less clear. TSN specifically degrades duplex RNA with I•U and U•I pairs, which are particularly unstable since duplex RNA containing single or double I•U/U•I pairs have much lower thermal stability compared to those with Watson–Crick base pairs . This fiCaenorhabditis elegans), plants (Oryza sativa), and mammals (Homo sapiens) into the expression vectors to express TSN proteins with an N-terminal His-tag in E. coli. Human, rice, and C. elegans TSN, named hTSN, osTSN, and cTSN, respectively, were purified by chromatography with a high homogeneity  that only adds Pate ends B. In sum32-labeled stem–loop RNA mimicking the primary miR142 precursor but in which the Dicer-cleavable loop region was replaced with a short loop (referred to as pre-miR142), as well as its inosine-edited counterpart (termed pre-miR142-AtoI)  of cTSN and RNA-binding affinity; a Kd of 235.8 ± 38.2 nM for unedited pre-miR142Δloop and 68.3 ± 7.1 nM for edited pre-miR142Δloop-AtoI, respectively. cTSN bound the RNA with the loop (pre-miR142) more strongly (twofold) than the RNA lacking the loop (pre-miR142Δloop)  . Taken tTo further clarify if cTSN prefers to target the ssRNA or ssRNA region imbedded in dsRNA, we also measured the binding affinity between cTSN and a 20-bp perfectly matched dsRNA (dsRNA) versus a 20-nt ssRNA (ssRNA). cTSN bound the dsRNA with an approximately twofold higher affinity (392.9 ± 51.5 nM) than the ssRNA (767.3 ± 132 nM), suggesting that cTSN prefers to bind dsRNA than ssRNA see . Taken tNext, we wondered if cTSN preferentially degrades A-to-I-edited RNA. We incubated the 5′-end fluorescein-labeled pre-miR142 with cTSN and the RNA cleavage patterns were revealed by gel electrophoresis see A. cTSN dA-to-I editing introduces unstable I•U or U•I base pairs, suggesting that this type of base-pairing induces structural changes . TherefoWe next prepared two mispaired dsRNAs with or without inosines in the middle of the chains: “mispaired dsRNA” containing AAUA/CGCC, and “mispaired dsRNA-AtoI” containing IIUI/UIUU. Notably, cTSN degraded both mispaired dsRNAs, i.e., with or without inosines B. These Structure-based sequence alignment shows that most putative catalytic residues are mutated in the SN domains of TSN when compared with those of staphylococcal nuclease . The metr approaches Dmax around 191 Å  has previously been characterized  domains. However, compared with its bacterial homolog, most catalytic residues in the SN domains are degenerated. In this study, we show that activity . As SN1–activity , deletioCaenorhabditis elegans was cloned by RT-PCR. The cDNA of Homo sapiens TSN was purchased from OpenBiosystems, whereas the cDNA of Oryza sativa TSN was purchased from the KOME database. The cDNA of each TSN was inserted into pET28a expression vector using XhoI and NdeI restriction cutting sites to express proteins with C-terminal 6xHistidine tags in E. coli. Site-directed mutagenesis constructs of cTSN were prepared by Pfu-Ultra II Fusion HS DNA Polymerase kit (Agilent Technologies). All constructs were expressed in E. coli BL21 (DE3) RIPL cells (Agilent Technologies) at an OD600 of 0.6 with 0.5 mM IPTG for 16 h at 18°C. Cells were harvested and lysed at 4°C in 50 mM HEPES (pH 7.0), 150 mM NaCl, 10 mM imidazole, 10% glycerol, 0.1 mM PMSF, 5 mM β-Mercaptoethanol, and 0.1% CHAPS by microfluidizer (Microfluidics M-110P). Cell lysate was passed through a His-Trap HP column  and TSN proteins were eluted with 50 mM HEPES (pH 7.0), 150 mM NaCl, 250 mM imidazole, and 5% glycerol. TSN protein samples were then passed through a HiTrap Heparin HP column  and eluted with a gradient of 0 to 1 M NaCl in PBS buffer (pH 7.4). Eluted protein samples were adjusted to a concentration of 2 mg/mL in PBS buffer (pH 7.4) with addition of 150 mM NaCl as stock for further experiments.The full-length cDNA of 280 of 1.0 in 50 mM HEPES (pH 7.0), 150 mM NaCl, 10 mM imidazole, and 1 mM β-mercaptoethanol. Sedimentation velocities of TSN were measured at 40,000 rpm (Beckman XL-A) at 4°C. Multiple scans (OD280) at