During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein.Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly.Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not. All in all the entire 5′UTR (335 nt) and approximately 300 nt of gag are important for packaging  of human immunodeficiency virus type 1 (HIV-1) is mediated by a specific interaction between the viral Gag protein and an RNA structure in the 5′ untranslated region (5′UTR) called encapsidation signal or Psi (Ψ). This association leads to incorporation of gRNA dimers into Gag/GagPol particles. Whereas the core encapsidation signal is composed of 110 nt partially overlapping the The viral Rev protein allows nuclear export of unspliced and singly-spliced HIV transcripts via interaction with an RNA structure called Rev-response element (RRE). Recently, we discovered that a Rev-mediated export from nucleus to cytoplasm is essential for a highly efficient encapsidation process of lentiviral vector and proviral gRNA cis-acting splicing regulatory sequences . In order to imitate the splicing pattern of HIV-1 we inserted a 345 bp fragment from NL4.3Re cis-acting regulatory sequence GAR (an exon splicing enhancer). The resulting plasmid VHgenomic and its transcripts are depicted in rev and tat expression plasmids into HEK293T cells cytoplasmic RNA was extracted and analyzed by RT-PCR. Fragments corresponding to singly-spliced and fully-spliced RNAs were detectable  and gag/gagpol in the absence or presence of a rev expression plasmid. Constant and high Gag/GagPol levels were provided by the codon-optimized and therefore Rev-independent gag/gagpol expression plasmid Hgpsyn. Similar amounts of Gag/GagPol are produced after cotransfection of Hgpsyn with or without a rev expression plasmid  and integration (wild type 5′ and 3′ ends after RT reaction). Consequently, two days after infection a mean titer of 3.3×10∧4 and 1.2×10∧4 GFU/ml in the presence of Rev could be detected for VHenv and VHnef, respectively . Previously, it could be shown that deletion or codon-optimization of these cis-acting sequences can reduce or prevent nuclear retention of the resulting transcripts even in the presence of splice donor and splice acceptor sites Hgenomic was 17-fold and 5-fold higher compared to the levels of the singly-spliced SD1-SA5 RNA and the fully-spliced SD1-SA5+SD4-SA7 RNA, respectively. The unspliced RNA is therefore the predominant RNA species in viral particles. Remarkably, high amounts of unspliced RNAs of VHenv and VHnef identical in sequence to the spliced transcripts of VHgenomic could also be detected in viral particles. Consistent with this finding, packaging of an RNA mimicking the spliced HIV env transcript was previously shown by others but not quantified in detail For all vector RNA species Rev had no statistically significant effect on cytoplasmic RNA levels . This ishis work  and 4 arhis work  that areenv transcript expressed from a proviral HIV construct was similarly low as for the multiply-spliced nef transcript lacking the RRE, it was previously concluded that Rev does not influence packaging of HIV env RNA env RNA could be inhibited by sequences not present in fully-spliced HIV RNAs. This negative effect could probably be overcome by a Rev-mediated nuclear export of env RNA leading to an encapsidation efficiency similar to that observed for the fully-spliced HIV transcript  confirms our previous results  and the encapsidation efficiency of the unspliced RNA of V results . TherefoHgenomic, Rev-dependent encapsidation of RRE-containing RNAs from VHenv correlates with an enhanced infectious vector titer in the presence of Rev. However, the titer was increased by a factor of 6 whereas packaging of RRE-containing RNAs was enhanced by two orders of magnitude. Both the infectious titer and encapsidation of Msd1-sa5+Msd4-sa7 RNAs from VHnef lacking the RRE are not affected by Rev. It is evident that relative high amounts of unspliced and spliced vector RNAs are packaged after transfection of VHenv and VHnef. Nevertheless, the infectious titer of both vectors in the presence of Rev is lower compared to VHgenomic. These results imply that some steps after cell entry may be not as efficient for small vector transcripts compared to the unspliced transcript of VHgenomic. These steps include the efficiency of reverse transcription, the formation of a functional preintegration complex, nuclear entry of the cDNA and finally integration into the cellular DNA.Similar to the situation observed for VHenv  and temporal (Rev and Tat-dependency) regulation of the encapsidation process and could lead to an increased packaging of spliced RNA.Encapsidation efficiencies of lentiviral vector transcripts lacking the RRE did not vary significantly with or without Rev , while feriments  and 3. HHgenomic and the unspliced Msd1-sa5 RNA expressed from VHenv, which are identical in sequence, were similar  Hgenomic contains the first 339 nt of the HIV-1 HXB2 gag gene . The sequence of HIV-1 pol is not present in this vector. An HIV-1 NL4.3 proviral fragment (nt 5915 to 6259 of GenBank entry AF324493) was inserted into the NotI site between SD1 and the RRE directly downstream of the remaining gag sequence. The first rev exon in this fragment was mutated to prevent expression (ATG to ATC: nt 5969 to 5971 and TAT to TAA: nt 6035 to 6037 of GenBank entry AF324493). All in all, 898 nt of env sequence including 39 nt of the 5′ env sequence as well as 859 nt containing the RRE, the SA7 and the splicing regulatory sequences ESE3, ESS3a and ESS3b are retained in VHgenomic. The first intron between SD1 and SA5 spans 468 nt and the second intron between SD4 and SA7 comprises 981 nt. No viral proteins are expressed from this viral vector. To clone VHenv and VHnef cytoplasmic RNA was extracted from HEK293T cells transfected with VHgenomic and spliced transcripts were amplified by RT-PCR  as detailed in Hgenomic were digested with KasI/NotI or KasI/EcoRI and ligated. In the resulting plasmid VHenv and VHnef DNA sequences of SD1 and SA5 or SD1 and SA5 together with SD4 and SA7 are fused, respectively. All generated plasmids and PCR fragments were controlled by sequencing.Expression plasmids for HIV-1 Rev (pcRev) tat, VSV-G and gag/gagpol expression plasmids with or without a rev expression plasmid the precleared  and filtered (0.45 µm filter) cell culture supernatant was used to infect HEK293 cells. Two days later green fluorescent cells were counted to obtain the infectious titer as GFP forming units per ml of supernatant (GFU/ml). Western Blot analyses were done essentially as described before HEK293 and HEK293T cells were cultured in Dulbecco's modified Eagle Medium (DMEM) with 10% fetal calf serum and appropriate antibiotics. Two days after cotransfection of HEK293T cells by the calcium phosphate coprecipitation method 2, 0.5% [vol/vol] Nonidet P-40, 1,000 U/ml RNase inhibitor, 1 mM dithiothreitol). Nuclei were pelleted by centrifugation for 2 min at 300xg and 4°C followed by careful collection of the supernatant as cytoplasmic fraction. Subsequently, the RNeasy Mini kit (Qiagen) was used to extract cytoplasmic RNA. Particle-associated RNA was prepared with the QIAamp Viral RNA Mini kit (Qiagen) after ultracentrifugation of the supernatants of transfected cells through a 30% sucrose cushion. All RNA samples were stored at −75°C until RT-qPCR analyses. Detection of unspliced RNA derived from VHgenomic was facilitated by sense primer p2 binding upstream of SD1 (5′-gtggaaaatctctagcagtggcgc-3′) and antisense primer p4 binding directly downstream of SD1 (5′-tctttccccctggccttaaccg-3′). Detection of singly-spliced SD1-SA5 RNA of VHgenomic and the unspliced Msd1-sa5 RNA of VHenv was possible by sense primer p3- overlapping SD1 and SA5 (5′-ggggcggcgactggaagaa-3′) and antisense primer p8+ binding directly downstream of SD4 (5′-tgattactatggaccacacaactattgc-3′). The fully-spliced SD1-SA5+SD4-SA7 transcript of VHgenomic and the corresponding transcripts with identical sequences Msd1-sa5+SD4-SA7 and Msd1-sa5+Msd4-sa7 of VHenv and VHnef, respectively, were detected using sense primer p3- together with antisense primer p10 binding downstream of SA7 (5′-ccgttcactaatcgaatggatctgtc-3′). All primers are identical or very similar to those used by Houzet et al. Determination of cytoplasmic and virion-associated RNA copy numbers of transfected HEK293T cells was done as described previously Materials and Methods S1Detailed description of RT-(q)PCR approaches and validation of the methods including additional data.(DOC)Click here for additional data file.


Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A); MagNA Pure LC2.0 Automatic extractor (B); KingFisher (C); and NucliSENS EasyMag (D) RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) of MS-2 coliphage spiked into each system’s lysis buffer served as an external control for both. Cycle thresholds (Cts) of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 RNA extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. Diagnosis of enteric viral infections by Real Time reverse transcription - polymerase chain reaction (rRT-PCR) of RNA extracted from stool suspensions is complementing and increasingly replacing diagnosis based on viral isolation and characterization in tissue culture The presence of inhibitory compounds in the extracted RNA and the extent of inhibition can be determined by semi-quantitative rRT-PCR of an RNA template that is present in the sample, added to it prior to extraction, or introduced into the rRT-PCR mix. Natural or modified, encapsulated coliphage MS2 RNA is one source of protected RNA that has been used as a non-competitive external RNA template control The efficiency of removing inhibitors in patient samples may be related to the intrinsic properties of the method used to extract the RNA P<0.001.Ninety-three stool suspensions were extracted by the four extraction systems after spiking the lysis buffer of each with MS2 that yielded similar final MS2 concentrations. Any contribution from endogenous MS2 RNA was ruled out since no MS2 RNA was amplified from any of these 93 stool suspensions upon extraction with protocol D when exogenous MS2 was omitted from the lysis buffer. Results for each extraction procedure by assigned levels of inhibition are shown in P<0.001.In order to further challenge the performance of the 4 extraction systems to exclude inhibitors of rRT-PCR, 92 stool samples with levels of inhibition ranging from a single cycle to complete inhibition were evaluated. These samples were selected from among archived stool samples previously tested for enterovirus and MS2 after extraction by QIAamp Viral RNA Mini Kit. A sufficient number of samples with high, intermediate, and low levels of inhibitors were chosen for re-analysis to enable comparison between extraction procedures at each of these levels of inhibition. The results of MS2 inhibition for each sample by extraction protocol is shown in 1, equivalent) and the second contained 64 fold (6 Cts) more enterovirus RNA . No significant difference between inhibition of MS2 rRT-PCR and enterovirus rRT-PCR was noted when equivalent amounts of enterovirus RNA was added to extracts prepared by protocols A, B and C , but not A (P>0.05).Analysis of variance, indicated that there were no significant differences between inhibition of Cts for MS2 and enterovirus RNA for Protocols A, B, and C, but not D, when the amount of enterovirus RNA was equivalent to that of MS2 (P>0.5), whereas there was significantly less inhibition when the 64-fold higher amount of enteroviral RNA was tested for RNA extracted by protocols B, C, and D  between the inhibition of rRT-PCR of the MS2 external control and the added enterovirus (P>0.05) for protocols A, C, and D. In protocol B the inhibition of the rRT-PCR for enterovirus was actually significantly higher than that for MS2 (P  = 0.045). Recovery of enterovirus RNA by the four different protocols was equivalent.Analysis of variance , part 2,One of the trends in pathogen identification is the increasing reliance on molecular methods to achieve high throughput, high sensitivity and specificity of results in clinically relevant times Four protocols for extracting RNA from stool samples were compared in this study. These four commercial extraction methods are among the most commonly used in the diagnostic laboratories. None of samples 1 to 93 contained MS2 RNA. Similarly Nonove et al (14) did not report any unusual amounts of MS2 RNA that would have indicated prior presence of MS2 in their samples when they added MS2 to 106 stool suspensions at concentrations two to eight-fold less than used in this study. MS2 coliphage was thus used as a non-competitive external control because of its absence from human clinical samples. MS2 is a better external control for rRT-PCR than plasmid or phage DNA since it also measures the efficiency of the RT reaction The number of stool suspension RNA extracts with inhibitors of MS2 rRT-PCR varied between RNA extraction protocols for randomly chosen stool suspensions. The amount of inhibitors in RNA extracts for individual stool suspensions also varied according to extraction procedures. This suggests that more than one type of inhibitor may be present and that different procedures do have different proficiencies in excluding them. Stool samples extracted by the KingFisher and the NucliSENS easyMag had fewer extracts with inhibitors than the QIAamp Viral RNA Mini Kit and MagNA Pure LC2.0 Automatic extractor. The results for samples 1 to 93 with the MagNA Pure LC2.0 Automatic extractor were similar to the 21% reported earlier by Ninove et al. The number of samples with inhibitors was low in the first group of samples (samples 1–93) that were used to compare RNA extraction procedures. In order to better compare the four extraction procedures, we selected from among previously tested samples, sufficient numbers of archived stool suspensions that had high, intermediate, and low levels of inhibitors after extraction by QIAamp Viral RNA Mini Kit (samples 101–192). The stool suspensions were re-extracted in parallel by all four protocols and evaluated for inhibition of MS2 rRT-PCR. As before, KingFisher and NucliSENS EasyMag semi were better than the QIAamp Viral RNA Mini Kit and MagNA Pure LC2.0 Automatic extractor.Differences in extraction efficiency might be at least as important as inhibitors, as Hata et al. The standard maximum volumes that could be extracted differ for the procedures: 140 µl for QIAgen; 100 µl for MagNA Pure; 50 µl for KingFisher; and, 200 µl for easyMag. The effect of using these manufacturers’ recommended volumes for stool suspensions was not evaluated in the present study, although to have done so would have meant that depending on the procedure used, there would have been a 2 to 4 fold increase in the amount of endogenous inhibitors in the starting volume without a concomitant increase in the extraction, washing and elution buffer volumes. Others have shown that increasing the starting volume increased the co-extraction of inhibitors The majority of rRT-PCR assays have a lower limit of quantitation of 10 target sequences per reaction with a Ct of approximately 35 cycles. A reaction with a three-fold shift upwards in Ct due the presence of inhibitors would still be positive, i.e. a specific signal would still be detectable below the 40 cycle cap recommended for the majority of rRT-PCR assays. A two Ct difference is within the statistical variation that may occur between repeated analyses of the same RNA. In contrast, a positive signal would no longer be detectible if there were a six-fold shift in Ct, which would produce a false negative outcome.In conclusion, inhibition is a complex process. All four extraction methods were suitable provided that an external control was used to identify problematic samples. rRT-PCR of MS2 RNA recovered from MS2 coliphage added to the lysis buffers of RNA extraction systems is a good predictor of inhibition of enteroviral RNA extracted from stool suspensions. More than one inhibitor may be present in the stool suspensions or added during extraction and their efficiency of removal differs between the extraction protocols. The correlation between the extent of MS2 rRT-PCR inhibition and enteroviral rRT-PCR inhibition increases inversely in relation to the amount of enteroviral RNA in the sample. In agreement with Dreier et al The Ethical Review Board of the Sheba Medical Center, Tel Hashomer approved this study (SMC-8859). The samples and results were stripped of all links to personal details pertaining to, or which could be used to identify individual patients. All data were analyzed anonymously. The Ethical Review Board exempted this study from a requirement for obtaining informed consent.Stool suspensions (N = 185) prepared for routine analysis of clinical stool samples sent to the Central Virology Laboratory (CVL) at Chaim Sheba Medical Center in Israel were used to evaluate the efficiency of four different RNA extraction systems in excluding inhibitors of rRT-PCR. Ninety-three of the stool samples contained unknown amounts of rRT-PCR inhibitors ; Fig. S1Small portions of fecal matter were vortexed for 15 seconds in stool suspension buffer, 2 ml 0.9% saline with glass beads (samples 1 to 93) or 5 ml of M199 containing 15.6 µg of dihydrostreptomycin, 15,625 U of Penicillin G, and 156 U of Mycostatin and 0.1 volume (v/v) of chloroform (samples 101–192). Suspensions were clarified by centrifugation at 2,500×g for 10 min and stored at −20°C until use.E. coli RNA MS2 coliphage E. coli Top 10F in NZYCM broth as described by Dreier et al 4, 50 mM Tris pH 7.5 and 0.01% (w/v) gelatin] and added to the respective extraction lysis buffers used in each of the extraction procedures before use. The amount of external control MS2 template added to the lysis buffers was adjusted to give a Ct of approximately 27  in the rRT-PCR mix upon addition of 5 µl of RNA.A natural Protocol A, manual extraction - QIAamp Viral RNA Mini Kit , 140 µl aqueous solution extracted and eluted into 50 µl.; Protocol B, automatic RNA extraction - MagNA Pure LC2.0 Automatic extractor with MagNA Pure LC Total Nucleic Acid Isolation kit-High Performance , 100 µl aqueous solution extracted and eluted into 50 µl; Protocol C, semiautomatic extraction-KingFisher , using an Ambion MagMAX Viral RNA Isolation kit , 50 µl aqueous solution extracted and eluted into 50 µl.; and Protocol D, semi-automatic extraction-NucliSENS easyMag semi-automatic extractor  using the easyMag extraction kit, 200 µl aqueous solution extracted and eluted into 55 µl. Extractions were performed according to manufacturers’ instructions except that RNA was extracted from 50 µl of stool suspension for all four protocols with addition of respective suspension buffer to reach the recommended aqueous volumes listed above. The RNA was stored at −70°C pending analysis and between analyses  was used for the amplification and detection of the MS2 and Enterovirus RNA by TaqMan technology as previously described Data were managed and analyzed using Excel (Microsoft) and SPSS (ver. 15) software. For each extraction protocol, the presence and relative amount of rRT-PCR inhibitor(s) was determined by comparing MS2 rRT-PCR Ct results obtained in the absence or presence of stool suspensions. Measured