different time intervals were then fitted to a continuous c(s) distribution model using the SEDFIT program.For AUC assays, the recombinant TSN proteins were concentrated to an ODTSN  was applied to an Agilent-Bio SEC-3 column (Agilent) connected to a DAWN HELIOS II-18 angle MALS (Wyatt Technology) detector with a wavelength setting at 658 nm. Samples were run in a PBS (pH 7.4) buffer containing 150 mM NaCl and 5 mM β-mercaptoethanol, with a flow rate of 0.2 mL/min using the ÄKTA-UPC 900 FPLC system . A 2 mg/mL sample of BSA (Sigma Lot#SLBH1159V) was used for calibration. Scattering data were analyzed using the ASTRA 6 software (Wyatt Technology).32-ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min. Reactions were stopped by sample loading buffers and the labeling results were analyzed on 15% TBE–PAGE.The pre-miR142 degraded by cTSN was used for terminal labeling assays. For 5′-end labeling, degraded RNA fragments, γ-P32P-labeled RNA substrates shown in Kd between cTSN and RNA by a one-site binding curve fitting using the equation Y = X/(Kd + X), where X is the cTSN concentration and Y is the percentage of bound RNA/total RNA.The 5′-end For the RNA degradation experiments shown in 2 at 25°C for 1 h (longer incubation time of 3 h was used for For the RNA degradation experiments shown in Rg estimation by the PRIMUS program (Dmax and P(r) distance distribution   was injected into an Agilent-Bio SEC-3 column at a flow rate of 0.02 mL/min. An X-ray wavelength of 0.8266 Å was used for data collection. Selected frames were merged and analyzed for initial  program , and the program  for Dmaxion 2.7) . Computaion 2.7) .


The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved. MGMT) promoter methylation status. Residual tissue, if existing, is routinely stored as formalin-fixed paraffin-embedded tissue (FFPE). Therefore, FFPE tissues represent an exploitable source of tumour material that can be used to perform the molecular studies in relation to clinicopathological information and known prognostic factors that are especially valuable in low-incidence diseases like GBM. RNA extracted from archival FFPE tissues has often suffered chemical modification, cross-linking, and degradation over time as a result of the fixation and archiving methods. Nevertheless, FFPE RNA has been successfully extracted from stored specimens [Genomic profiling studies of glioblastoma (GBM) have established that GBM can be sub-classified into different intrinsic subtypes according to gene expression. Molecular classifications of GBM might better explain differences in outcome and response to treatment rather than morphological or clinical factors –3. Gene pecimens , 5 and upecimens , 7. Recepecimens –17. HoweWe have performed a pilot study to determine whether gene expression data obtained from FFPE GBM tumour samples was comparable to that obtained from paired FF samples from the same tumour when assessed by RNA-Seq using the Illumina platform.This study was approved by the Institutional Review Board of the Hospital Germans Trias i Pujol (PI-14-016) and by the Ethics Committees of all the participating institutions and conducted in accordance with the Declaration of Helsinki.We selected eleven cases from a database of 432 GBM patients for whom we had both FFPE and FF tumour samples. All patients had primary glioblastomas, as confirmed by pathological review . Two samples had been obtained from the same tumour from each patient, one of which had been stored as FFPE and one as FF.The RNA extraction of FF and FFPE tumor specimens was performed on five 15μm-deep tissue cuts using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s recommendations. RNA quantity and purity were measured with the NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA integrity, determined by the RNA integrity number (RIN), was determined with the 2100 Bioanalyzer (Agilent).Samples were sequenced at Centro Nacional de Análisis Genómico . A modified TruSeq™ Stranded Total RNA kit protocol (Illumina Inc.) was used to prepare the