MS2 Cts results were assigned to one of five levels of inhibition: no inhibition, inhibition of 1 to 3 Cts (2 to 8 fold), inhibition of 4 to 6 Cts (16 to 64 fold), inhibition of 7 to 9 Cts (128 to 512 fold) and inhibition of ≥10 Cts (≥1024 fold). Similarly, the presence and relative amount of inhibitors of enterovirus RNA rRT-PCR was determined by comparing enterovirus rRT-PCR Ct results obtained in the absence or presence of stool suspensions for enterovirus added before extraction or enterovirus RNA added after extraction. The inhibition for suspensions that gave Cts for MS2 rRT-PCR below that for MS2 in suspension buffer alone was set to 0. The upper limit for rRT-PCR inhibition was capped at 29 Ct.The non-parametric Friedman Test was used to determine to determine whether there were significant differences between results among the four different extraction protocols. Post-hoc analysis of pairwise differences between protocols was performed using a Wilcoxon Signed-Rank Test (SPSS) with a Bonferroni correction that set the significance level for the pairwise comparisons to P<0.008. Comparison of MS2 rRT-PCR and enteroviral rRT-PCR Cts was by repeated measurements, analysis of variants.Figure S1MS2 rRT-PCR inhibition in RNA extracted from stool suspensions using four different RNA extraction protocols. Equal amounts of stool suspensions chosen randomly from among samples sent to the laboratory were extracted by four protocols: (A) QIAgen, (B) magNA Pure, (C) KingFisher, and (D) easyMag as described in Methods. MS2 coliphage calculated to give 27 Ct by rRT-PCR was added to the extraction buffer. rRT-PCR values for MS2 in RNA extracted from buffer controls were subtracted from the values for MS2 in RNA extracted from stool suspensions. This difference, the number of Cts of inhibition, is shown in the box to the right of the sample numbers. Negative values were set to 0 and the maximum values for inhibition “C” were capped at 29 Cts. Samples with inhibition >10, 7 to 9, 4 to 6, and 1 to 3 Ct are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. Blank white boxes indicate no inhibition. The numbers of samples in each category and the significance in differences are shown in (PDF)Click here for additional data file.Figure S2MS2 rRT-PCR inhibition for RNA extracted from stool after re-freezing and thawing by extraction protocol. 1. Three repeated measurements of MS2 rRT-PCR were performed for the 23 samples by protocols: (A) QIAgen, (B) magNA Pure, (C) KingFisher, and (D) easyMag as described in Methods. The RNA was stored at −70°C between tests. MS2 coliphage was added to the extraction buffer. rRT-PCR values for MS2 in RNA extracted from buffer alone were subtracted from the values for MS2 in RNA extracted from stool suspensions. This difference, the number of Cts of inhibition, is shown in the boxes to the right of the sample numbers. Negative values were set to 0 and the maximum values for inhibition “C” were capped at 29 Cts. Samples with inhibition ≥10, 7 to 9, 4 to 6, and 1 to 3 Ct are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. Blank white boxes indicate that the samples were not inhibited. Samples were stored at −70°C between measurements. 2. The means for each of the three repeat tests the square of the means (M Sq), the standard deviation of each run (S.D.), and the significance of differences between the means of the repeated measurements by extraction protocol are presented. There were no significant differences between the repeat measurements for each extraction protocol. 3. Friedman test values for comparison among the 4 extraction methods. There were significant differences among the extraction protocols (shaded yellow boxes). 4. The Wilcoxon Singed-Rank Test (SPSS) with Bonferroni correction (Significance if P<0.008) for the pair-wise comparison of the means of the three measurements of the rRT-PCR results from the different extraction protocols. Shaded yellow boxes indicate where pairwise differences between extraction protocols were significant.(PDF)Click here for additional data file.


Massively parallel sequencing technology now provides the opportunity to sample the transcriptome of a given tissue comprehensively. Transcripts at only a few copies per cell are readily detectable, allowing the discovery of low abundance viral and bacterial transcripts in human tissue samples. Here we describe an approach for mining large sequence data sets for the presence of microbial sequences. Further, we demonstrate the sensitivity of this approach by sequencing human RNA-seq libraries spiked with decreasing amounts of an RNA-virus. At a modest depth of sequencing, viral transcripts can be detected at frequencies less than 1 in 1,000,000. With current sequencing platforms approaching outputs of one billion reads per run, this is a highly sensitive method for detecting putative infectious agents associated with human tissues. Infectious agents (IAs) are a common cause of acute and chronic human diseases. For example, it is estimated that approximately 20% of all human cancers are caused by an infectious agent Previous studies have focused on a targeted approach using RT-PCR or conventional detection methods to confirm disease associations with known IAs. The expansion in sequencing capacity enabled by massively parallel sequencing platforms allows for the first time complete and unbiased surveys of the human genome, transcriptome and microbiome. Several recent studies have demonstrated the utility of this approach for detecting IAs; examples include the discovery of a novel polyomavirus in Merkel Cell carcinoma Although many investigators are using metagenomic sequencing for the purpose of detecting novel IAs, a rigorous empirical study of IA detection level in human tissues is missing. This kind of experiment is necessary to determine sequencing depth and to establish precisely the amount of IA expression that can be reliably detected. Such an experiment also provides a proof of principle for the methodology, by illustrating that known IAs can be detected if present at a certain level of abundance. This information will be useful for any group attempting IA detection using sequence data generated on a massively parallel platform. The data presented here establishes a detection limit using RNA sequencing data from a human colorectal tumour biopsy tissue spiked with viral RNA.th the number of bases (10 Gb) vs. the 100 Gb required for whole genome sequencing. Transcripts at only a few copies per cell are readily detectable, allowing the discovery of even low abundance viral and bacterial transcripts. Transcriptome analysis also has the advantage of only reporting actively expressed transcripts rather than just the presence of a particular sequence. In the case of IA gene expression, this implies a much greater likelihood of disease involvement, especially if expression levels can be compared to corresponding normal tissue from the same individual.We have implemented a bioinformatics strategy  for the Heterosigma akashiwo (Raphidophyceae) The capability to detect candidate infectious agents in a human sequencing library was evaluated using spiked-in RNA virus and computational host sequence subtraction. In order to determine sensitivity experimentally, a known IA was required as a template. RNA isolated from a single stranded RNA virus was chosen to enable testing of the entire procedure, including cDNA synthesis and sequence library construction. A non-human virus was selected to ensure all detected reads would be derived from the spiked material and not from native expression. The virus chosen was Heterosigma akashiwo RNA virus (HaRNAV), which infects the toxic bloom-forming unicellular alga, HaRNAV RNA was quantified by Nanodrop® and added over 4 log dilutions to aliquots of total RNA extracted from a snap-frozen human biopsy sample  and 3,536 (72.6 Mbp) viral refseq sequences . It is publicly available for download at ftp://ftp.bcgsc.ca/supplementary/IADB/. For alignments to the IAdb, Novoalign (http://www.novocraft.com/) was used  which reports all locations and genomes mapped equally well by a given read pair. Novoalign is a more permissive but slower short read aligner than bwa that takes into account quality scores. These features make it more suitable than bwa for identifying metagenomic sequences, which may have limited similarity to the reference sequences in IAdb. Counts of read pairs matching each Genbank accession in our IAdb were obtained. However, for the purpose of this study, we only tallied read pairs that mapped unambiguously, that is, where both members of the mate pair matched the same accession and with the correct pairing distance and orientation.Read pairs that remained unaligned to any of the human sequence databases were used to interrogate a custom database that contains all completely sequenced bacterial and viral genomes from Genbank. This in-house database, which we call IAdb is frequently updated to remain current. The version of IAdb used for this manuscript was composed of 27,699 sequences totaling 3.9 Gbp, including 23,163 sequences from 1,016 bacterial genomes.  it speeds up the final alignment to IAdb, by weeding out decreasing number of pairs aligned against increasingly larger sequence databases and 2) it helps remove the background noise. For the present study alignments were run on a single 3 GHz 8 CPU Intel(R) Xeon(R) 64-bit 61 GB RAM computer running CentOS release 5.4.These data illustrate robust recovery of both the HaRNAV RNA and the phage DNA in terms of positive hits with the expected pairs per million being proportional to those observed (Pearson correlation >0.99). However the number of RNA viral read pairs in each library was about 1/3 of that expected, based on the amount of material spiked into the library. This discrepancy may be due to the difficulty of accurate quantification of ssRNA viruses coupled with a preferential capture of smaller RNA products in the library preparation. We have demonstrated with the phage spike in that recovery was close to expected after the cDNA stage again indicating that the bias was at an early stage of library preparation. From the simulated data presented in Nevertheless, minute amounts of viral RNA were detected, corresponding to an average of less than one copy per cell  and DNA virus Human herpesvirus 4 (NC_009334.1) genomes, respectively and ran these faux read pairs through the IA detection pipeline. The simulation tested the effect of read lengths of 36, 50, 76 and 100 nt and error rates by simulating HaRNAV and herpesvirus genomes having up to 5% simulated error.We observe that mapping is minimally impacted by errors, consistent with the permissive limit on the mapping accuracy of Novoalign, the aligner used to search IAdb . Becausepol) and its closest known viral genome homologue (FJ979639.1) deposited in the Genbank NT database, a region of the Simian retrovirus 4 (SRV4) strain having 60% sequence identity with HERV-K, were chosen for the simulation. When reads were simulated with no errors, none of the HERV-K pairs passed the pipeline beyond human transcriptome alignments and all the SRV4 were recovered. When we applied a 5% simulated mutation rate to both sequences and simulated 76 nt paired reads, we observe that in average 76+/−35.4 read pairs escaped the transcriptome filtration for HERV-K when testing 1000 pairs, due to corresponding read pairs that accrued more than bwa's maximum allowable limit of 3% errors. The number of unfiltered pairs is low despite a substantial (5%) mismatch rate, because when at least one read within a pair still maps to a human sequence target with more than 97% accuracy the pair is removed. This is in fact the case for 92.4% of pairs simulated from an HERV-K sequence having 5% randomly distributed errors. In contrast, none of the SRV4 faux pairs are subtracted, since the sequence is sufficiently divergent from human sequences, including HERVs. The pipeline is thus conservative in that it is designed to stringently remove as much of the host “noise” as possible before the final, more permissive mapping to viral/bacterial genomes.The mapping accuracy of bwa (<3%) could be limiting in identifying retroviruses whose identity is close to that of human endogenous retroviral (HERV) sequences, for instance, but unrestrictive if sequences are sufficiently (>3%) divergent. To test this hypothesis, we challenged the IA detection pipeline in identifying and subtracting HERV-derived read pairs while detecting HERV-like viruses-derived pairs. HERVs are remnants of ancient retroviral genomes integrated in human germline DNA, some of which are transcribed. A HERV-K sequence (DQ821442.1) encoding a reverse transcriptase  and herpesvirus 4 (NC_009334.1) genome sequence entries, respectively. Whereas we do not detect any HaRNAV reads in the former experiment because of lack of similar entries in IAdb, we detect herpesvirus pairs, due to the overrepresentation of highly similar herpesvirus species in public sequence data repositories . This hiThere is a worldwide effort towards sequencing the human microbiome In contrast to previous metagenomic IA detection approaches that used pyrosequencing Finally, it is well understood that the discovery of an IA association with disease is only the first stage in determining causality. Further validation studies to determine statistical significance in larger sets are needed as well as independent evidence of pathogenicity.Heterosigma akashiwo (NEPCC 522). Cultures (400 mL) were grown in 1 L Erlenmeyer flasks  in a 14∶10 light:dark cycle  at 19°C, and the viruses amplified as outlined in Tai et al. (2003).HaRNAV was amplified using the Raphidophyte 2 and incubated with 0.1 U/µL of RNase A (Invitrogen) and 1 U/µL of Amplification Grade DNAse 1 (Invitrogen) for 3 h at room temperature. The viruses were extracted with Trizol (Invitrogen) as per manufacturer's instructions and resuspended in 20 µL RNaseAway (Invitrogen).HaRNAV was concentrated by ultracentifugation of 78 mL of lysate at 108 000 x g for 5 h, and the pelleted virus resuspended in 75 µl of supernatant. The concentrated virus was brought to a final concentration of 5 mM MgClSynechococcus strain DC2 (WH7803) as the host −2 s−1. The viruses were amplified by adding the bacteriophage to 500 mL of exponentially growing Synechococcus at a virus to host ratio of 1 Bacteriophage S-PWM3 was isolated from the Gulf of Mexico using the cyanobacterium Sample tissue, a colorectal tumour biopsy, was acquired from the BCCA Tumour Tissue Respository (TTR) as a frozen biopsy. A maximum of 30 mg of tissue was homogenized in 600 µl RLT buffer (Qiagen) and passed 5 times through a syringe fitted with a 20 G needle. RNA was purified using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. Genomic DNA contamination was reduced using an on-column DNase I treatment according to the kit protocol.Total RNA was quantified using Qbit. RNA quality was assessed using Agilent Bioanalyzer 2000 RNA nanochip. 2 µg of total RNA was used in each library construction. Briefly, ribosomal RNAs (rRNAs) were depleted from total RNA by following the protocol of RiboMinus Eukaryote Kit for RNA-Seq . Libraries were then constructed according to BCCA Genome Sciences Center RNA-Seq paired-end library construction protocol with an approximate 200 bp inset size Libraries were sequenced on the Illumina GAIIx system using a paired end strategy at a read length of 75 bases.http://www.novocraft.com).Alignments were performed using BWA v0.5.4 [sample -o 1000] In order to determine the detection level, the size of the viral RNA genome (4.4 Kb) was used to calculate the mass of the genome, producing an estimate that 1 pg of viral RNA constitutes approximately 150,000 copies. In order to estimate the number of viral genomes per cell, we assumed that an average cell has 20 pg of total RNA. Therefore 2 µg of total RNA, as used in our library preparation, equates to approximately 100,000 cells.


Self-Rated Health (SRH) correlates with risk of illness and death. But how are different questions of SRH to be interpreted? Does it matter whether one asks: “How would you assess your general state of health?” or “How would you assess your general state of health compared to persons of your own age?”(Comparative SRH)? Does the context in a questionnaire affect the answers? The aim of this paper is to examine the meaning of two questions on self-rated health, the statistical distribution of the answers, and whether the context of the question in a questionnaire affects the answers.Statistical and semantic methodologies were used to analyse the answers of two different SRH questions in a cross-sectional survey, the MONICA-project of northern Sweden.The answers from 3504 persons were analysed. The statistical distributions of answers differed. The most common answer to the General SRH was “good”, while the most common answer to the Comparative SRH was “similar”. The semantic analysis showed that what is assessed in SRH is not health in a medical and lexical sense but fields of association connected to health, for example health behaviour, functional ability, youth, looks, way of life. The meaning and function of the two questions differ – mainly due to the comparing reference in Comparative SRH. The context in the questionnaire may have affected the statistics.Health is primarily assessed in terms of its sense-relations (associations) and Comparative SRH and General SRH contain different information on SRH. Comparative SRH is semantically more distinct. The context of the questions in a questionnaire may affect the way self-rated health questions are answered. Comparative SRH should not be eliminated from use in questionnaires. Its usefulness in clinical encounters should be investigated. When questions about self-rated health (SRH) were first included in questionnaires, this was done in parallel to the social greeting “how are you?” This implied a conversational way of introducing questions about health matters, intended to form a relationship and show interest and care . When coHowever, this simplistic, straightforward interpretation has subsequently been revised. The answers to SRH questions correlate with life habits, disease, physical functional ability, symptoms, education, income, wealth, social capital, age, sex, parental health, attitudes etc -9. InterTwo alternative formulations are often used in questionnaires. These could take the form: “How is your health in general?”  and “How would you judge your health compared to other people of your age?” (Comparative SRH-question) . It has Our research questions are: 1) Are their differences in the statistical distribution of the answers to the two formulations of the question? 2) What are the semantic meanings of a General SRH-question and of a Comparative SRH-question, and how do these meanings differ? 3) Does the context for the question influence the answers given?Questionnaires from the MONICA project  in North Sweden issued in 1990, 1994, 1999, were investigated . The MONThe questionnaire was divided into two parts. Part 1 was answered at home and returned by post. Part 2 was answered in combination with a medical investigation and sample testing at the nearest health care unit. Forty units were involved. Two questions on self-rated health were included. In 1990 and 1994, these were worded:” How would you assess your general health condition – good, bad, or somewhere in between”  and” How would you assess your general health condition compared with other persons of the same age - better, worse, similar?” (Comparative SRH). Since answers to both SRH questions were separated into three alternatives for those years, they could be used without regrouping the primary data. The response patterns were analysed statistically. A frequency calculation was carried out on answers to the SRH questions, and the Spearman Rank-ordered correlation coefficient calculated using SPSS18.The answers from questionnaires in 1994 and 1999 were used to study the influence of a different context for the Comparative SRH question, since this had been changed in the meantime. The General SRH question was not analysed as it was reformulated in 1999 to a five-grade scale making comparison with previous years less meaningful. Statistical significance was estimated with the Chi-2 test.For the semantic analysis a basic semantic methodology was employed . SemantiThe investigation was carried out within the approval for the Northern Sweden MONICA-study by the Research Ethics Committee of Umeå University. All subjects gave informed consent to participate.There were 1583 responses to the MONICA questionnaire in 1990, and 1921 responses in 1994. The response rates were 79% and 77% respectively. Table Table Figure The response distribution to the Comparative SRH question changed significantly between 1994 and 1999. In 1994, 1921 persons answered while in 1999, 1823 persons answered. They were aged 25-74 years and selected in the same manner in 1994 and 1999. The response rates were 77% and 73% in 1994 and 1999 respectively. The proportion selecting the alternative “better” increased from 15% to 22%, i.e. a 7 percent units increase, between the two years .The chosen regions of North Sweden were homogeneous from a linguistic and cultural perspective. Economic, social, and educational differences were small [  in relation to (something else) to find the similarities or differences. Its sense-relations are coupled to words like