RNA-Seq libraries from FFPE samples. Ribosomal RNA (rRNA) was depleted from 0.5–1.0 ug of total RNA using the RiboZero Magnetic Gold Kit . rRNA-depleted RNA samples were purified using Agencourt RNA Clean XP beads (Beckman Coulter Genomics) and RNA was eluted with the Elute, Prime, Fragment Mix from the TruSeq Stranded Total RNA kit. The RNA fragmentation time was shortened to 2.5 minutes due to the low quality of the initial total RNA . Following the fragmentation, first and second strand synthesis, the Illumina bar-coded adapters were ligated at 1/10 dilution of the recommended concentration. Libraries were enriched with 15 cycles of PCR. The size and quality of the libraries were assessed in a High Sensitivity DNA Bioanalyzer assay (Agilent).The starting input material for the libraries construction was DNA free total RNA from FF using the TruSeq™ Stranded Total RNA kit protocol (Illumina Inc.), according to the manufacturer’s protocol with some modifications for the FFPE samples, and the final library was quality controlled in Agilent DNA 7500 Bioanalyzer assay (Agilent).Each library was sequenced using TruSeq SBS Kit v3-HS (Illumina), in paired-end mode with a read length of 2x76bp. We generated minimally 65 million paired-end reads passing filter for each FFPE RNA-Seq library or at least 54 million paired-end reads passing filter for each FF RNA-Seq library in a fraction of a sequencing lane on HiSeq2000 (Illumina) following the manufacturer’s protocol. Image analysis, base calling and base quality scoring of the run were processed by integrated primary analysis software—Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA 1.8.The bioinformatic analyses included alignment and quantification, sample quality metrics, differential gene expression analysis, gene variant calling, and prediction of GBM molecular subtype.RNA-Seq reads were aligned to the human reference genome (GRCh38) using STAR (version 2.5.1b)  with ENChttp://gemtools.github.io/). Gene body coverage, GC content, paired-end inner distances, median transcript integrity number (TIN) across all the transcripts and distribution of mismatches across reads were computed with RSeQC [Several quality metrics were calculated to evaluate the differences within each FF-FFPE pair and across the different preservation conditions. For categorical data,a Fisher’s exact test was applied for each pair. For differences in means between the two conditions, a t-test was applied. PCR duplicates were calculated with sambamba . The numth RSeQC , 22. Theth RSeQC . CorrelaRSEM read counts were used as input for DESeq2 (version 1.10.1) , 25. TheWe counted the number of mismatches with respect to the reference genome for each GBM-associated gene directly from the mpileup generated by samtools without any filter , 28. Varhttp://mksamur.github.io/RTCGAToolbox/). The core function ‘getFirehoseData’ with ‘dataset = GBM’ and ‘runDate = 20151101” was used to access and download the data. The associated clinical annotation for each sample was downloaded using the cgdsr R package . The TCGA RNA-seq dataset comprised 145 RNA-seq samples grouped into the five established GBM molecular subtypes . Prediction was made for the four FF and the three informative FFPE samples (excluding FFPE_AA6365) using the largest value of lambda such that error was within 1 standard error of the minimum. Read counts were transformed with the variance stabilizing transformation using DESeq2.Batch effect correction between the RNA-seq datasets was carried out with the sva R package [The glmnet R package  was used package . Genes whttps://www.ncbi.nlm.nih.gov/bioproject/342811.All data underlying the findings described in the manuscript are fully available without restriction from the BioProject database: We had paired FF and FFPE samples from 11 patients. Of the 11 FF samples, only four met the requirements to ensure informative results from RNA-Seq . All analyses were carried out in the samples from these four patients. All FFPE RNAs had very low RIN values (≤2.6), but interestingly, RIN values were not related to storage time .RNA fragmentation is a major effect of FFPE environments. A good proxy to evaluate if the RNA molecule length is affected is to calculate the paired-end inner distances for each RNA-seq experiment. As expected, FFPE specimens disclosed smaller distances between read pairs than FF samples , P<0.001-16, 2x10-16 except pair 3, -16 except pair 3, Investigating the library diversity captured by sequencing FFPE material can help identify any loss of informative RNA-Seq reads due to poor sampling of the RNA molecules , 32. We -16, -16, As previously reported , mapping-16, Differential gene expression analysis revealed 2133 differentially sampled genes with FDR<0.05 . In FFPE2~0.9), with the exception of FFPE_AA6365, the most highly degraded sample (R2~0.35)  . A princR2~0.35) .IDH1, IDH2, NF1, PTEN, PDGFRA and TP53) [The FFPE mismatch profiles diverged substantially from their paired FF samples . Specifind TP53)  also harnd TP53)  reportedThe prediction of molecular subtype with Lasso regularization showed that all but one FFPE sample AA6365) could be classified in one of the five GBM molecular subtypes 65 could . The meshttp://www.rarecancerseurope.org//About-Rare-Cancer, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789814/) with an incidence in adults of 3.19 per 100,000 inhabitants and a high mortality rate[GBM is a rare disease Click here for additional data file.S2 FileThere are five tabs: (A) Results of the differential gene sampling analysis. Genes are sorted by significance. (B) List of differentially sampled genes with FDR <0.05. (C) List of over-sampled genes in FFPE samples sorted by gene name. The majority are non-coding RNA belonging to specific gene families and have no functional annotation. (D) List of down-sampled genes in FFPE sorted by gene name. The majority are protein-coding genes. (E) Gene ontology enrichment(XLSX)Click here for additional data file.S1 TableNumber of mpileup substitutions with respect to the reference genome. Gray shaded areas indicate C>T and G>A changes.(DOCX)Click here for additional data file.S2 TableNumber of SNP artefacts originated by C>T and G>A changes. We counted cases that were CC (GG) homozygous in the FF sample and CT (GA) or TT (AA) in the paired FFPE sample.(DOCX)Click here for additional data file.S3 TableNumber of overlapping SNPs (shaded) and non-overlapping SNPs (non-shaded) for each FF-FFPE pair and each gene. 0/1 indicates a heterozygous SNP. 1/1 indicates a homozygous alternative. NA indicates not assessed.(DOCX)Click here for additional data file.S4 TableGrey shaded areas indicate genes included in Verhaak’s gene signature.(DOCX)Click here for additional data file.


Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments. Herein we report the design and synthesis of novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner. The technique relies on hijacking a bacterial tRNA-guanine transglycosylase (TGT) enzyme.interest .2529 TO's fluorogenic interaction with nucleic acids can give up to 1000-fold fluorescent enhancement, and TO derivatives have been widely adopted in a variety of PNA and DNA forced-intercalating (FIT) probes,32 ECHO probes,35 an RNA GTP sensor,28 and fluorogenic RNA aptamers such as RNA mango.15 In our previous work, we chemically modified TGT's natural substrate, preQ1, with a TO moiety to yield 1a   are wellyield 1a  and obsel Fig. S1. To addr RNA-TAG . The nuc1-TO derivatives by incorporating bulky side chains on the TO scaffold at the benzothiazole's N3 or quinoline's N1 positions , as well as the alkyl groups at the TO moiety to an ethyl, propyl, isopropyl, and benzyl group, thus gradually increasing the steric bulk (5c–f). With these derivatives in hand, we first investigated probe background fluorescence in the absence of enzyme at various concentrations of in vitro transcribed mCherry mRNA, which contained a 17 nt TGT recognition element within the 3′ UTR. We observed that as steric bulk on the TO moiety increased, the