grading, order, assessing, and classifying. The word “persons” directs attention to individuals, not an anonymous group of people. The direction to compare with other people of one’s own age leads to domains of significance such as social comparison, performance, profession, income, and achievement in life. Such comparisons are coupled to ranking, and the creation of hierarchies and value structures. Comparisons are used frequently in both everyday speech and writing. Comparative SRH provides the respondents with a reference system, namely “compared with other persons of the same age”. This gives the answers a more determinate character and provides less room for purely subjective evaluation. The alternative replies to the Comparative SRH question consist of the comparing adjectives, “better”, “worse” and “similar”. Thus, the answer is influenced by the double challenge to compare, firstly what is given by the question itself, and secondly by the alternatives provided.In 1994, the SRH questions were placed in the section that was sent out by post and answered at home. The SRH questions followed each other in the questionnaire, and were placed in a context concerning diseases and cardiovascular mortality of close relatives. A drawing implying old grandparents accompanied the questions. In 1999, the SRH questions were moved to the section that was completed in combination with the visit to the health care unit for medical investigation. The drawing of grandparents was removed and the SRH questions were opening questions, implying that the other questions – which might have reminded the respondents of negative health outcomes or hereditary disease in the family - had less influence on how they were understood and answered.Statistical partitioning between the alternative answers differed significantly for the two questions. The study replicated already known differences in answers to General and Comparative SRH. The General SRH question did not imply comparison whereas the Comparative SRH question did, both directly and by the answering alternatives given. The comparison involved “sense-relations” coupled to the word “health”. The word “persons” steered the question towards concrete comparisons. Both words might, in this context, imply a comparison involving a stratification of social status, which are well-known to influence health . The ansThe changed response distribution between 1994 and 1999 to the Comparative SRH question indicates that the answers might have been influenced by the considerable change in the context that had taken place between those years.The study was based on good quality data. The data could be processed without recoding alternative answers, and connects directly with the choices made by participants. This helps in understanding how the questionnaire functioned for the participants. The semantic method was based on well-established principles. The validity of the analysis must be judged with criteria appropriate for the method, namely proper use of concepts, logical consistency, internal coherence and plausibility. Our conclusion about a semantic difference between the General and Comparative SRH questions was corroborated by statistical analysis of the answer distribution. In addition, the semantic analysis provided an explanation of the numerical outcome, which further empirical investigations would not have yielded. That the outcome of the Comparative SRH question changed between 1994 and 1999 also satisfies semantic theories about the influence of situation and context for the interpretation and function of utterances and questions.Semantic theory is applicable to all natural languages. Therefore we argue that the semantic method can be generalised to other languages and cultures. However, the numerical distribution of answers and correlations from this study cannot be generalised to other languages and contexts.Findings from earlier studies are reproduced in our material both as of correlation to age and distribution of answering alternatives. A relationship between education and SRH has also been demonstrated in earlier studies ,11. In aOur material in 1994 and 1999 revealed a statistically significant increase in the number of people giving the answer “better” to the Comparative SRH question. The placing of the question was changed between the two years (see Results/Context for the questionnaire). The change in the answer distribution might be explained by a health improvement in the population sample. However no similar increase of “better” was observed in the neighbouring county of Västerbotten, where the positioning of the Comparative SRH question remained unchanged in a similar questionnaire. The response rate in the MONICA study fell by 4% between 1994 and 1999. This might have resulted in a shift towards the alternative “better” in 1999. However, the increase remained in the following surveys, indicating that the lower response rate in 1999 is not the main explanation for the shift to the alternative “better”. The results suggest that part of the explanation for the 7 percent unit increase between 1994 and 1999 of the answer “better” was the changed context for the SRH-questions.Our semantic analysis highlights that the SRH questions are unclear and imprecisely formulated from a scientific point of view, with concepts poorly defined. It may be thought strange for such questions to have found their way into a scientific questionnaire. Their status in the scientific context is defended by their empirically firm link to important health outcomes. Nevertheless, there are theoretical contradictions in how the concept of self-rated health is treated in the scientific literature. Objective health is often regarded as the norm and target for subjective health assessments, while subjective assessments of health are regarded as less accurate rather than different. The correlations of SRH questions with health outcome show that simple everyday speech should not be underestimated as a source of information, in spite of its apparent vagueness.Health comparisons are frequently made downwards, i.e. with people in worse condition ,18,19. TOur analysis showed that the General and Comparative SRH questions have semantically different meaning. Consequently one cannot assume that the answers represent parallel judgements of self-rated health. Instead, they are different ways of making an overall health assessment. Similar conclusions were reached by Sargent-Cox et al. in a multiple regression analysis. They state “These results show that the three SRH items are not equivalent measures of health and cannot be used interchangeably” .Vuorisalmi et al. claimed that the age-compared estimate did not measure objective health conditions in the same way for different age groups . They coSemantic analysis brings insight into how questions of Self-Rated Health could be interpreted.Health is not assessed primarily in terms of the dictionary denotation of health, but of its sense-relations (associations).Comparative SRH and General SRH contain different information on SRH, comparative SRH is semantically more distinct but both can be used in questionnaires.Contexts surrounding the questions in a questionnaire might be or importance for the answers and should therefore be explored in further studies.Investigate the usefulness of SRH in clinical encounters and its function as a key question.The authors declare that they have no competing interests.GW conceived of the study and wrote the manuscript. PT discussed the semantics and philosophical part and drafted the manuscript. UJ supervised the statistical and epidemiological part and the use of the MONICA-database. KH read several drafts of the manuscript and helped in focusing on more essential parts. AF supervised the project, continually read, criticized and made alternative texts. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/12/154/prepub


A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1) the fundamental veto on "foresight evolution", 2) modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3) the updated library of aa-binding sites in RNA aptamers successfully selected in vitro selection of aa-specific RNA aptamers apparently had nothing to do with translation, this striking preference provides a new strong support to the notion of the genetic code emerging before translation, in response to catalytic (and possibly other) needs of ancient RNA life. Consistently with the pre-translation origin of the code, we propose here a new model of tRNA origin by the gradual, Fibonacci process-like, elongation of a tRNA molecule from a primordial coding triplet and 5'DCCA3' quadruplet (D is a base-determinator) to the eventual 76 base-long cloverleaf-shaped molecule.The aa-binding sites of arginine, isoleucine and tyrosine contain both their cognate triplets, anticodons and codons. We have noticed that these cases might be associated with palindrome-dinucleotides. For example, one-base shift to the left brings arginine codons CGN, with CG at 1-2 positions, to the respective anticodons NCG, with CG at 2-3 positions. Formally, the concomitant presence of codons and anticodons is also expected in the reverse situation, with codons containing palindrome-dinucleotides at their 2-3 positions, and anticodons exhibiting them at 1-2 positions. A closer analysis reveals that, surprisingly, RNA binding sites for Arg, Ile and Tyr "prefer"  the anticodon(2-3)/codon(1-2) tetramers to their anticodon(1-2)/codon(2-3) counterparts, despite the seemingly perfect symmetry of the latter. However, since Taken together, our findings necessarily imply that primordial tRNAs, tRNA aminoacylating ribozymes, and (later) the translation machinery in general have been co-evolving to ''fit'' the  genetic code, rather than the opposite way around. Coding triplets in this primal pre-translational code were likely similar to the anticodons, with second and third nucleotides being more important than the less specific first one. Later, when the code was expanding in co-evolution with the translation apparatus, the importance of 2-3 nucleotides of coding triplets "transferred" to the 1-2 nucleotides of their complements, thus distinguishing anticodons from codons. This evolutionary primacy of anticodons in genetic coding makes the hypothesis of primal stereo-chemical affinity between amino acids and cognate triplets, the hypothesis of coding coenzyme handles for amino acids, the hypothesis of tRNA-like genomic 3' tags suggesting that tRNAs originated in replication, and the hypothesis of ancient ribozymes-mediated operational code of tRNA aminoacylation not mutually contradicting but rather co-existing in harmony.This article was reviewed by Eugene V. Koonin, Wentao Ma (nominated by Juergen