derivative emitted less fluorescence (Me > Et > Pr > iPr ≈ Bn)  demonstrated little improvement over our first generation probe 1a. However, 5f, substituted with an aromatic benzyl group, demonstrated an improved 35-fold turn-on. We also evaluated the importance of the point of attachment on the TO moiety for methyl and ethyl substitutions. We found similar increases in fluorescence between methyl  and ethyl  substitutions of the two regioisomer pairs indicating that the substituent identity, rather than their location, is the dominating factor in minimizing non-specific background fluorescence. From this data we can conclude probe 5f significantly lowers the fluorescent background from nonspecific interactions with nucleic acids while maintaining high fluorescent intensity once covalently linked through RNA-TAG enzymatic transglycosylation.We initially prepared a small collection of preQ) Fig. S2 likely dat 37 °C . Our res5a–f, we next explored an alternative linker to improve kinetics of transglycosylation. We examined what effect replacement of the hydrophilic PEG3 linker by a more hydrophobic 6-carbon alkyl linker would have on labeling efficiency. Using HPLC to quantify reaction completion, we observed that when 10 μM 17-nt ECY-A1 hairpin was treated with 1 μM (0.1 eq.) TGT and 10 μM 1a, approximately 65% of the substrate was labeled after 2 h. However, use of 1b, which employed an alkyl linker  cells were transiently transfected with the mCherry construct plasmid. After overnight culture, the cells were fixed, permeabilized, and subsequently treated with 0.5 μM 6f and 0.5 μM TGT. Cells were then incubated for 3 h and subsequently imaged without a wash step. Significantly greater staining was observed for cells treated with TGT and 6f compared with control cells that were; (1) not treated with TGT, (2) not transfected with the mCherry construct, and (3) only treated with 6f in the absence of TGT and mRNA expression  to be recognized by the enzyme efficiently. We support this hypothesis with evidence from a fixed cell imaging experiment with elevated probe concentration demonstrating that when using 2 μM of 6f (4× our previously used concentration), RNA could not be efficiently imaged due to a high fluorescent background (Fig. S7KM for the RNA and preQ1 probe through directed evolution. We believe this could greatly benefit the labelling of the RNA in vivo. In order to increase the signal-to-noise ratio for the in vivo fluorescently labeled RNA, we also plan to construct multiple TAG sequence repeats genetically engineered in the target mRNA to increase the chances of labeling and abundance of signal per RNA.In our effort to image mRNA in live cells using RNA-TAG, we transiently co-transfected HeLa cells with a plasmid capable of expressing TGT in mammalian cells and the mCherry construct plasmid used in our previous experiments Fig. S6. The TGTd Fig. S6. We therd Fig. S7. Live ce1-TO fluorogenic probes for imaging mRNA in mammalian cells using RNA-TAG. The advantages of using RNA-TAG to image mRNA in cells include fast labeling kinetics, an easy preparation of the fluorogenic probes, and a submicromolar working concentration of both enzyme and probe. These novel preQ1-TO probes greatly reduced the fluorescent background and maintained a high fluorescent intensity after labeling. By exploring the structure activity relationship of preQ1-TO probes, we were able to optimize fluorogenic RNA-TAG labeling for cellular imaging. Our optimal probe, a benzyl substituted TO probe linked to preQ1via a C6 linker, demonstrated greatly improved labeling kinetics and a 100-fold increase in fluorescence intensity upon covalent incorporation to a full length mRNA transcript. We believe that this robust and versatile technology will provide a powerful tool to detect and image RNAs of both fundamental and practical interest. We are currently exploring expanding this technology to enable live-cell detection of expressed mRNAs.We have developed a new class of preQThere are no conflicts to declare.