Brosius) and Anthony Poole. Genetic Code refers to a nearly universal assignment of triplets (codons) of nucleotides (nts) to amino acids (aas), linking hereditary entities to the functional blocks of life Figure . In pracUnraveling the origin of the genetic code and translation machinery is an inherently difficult problem . On one Furthermore, the very existence of the two languages  implies an evolutionary motivation. This is a great challenge to evolutionists, even at the most abstract conceptual level of reasoning. When seeking, for example, the arguments for perhaps the most fateful transition in life history - from ribozymes to enzymes  - we canvia a closer analysis of:The detailed treatment of the pre-translational origin of the genetic code is a whole separate topic that will be reviewed elsewhere . In this report, we consolidate our arguments for the "pre-translational" code 1. The fundamental veto on "foresight evolution"2. The recently updated library of direct RNA-aa binding sites --- possibly pointing at the earliest " era" in the history of the genetic code , andper se . The two are radically different when it comes to the veto on "foresight evolution". The first ("key-lock") idea assumes some sort of direct stereochemical affinity between amino acids and oligonucleotides that are similar to, or just contain, anticodons (or codons) -16. AppaThis is clearly not the case with the other ("adaptor") idea, stipulating existence of intermediate molecular agents capable of recognizing both an amino acid and a corresponding codon in mRNAs simultaneously . The logad infinitum )The rest of the paper is a rather involved discussion of the potential relics of the pre-translation stages in the tRNA structures, in part, recapitulating the previous publications of the authors. The idea with the Fibonacci-like iterative process of tRNA evolution is of course very elegant and numerologically appealing but the entire scheme is quite speculative.Translation without code does not make sense, but code without (and before) translation does". I think this is good logic. I also applaud with the authors' approach of combining many different lines of evidence in approaching this formidable evolutionary puzzle. However, my overall position is that we still have not developed the right way to approach this problem, so all current scenarios are likely to be wrong in most details. In this situation, what matters is the quality of the discourse, and in this respect, the current paper will be helpful to any researcher interested in the problem.So does this paper report progress in our understanding of the origin and evolution of translation and the code? It is hard to give a 'yes' or 'no' answer. The paper ends with the overall conclusion that "We have greatly appreciated the reviewer's comments and critique; our response is structured in the following three sections:in vitro experiments with aa-specific RNA aptamers), the crucial "key-lock vs. frozen accident" alternative. Naturally, in vitro selection of RNA aptamers (aimed at an increasingly more specific recognition of a particular amino acid) has little, if anything, in common with shaping of the genetic code that actually took place in early evolution. Apparently, the real code developed stepwise following the classic scheme of gene duplications with subsequent specifications, regardless of whether it have started with a very specific stereo-chemical "key-lock", a true "frozen accident", or possibly something in between --- a "frozen stereo-chemical accident" /codon(1-2) tetraplets in particular --- are all the more impressive.Just like the singularity point in the origin and evolution of universe, the actual start of bilingual life  was, is, and probably will always remain obscure. In such an "agnostic" (in Huxley's meaning) situation, one of the very few intellectual pursuits that a theoretician could afford to accept as not being hopelessly speculative would be to examine, by all conceivable means  ones being: (1) was this interaction absolutely random, or stereo-chemically selective (at least weakly), and (2) did this interaction have anything in common with the real genetic coding?how to account for this striking level of significance by any other means?The similarity of aa-binding sites in RNA aptamers independently selexed for a given amino acid casts doubt on the randomness. Moreover, the significant excess of cognate anticodons within these sites strengthens the hypothesis of underlying stereo-chemical preference. The RNA aptamer data are statistically compelling in this regard , and thPreviously, one of us (ES) has formulated this precisely: logic dictates the hypothesis of direct interactions between RNA and amino acids in the emerging RNP life, but corresponding binding sites may not have anything to do with the current cognate triplets . In thisTwo of us (SNR and ASR), just like the Reviewer 1 (and for similar reasons), initially also made light of the stereo-chemical hypothesis until (1) revealing the sub-code for two modes of tRNA recognition by aaRSs Figure , and (2)per se). Quite the opposite, in a series of papers [in vitro selection of aa-specific RNA aptamers [Aminoacylation of a tRNA molecule from two, major and minor grove, sides Figure  is suppof papers -20,35,36f papers ,26 and tf papers -20,35,36f papers ,26 and iaptamers  complemeAs far as Ala, Gly or any other (presumably) primordial amino acids are concerned, at this time we are not aware of any reports stressing not the excess (or deficiency) of their cognate anticodons (codons) in aa-binding sites of RNA aptamers, but rather the successful selection of such RNA aptamers at all. Thus, it remains unclear whether such excess in fact exists; further, more refined, experimental attempts with selection of RNA aptamers for such amino acids would be most welcome.Wentao Ma, College of life sciences, Wuhan University, P.R. China, nominated by Juergen Brosius.The origin of genetic code and translation system is a problem (or two related problems) full of controversies. The reason is that the translation system is very complicated and the coding principle is not clear . According to the Darwinian Continuity principle (i.e. "evolution has no foresight") , the emeBased on the analysis on aptamer data of three amino acids containing both their anticodons and codons, the authors asserted reasonably that such cases should be associated with the palindrome-dinucleotides. Together with data of other amino acids in  and also"For amino acids encoded by dinucleotide-palindrome-containing triplets, their binding sites in RNA aptamers "prefer" the codon(1-2)/anticodon(2-3) motifs over the codon(2-3)/anticodon(1-2) ones in spite of their seemingly perfect symmetry." This conclusion seems also to be supported by the data in . Indeed,"The motif 5'-CGCG-3' is of a special interest Figure , becauseI think that this description has some problems. The corresponding motif should be 5'-GCGC-3' when CGC is a codon(1-2) and GCG an anticodon(2-3) for CG palindrome, while the corresponding motif should be 5'-CGCG-3' when CGC is a codon(2-3) and GCG an anticodon(1-2) for GC palindrome."This ambiguity is fraught with confusion for coding. Very telling, therefore, is the fact that Arg-binding sites do not contain the 5'-CGCG-3' motif at all, in contrast to the above three motifs of the codon(1-2)/anticodon(2-3) type. This fact becomes even more telling if we take in account presence of 5'-CGCG-3' beyond aa-binding sites /anticodon(1-2), thus not appearing. If so, a directly contrary motif should be 5'-GCGC-3' (as I mentioned above), which should be abundant. However, the analysis or comment on 5'-GCGC-3' does not appear in the manuscript.We found the reviewer's comments and critique particularly incisive; the two major issues are discussed below.including 5'-GCGC-3'.First of all, one should take into account that for arginine these tetraplets, 5'-CGCG-3' and 5'-GCGC-3', are both codon(2-3)/anticodon(1-2) relative to the GC palindrome and, simultaneously, are both codon(1-2)/anticodon(2-3) relative to the CG palindrome, the only difference being the dinucleotide with which anticodon GCG and codon CGC overlap --- GC in the 5'-CGCG-3' case, and CG in the 5'-GCGC-3' case. Accordingly, if anticodon GCG is a driver, and GC palindrome is under consideration, then the corresponding tetraplet containing Arg's codon is 5'-CGCG-3' (and only 5'-CGCG-3'). On the contrary, if anticodon GCG is again a driver, but it is CG palindrome that is under consideration, then the corresponding tetraplets are 5'-GCGN-3', deliberately focus our description on 5'-CGCG-3'. The result - absence of this motif in Arg-binding sites - speaks for itself. Remarkably, in independently selexed Ile-binding sites we also do not see (and probably exactly for the same reason) the 5'-AUAU-3' tetraplet in which anticodon UAU supposedly drives codon AUA as a hitch-hiker but via the central Ile-irrelevant UA palindrome .Importantly, it is the CG at 1-2 positions of codon (2-3 positions of anticodon) that actually specifies Arg, whereas the GC at 2-3 positions of codon (1-2 positions of anticodon) is irrelevant (specifying Ala). It thus appears that codon CGC might have "formally" accompanied (as a hitch-hiker) anticodon GCG in 5'-CGCG-3' via the Arg-unrelated GC dinucleotide, and that is precisely why we chose to complete absence of all others, including the 5'-GCGC-3'. Again, one can see exactly the same pattern in Ile-binding sites with anticodon UAU as a driver: a great excess of the 5'-UAUU-3' and complete absence of all others, including the 5'-UAUA-3'  excess of 5'-GCGG-3' and (2) ' Figure .coding triplets in Ile-binding sites preclude confusion with Tyr (and vice versa) as if the RNAs selected for direct affinity to a specific amino acid have already been protected from confusion with its complementarily encoded partner. This is surprising, because the RNA aptamers have not been specifically selected to avoid non-cognate amino acids. We have decided to address this issue in a more nuanced fashion elsewhere ; however, the reviewer's remarks led us to restoring the original paragraph of the manuscript here, in the present discussion.Furthermore, the UAU anticodon of Ile is a codon for Tyr. However, the latter is not significantly overrepresented in Tyr-specific sites of RNA aptamers. Symmetrically, AUA is a significantly overrepresented triplet, as an anticodon, in Tyr-binding sites but not, as a codon, in Ile-binding ones. Thus, it appears that these coding tetraplets, 5'-UAUU-3' and 5'-AAUA-3', cannot be confused. In contrast, coding tetraplets (in putative r-aaRSs) formed by complementary anticodon and codon, are indeed confusion-prone simply because any such tetraplet is a perfect self-complementary palindrome. For example, Ile's anticodon UAU and its complement AUA form the 5'-UAUA-3' palindrome, the complementary image of which is, again, 5'-UAUA-3'. Same holds for tyrosine as well as complementarily encoded arginine and alanine. In such cases, the two complementary r-aaRSs might have had virtually the same aa-coding sites, leading to the high risk of wrong aminoacylation --- accordingly, they would have been selected against.Tyr-binding sites show overrepresentation of the characteristic 5'-RAUA-3' motifs (with AUA anticodon): 5'-GAUA-3' and 5'-AAUA-3', which is a precise complement of the characteristic 5'-UAUU-3' motif (with UAU anticodon) that happens to be overrepresented in Ile-binding sites . Therefore, the detection of these mutually complementary and "non-confusable" tetraplets, 5'-UAUU-3' and 5'-AAUA-3'  within Ile- and Tyr-binding sites of independently selected RNAs is all the more meaningful. At this time, we do not suggest any mechanistic explanation for this somewhat surprising result but it shows us that there might have been, indeed, certain structural prerequisites for two complementary modes of tRNA recognition by aaRSs, firstly ribozymes then enzymes. Moreover, this result indirectly supports, in our opinion, two basic hypotheses: (1) that primary stereo-affinity did play an essential role in the origin of the genetic code, and (2) that even before translation amino acids might have been engaged in coding by complementary pairs rather than one by one. The above immediately suggests a relatively simple idea for the selex experiment control - to use both plus and minus RNA sequences in selection of aa-binding RNA aptamers, so that selection for one aa-binding site would facilitate starting conditions for selection of its complementary partners.Obviously, the correct logical flow should be in reverse: selection for stereo-affinity of a given amino acid  to the oligonucleotides containing this aa-cognate anticodon UAU within the 5'-UAUU-3' tetramer automatically entails selection for the presence of AUA within 5'-AAUA-3' in the complementary sequence which happens to be preferred by Tyr - the Ile's complementary partner To our knowledge, there were no reports of successful selection of RNA aptamers for Ala, the complementary partner of Arg  the foresight evolution. And, the risk of ambiguous anticodon-to-aa assignment, while originally of crucial importance for primordial coding in the RNA world, was of less, if any, relevance for translation which most likely evolved later. In the RNA world, errors of RNA aminoacylation might have been serious enough to be selected against. In conclusion, this remains a high priority task on our agenda - to find out which component of the genetic coding ambiguity has been minimized before (and outside of), and which --- after (and in co-evolution with) the development of translation machinery."Literally: 3 + 4 = 7, 4 + 7 = 11, 7 + 11 = 18, 11 + 18 = 29, 18 + 29 = 47, and finally 29 + 47 = 76!" This seems to be a coincidence. The Fibonacci-like iterative process in the model of tRNA growth Figure  is not bActually, there are quite a lot other interpretations based on the authors' previous work that seems to be problematic because there is not a consideration on those intermediates steps involve in the origin of translation system, for example, those concerning r-aaRSs and the operational code. The r-aaRSs ("implementers") should be a set of functional RNA molecules in addition to the set of tRNAs ("adaptors"). If they should have existed, what is the original source of these RNA molecules before they were recruited into the translation system? In addition, what should their structure be like to implement their function? Similarly, those interpretations concerning the operational code are also obscure. If the operational code should indeed have worked in the RNA world, and have occurred before the emergence of the anticodon loop, what advantage would drive the later emergence of the anticodon loop? On these points, I do not mean that the events involved is impossible, instead, I tend to agree the view of Koonin and Novozhilov , namely, "a real understanding of the code origin and evolution is likely to be attainable only in conjunction with a credible scenario for the evolution of the coding principle itself and the translation system". The extended discussion in the present manuscript on these events seems to give readers an impression that there are too many assumptions without a detailed interpretation solidly based on a scenario according to the Darwinian Continuity principle. Perhaps a better choice of the manuscript is to focus its discussion on its formal schemes mentioned above, and extend the discussion only a little to the issues concerning the authors' previously work on r-aaRSs, the operational code and others.We are certainly aware of the translation problem, and we are not  staking a claim to the full, comprehensive solution --- rather, we suggest a number of clues to that effect.As far as possible intermediate advantages in evolution of the operational code, putative r-aaRSs, and tRNAs are concerned, we feel that the reviewer might, figuratively speaking, be asking too much --- and not just from the proposed scenario, but from the field of the genetic code origin research in general. Moving on to the specifics of the Fibonacci-like iteration coincidence, we would like to touch briefly on the following two aspects:First, the internal periodicity of tRNA sequences and certain other considerations suggest to many investigators that tRNA precursors were at first much shorter (why would they be longer?), but have grown afterwards. If so, the following important questions arise: (1) how is it possible to achieve the same structure in the simultaneously growing molecules? and, (2) how could this growth reflect the principle of evolutionary continuity, where the next stage inherits useful functions gained at the previous stage? For (1), we show how this could be perfectly possible, and for (2) we demonstrate that the process of Fibonacci-like iterative growth fits the continuity principle (at least outwardly). To tell the truth, it was not the yet another example of the golden ratio implementation in Nature that held particular appeal to us, but rather the idea of the regularized and coordinated growth of the initial coding tri- and tetra-nucleotides "towards" one and the same final cloverleaf, a process appealingly consistent with the continuity principle.ab simplecioribus ad complexiora, it goes without saying that, in order to make any stepwise evolutionary scenario workable, one should think about "Darwinian" motivation for each step. Gradual shaping of code adaptors into the final tRNA cloverleaf is no exception; however, we can only guess which specific agents in the "late" RNA world  could drive this process. This said, the tRNA molecule is truly a molecule "for all seasons" [Second, when studying evolution seasons"  --- it cAnthony Poole, Stockholm University, Stockholm, SwedenThis paper is a wonderful piece of detective work that at the same time synthesizes many separate observations and theories on the origin of the genetic code and tRNAs. I really found nothing in here that requires substantial comment or clarification, other than to say it is a thoroughly interesting read - real food for thought. The analysis of results of amino acid binding sites in vitro selected RNA aptamers is thorough, and the Fibonacci process-inspired model for the evolution of tRNAs is truly insightful and thought-provoking. Perhaps one of the most interesting points of this process is that it yields a very precise stepwise model wherein tRNAs can converge independently from unrelated short RNA aptamers that bind amino acids upon a common length (and structure), ultimately containing both operational and genetic codes.We are very grateful to Reviewer 3 for this encouraging, succinct and yet precise formulation of what we have actually set out to achieve when writing the manuscript.


Padina sp., Amphiroa sp. and Turbinaria sp.) and the overlying water column to natural changes in reefal carbonate chemistry. We compared macroalgal intracellular DMSP and water column DMSP+DMS concentrations between the environmentally stable reef crest and environmentally variable reef flat of the fringing Suleman Reef, Egypt, over 45-hour sampling periods. Similar diel patterns were observed throughout: maximum intracellular DMSP and water column DMS/P concentrations were observed at night, coinciding with the time of lowest carbonate saturation state. Spatially, water column DMS/P concentrations were highest over areas dominated by seagrass and macroalgae (dissolved DMS/P) and phytoplankton (particulate DMS/P) rather than corals. This research suggests that macroalgae may use DMSP to maintain metabolic function during periods of low carbonate saturation state. In the reef system, seagrass and macroalgae may be more important benthic producers of dissolved DMS/P than corals. An increase in DMS/P concentrations during periods of low carbonate saturation state may become ecologically important in the future under an OA regime, impacting larval settlement and increasing atmospheric emissions of DMS.Oceanic pH is projected to decrease by up to 0.5 units by 2100 , reducing the calcium carbonate saturation state of the oceans. The coastal ocean is expected to experience periods of even lower carbonate saturation state because of the inherent natural variability of coastal habitats. Thus, in order to accurately project the impact of OA on the coastal ocean, we must first understand its natural variability. The production of dimethylsulphoniopropionate (DMSP) by marine algae and the release of DMSP’s breakdown product dimethylsulphide (DMS) are often related to environmental stress. This study investigated the spatiotemporal response of tropical macroalgae ( Since the Industrial Revolution, atmospheric CO2 concentrations have increased from 280 ppm to ∼390 ppm; CO2 concentrations have been increasing by ∼2 ppm yr−1 since 2000 2 will cause the pH of the oceans to drop by 0.3–0.5 units by 2100 The carbonate chemistry of the oceans is regulated by a carbonate equilibrium that is driven by the dissolution of atmospheric COpCO2 in atoll and barrier reef lagoons may be higher than offshore waters Coastal habitats such as coral reefs are characterised by more extreme natural variations in carbonate saturation state compared to the open ocean, reflecting diurnal and seasonal cycles driven by biological and physical processes 2 (e.g. for photosynthesis) Lithothamnion glaciale was significantly reduced at high CO2 , whilst only the very high CO2 treatment (1500 ppm) induced