RNA molecules are essential players in many fundamental biological processes. Prokaryotes and eukaryotes have distinct RNA classes with specific structural features and functional roles. Computational prediction of protein structures is a research field in which high confidence three-dimensional protein models can be proposed based on the sequence alignment between target and templates. However, to date, only a few approaches have been developed for the computational prediction of RNA structures. Similar to proteins, RNA structures may be altered due to the interaction with various ligands, including proteins, other RNAs, and metabolites. A riboswitch is a molecular mechanism, found in the three kingdoms of life, in which the RNA structure is modified by the binding of a metabolite. It can regulate multiple gene expression mechanisms, such as transcription, translation initiation, and mRNA splicing and processing. Due to their nature, these entities also act on the regulation of gene expression and detection of small metabolites and have the potential to helping in the discovery of new classes of antimicrobial agents. In this review, we describe software and web servers currently available for riboswitch aptamer identification and secondary and tertiary structure prediction, including applications. Fifty years ago, the central dogma of molecular biology proposed a preferential flow of information, stating that DNA is transcribed into RNA, which in turn is translated into proteins with structural or catalytic functions Crick, . Since tS-adenosylmethionine (SAM)    or the introns within an mRNA sequence. These sensors are capable of binding a great variety of small molecules, such as vitamins, amino acids, and nucleotides   to create a position-specific score matrix based on primary sequence conservation . Infernal has many functions that are based on analogous ones in HMMER, such as output formatting, which is very similar to the two software packages. Infernal is available at http://eddylab.org/infernal/.Infernal (INFERence of RNA ALignment) was first created by Sean Eddy in 2002  and a putative secondary structure alignment. The secondary structure and MFE are calculated using the Vienna RNA package    in the search for ten riboswitches families on Rfam or RefSeq databases .Although we agree that HMMER is faster than Infernal, our experience in searching for riboswitches in genomes does not corroborate their report of similar searches. In our case, most of the candidate regions found while using HMMER were not identified by Infernal, and the ones that were in common were discarded according to the following threshold filters: positive values for HMMER and values above the Rfam gathering score for Infernal, or Actinobacteria  of a single RNA sequence. On the other hand, the multiple sequence analysis has the advantage of providing higher prediction accuracy compared to single sequence analysis because it incorporates evolutionary information. Nonetheless, this approach is not always suitable, given that knowledge of a set of homologous sequences is required. Below, we list a few programs that perform these analyses.http://unafold.rna.albany.edu/?q=mfold/rna-folding-form.The Mfold software for RNA folding was published as a stand-alone option Zuker, . The fir3) time dynamic programming algorithm. This methodology allows computation of the partition function of a nucleotide sequence over all possible unpseudoknotted secondary structures. RNAfold is available as a free software Vienna RNA package as well as web server -based prediction of RNA secondary structures with a broad class of pseudoknots  modeling, for instance, which is based on sequence similarity, works properly when there is an experimentally elucidated structure to be used as a template  from existing RNA structures whose sequences match subsequences of the target RNA. The Fragment Assembly of RNA (FARNA) .FARNA/FARFAR builds http://rna.physics.missouri.edu/.The Vfold model  offers a user-friendly interface that permits the input of a sequence to fold RNA using de novo methods. Alternatively, the user can provide secondary structure and distance restraints and a 3D structure in the PDB format to jump-start the modeling close to the expected final outcome.SimRNA  helix in the 3D structures  and L-glutamine riboswitch (61-nt)—with approximately 6 Å of RMSD with the crystallographic structure. Although the tools are less accurate, they can correctly predict the overall global folding. Thus, the larger the targets without a template—ydaO riboswitch (108-nt)—, the less accurate will the predictions be (10 Å best-case RMSDs).In this review, we focused on the most frequently used software and web-based tools for riboswitch prediction that encompass RNA secondary and tertiary structures. Moreover, the study of riboswitches contributed to the analysis of computational methods used for the structural prediction of RNAs. The Rfam database, used by various RNA motif prediction tools, is maintained and extended by the Infernal software. Prediction of the secondary structure is useful not only for the functional analysis of RNAs but also to improve the search for structural RNAs in genomes and build 3D models. Although the prediction of single long sequences is still limited, comparative approaches like RNAalifold increase the prediction accuracy. For tertiary structure prediction, the availability of a homologous structure increases the quality of the predicted models. RNA-Puzzles experiments have shown that, in the absence a homologous structure, targets greater than 100-nt have less accurate models, although it is possible to predict the overall folding.DA and NJ: participated in the literature research, coordinated the structuring of the paper, and wrote the article; EC and FP: proposed and idealized this work, discussed topics, helped writing the article and supervised the organization of the whole process.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.