a decrease in calcification in the summer L. glaciale may also be reduced under high CO2 (589–1080 µatm) 2 conditions (700 ppm), net dissolution exceeded net calcification in Lithophyllum cabiochae2 conditions (550–760 ppm) 2 (1000 ppm) and UV radiation (particularly UVB) may act synergistically to inhibit growth, photosynthetic O2 evolution and calcification in the geniculate coralline alga Corallina sessilisThe response of calcified organisms to reduced carbonate saturation state is varied depending on their carbonate structure, their method of calcification and their ability to benefit from an increase in COpCO2 conditions Padina spp. (Ochrophyta: Dictyoaceae), one of only two known calcifying brown algae, appear to thrive in CO2 vent systems, albeit with reduced calcification in the more acidified areas 2 availability 3 mesocosms dominated by macroalgae) suggest that although calcification appears to decline under high CO2 conditions, net organic production does not change In the field, calcifying organisms may decrease in abundance under high, variable Dimethylsulphoniopropionate (DMSP) is a sulphur compound produced by many marine algae and is the major precursor to dimethylsulphide (DMS), a gas that may be linked to local climate regulation through aerosol production and cloud formation Ulva lactuca and U. clathrata was not affected by elevated pCO2 conditions (up to 1514 µatm) L. glaciale, reduced pH (pH 7.7), particularly when variable, led to an increase in intracellular DMSP concentrations Emiliania huxleyi under high temperature and high CO2 (+4°C/1000 ppm) Thalassiosira pseudonana and Phaeodactylum tricornutum under high temperature and CO2 (+6°C/790 ppm) Information on the effect of reduced carbonate saturation state on intracellular DMSP concentrations in algae is limited and has provided variable results. Intracellular DMSP concentrations in the green macroalgae In tropical reef environments, intracellular DMSP may be important as an antioxidant, grazing deterrent and/or compatible solute. Recent studies also suggest that intracellular DMSP may play a role in improving tolerance to variable carbonate chemistry conditions Research was conducted on the fringing Suleman Reef, Gulf of Aqaba, northern Red Sea, Egypt  in August 2011. This reef is characterised by four distinct zones: seagrass beds nearest the shore, the reef flat 40–60 m from the shore, the reef crest 100–120 m from the shore and the reef slope which extends away from the reef crest to a depth of ∼10 m. On average, water depth was uniform across the reef platform (∼0.8 m). All research was approved by the Dahab Marine Research Center and the Marine Environmental Center, Suez Canal University, provided the permit to conduct the research.In situ water temperature, salinity and dissolved oxygen were recorded at each timepoint using a YSI Pro 2030 instrument. In situ photosynthetically active radiation (PAR) at the sea bed was recorded hourly at the reef flat and reef crest using an Apogee QSO-E underwater quantum sensor and Gemini voltage data logger.Water and macroalgal samples were taken at the reef flat  and the reef crest  over two 45-hour experimental periods, at T14, T21, T29, T38 and T45 hours (Where T0 was 00∶00 on day one). 22 Coulometer  with acidification module  and methods described by Dickson et al. In situ TA, DIC, temperature and salinity measurements were used to calculate pH, pCO2, HCO3− and CO32− concentrations and calcite (ΩCa) and aragonite (ΩAr) saturation states using CO2SYS 4] using Dickson At each timepoint, water samples were preserved for carbonate chemistry with MgClPadina sp. , Amphiroa sp.  and Turbinaria sp. . Both Padina sp. and Amphiroa sp. were found on the reef crest and reef flat; Turbinaria sp. was found only on the reef crest. Macroalgal samples (∼0.2 g) were stored in 10 M NaOH in gas-tight glass vials sealed with Pharma-Fix (Grace Alltech) crimp lids to hydrolyse all intracellular DMSP into DMS. Vials were stored in the dark until DMS quantification.Three macroalgal species were analysed for intracellular DMSP : At each sampling timepoint, water samples were collected from the reef flat and reef crest (n = 5 per location per timepoint), from 15 cm above the seabed. 50 ml water samples were filtered using a 0.7 µm depth filter (Millepore). The filtrate and filter paper were stored in glass vials sealed with Pharma-Fix septa at a final concentration of 0.33 M NaOH. These samples represented the total dissolved and particulate DMS+DMSP pool (DMSPt). Logistical constraints limited DMSPt sample collections to the first 45-hour sampling period only.In addition to the reef flat/reef crest diel comparison, water samples (n = 3 per location) were also taken at one timepoint (16∶00) along a transect perpendicular to the shore at five locations: at the seagrass bed (10 m from the shore), on the reef flat (50 m from the shore), crest (100 m from the shore) and slope (120 m from the shore) and 270 m offshore . Samples were prepared for DMSPt using the methods described above.All DMSP samples were analysed using a Shimadzu 2014 gas chromatograph (GC) fitted with a 25 m capillary column  and a sulphur-specific FPD detector . Samples were analysed within 2 months of collection. Intracellular DMSP samples were analysed by direct injection of the vial headspace into the GC injector port. Water column DMSPt samples were pre-concentrated using the purge and cryotrap technique Padina sp. and Amphiroa sp. from the reef crest and flat that was calcified was determined by recording the mass of samples before and after overnight storage in 10 M NaOH to break down the organic material. The mass remaining represented the calcified portion, which was expressed as a percentage of the original mass. The proportion of Amphiroa sp. from the reef crest that was calcified could not be calculated as the carbonate structure broke down in the NaOH preventing final mass assessment.The proportion of Intracellular DMSP measurements could not be transformed to achieve normality and homogeneity of variance, thus a non-parametric multi-comparison Kruskall-Wallis test was performed to compare the data sets. Transect DMSPt data were analysed using general linear models (test assumptions met). Analyses were conducted using Minitab V15.Generalised Additive Models (GAMs) were used to assess the relationship between the reefal abiotic parameters and measured macroalgal and water column DMSP concentrations. GAMs were conducted in R (V2.14.2) using the ‘mgcv’ package −2 s−1 at 14∶00 on day one and day two of the 45-hour experimental periods.Both the reef crest and reef flat exhibited a diel trend in water temperature: highest water temperatures were in the afternoon and lowest temperatures were at dawn . Whilst −1) or flat  was lower than the reef crest (∼2100 µmol kg−1) at all daytime sampling points; at night (21 h and 45 h) DIC on the flat and crest were similar  are available in TA did not exhibit a strong diurnal oscillation on the reef crest  was significantly lower than the crest , although this was not significant (Z <3.40) and was characterised by a relatively high variability at 21∶00 (T21+T45) and 07∶00  and low concentrations at 14∶00 T14+T38, . An exce∶00 T29, . DMSPt dAmphiroa sp. , Turbinaria sp. (reef crest) and water column DMSPt   and slope (14.67±1.11 nmol L−1) were significantly lower than the seagrass (23.38±2.04 nmol L−1) and offshore (23.89±1.29 nmol L−1) sites. The reef flat exhibited an intermediate DMSPt concentration (18.32±0.66 nmol L−1).A significant difference in DMSPt was observed between the five transect sampling sites (F= 0.001) . DMSPt oPadina sp. samples from the reef crest  and flat . The proportion for Amphiroa sp. on the reef flat that was calcified was 43.6±5.0%. The proportion of Amphiroa sp. on the reef crest that was calcified could not be determined, although visually these samples appeared to be more heavily calcified.No significant difference in the proportion of thallus that was calcified was observed between 2 utilisation and release was likely higher on the reef flat than the crest due to the high proportion of macroalgal cover on the reef flat. CO2 uptake for photosynthesis during the day (which reduces DIC) and release by respiration at night (which increases DIC) were the probable drivers of the diel pattern in DIC concentrations. This proposed biological control mechanism was also reflected in the dissolved oxygen concentrations, which were ∼160% saturated during the day (release from photosynthesis) and <60% saturated during the night (uptake for respiration) on the reef flat >Amphiroa sp. (flat) >Padina sp. (crest and flat)>Turbinaria sp. (crest). Grazing pressure may have contributed to the species-specific and diel patterns in intracellular DMSP concentrations, as grazing pressure is typically highest at night on tropical reefs Intracellular DMSP concentrations of Red Sea tropical macroalgae in this study were higher than intracellular DMSP concentrations reported for the same genera from Australia The production of DMSP by algae is energetically costly Amphiroa sp. This suggests that either DMSP was utilised during the day and replenished at night or there was a lag in the response of intracellular DMSP in Amphiroa sp. to ROS production (perhaps due to the energy outlay required) DMSP and its breakdown products have been proposed as an ‘antioxidant cascade’ Padina sp. and Turbinaria sp., suggesting that DMSP may be up-regulated during periods of low oxygen saturation.Dissolved oxygen levels remained well above the suggested level for hypoxic impact (∼30% saturation) Tropical corals have been described as one of the most important benthic sources of DMSP and DMS in the coastal zone Amphiroa sp.  was visibly reduced on the reef flat, suggesting that the algae were, relatively speaking, partially decalcified in the more variable conditions. This could not be quantitatively confirmed but changes to the calcite structure of red coralline algae in response to variable carbonate saturation has been observed in the temperate coralline alga Lithothamnion glacialePadina sp. (aragonite) was not significantly different between the reef flat and reef crest, despite previous reports of decalcified Padina sp. in naturally low pH environments in Italy and Papua New Guinea Calcification in 2 conditions In coastal regions, the effect of OA will be superimposed on the naturally variable environment, resulting in new carbonate extremes Figure S1Diel pattern in abiotic parameters of Suleman Reef, Egypt.In situ A: dissolved oxygen (%), B: photosynthetically active radiation , C: pCO2 (µatm), D: HCO3− concentration (µmol kg−1), E: CO32− concentration (µmol kg−1), F: pH.(TIF)Click here for additional data file.