After years and years of consensus expert opinion as to mean arterial pressure (MAP) target and vasopressor choice in septic shock management, literature is now emerging that supports the MAP target of 65 mm Hg and norepinephrine as the vasopressor choice. However, the literature remains sparse as to the timing of vasopressors relative to fluid resuscitation and how MAP support is balanced between the choices of vasopressor versus fluid resuscitation. Bai and colleagues report data that reveal an association between earlier vasopressor initiation in septic shock and better outcome. Whether this is a linkage to better care, is related to improved early tissue perfusion, or relates to sparing of fluids to reach the MAP target is not yet known. Critical Care, Bai and colleagues [In a previous issue of lleagues  report tSepsis is a disease of common occurrence with vexingly high mortality rates despite all of the advances in diagnosis, process changes, and treatment modalities ,3. BasedBai and colleagues performed a retrospective study of timing of initiation of norepinephrine for treatment of septic shock. They analyzed outcomes of 213 patients admitted to one of two intensive care units. The authors found a strong relationship between delayed initial norepinephrine administration and 28-day mortality.These findings strengthen the mantra of early initiation of protocolized management of septic shock that includes vasopressor therapy for the early achievement of an MAP target. Perhaps this target should join early diagnosis, early antibiotics, early and appropriate fluid resuscitation, and source control as the essentials of septic shock management .Earlier initiation of vasopressor therapy raises the MAP to a level that facilitates tissue perfusion and prevents onset or progression of organ dysfunction. It is well recognized that organs require a critical MAP to maintain adequate perfusion. When the MAP is allowed to stay below that organ’s critical perfusion pressure, organ injury may occur.Earlier initiation of vasopressor therapy is linked to better knowledge and better management skills for severe sepsis by the treating providers. It may be that earlier vasopressor therapy is a marker of quality of delivered care and this alone would lead to improved outcomes regardless of the impact of the vasopressor alone on outcome.Earlier administration of vasopressor therapy leads to a decrease in the amount of fluids administered over the first 24 hours or more of therapy of septic shock. There is concern that the use of aggressive early fluid resuscitation in septic shock  leads to potential detrimental increased tissue edema down the road in the patient with septic shock, although this concern is not currently supported by solid evidence. It may be that this edema is associated with organ dysfunction, need for organ support, and the ability to wean organ support. This could lead to longer days on mechanical ventilation, longer days in the intensive care unit, and the potential for later-stage increases in morbidity and mortality. It is possible that aggressive fluid resuscitation saves lives on the front end but a price is paid on the back end. There likely is a fine balance between the use of vasopressors to maintain MAP versus the use of continued fluid resuscitation in the presence of capillary leak to maintain MAP. Much additional research will be required to offer guidance to the treating clinician. In the meantime, one should not err on the side of fluid restriction for the sole purpose of decreasing third spacing down the road. Patients typically autodiurese this fluid when septic shock has resolved and the patient is improving. Whether facilitating this fluid exit with diuretic therapy would be beneficial is currently unknown.In our mind, there are three potential reasons why earlier initiation of vasopressor therapy is linked to improved outcome. Each one deserves attention as a potential link. Other potential reasons may yet be brought forward.This trial raises some important questions that need to be answered with large prospective randomized studies: is there an optimal time for initiation of intravenous pressors, and are some of the components of a sepsis protocol more important than others?


Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles.in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method.We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. Viruses are the most ubiquitous and abundant biological entities on Earth . They ini.e., infectious) viral particles from their environment, protected from the action of nucleases .–35.32–35i.e. MS2 and CoxB3), no difference was observed between the viral titer obtained after either sucrose or CsCl purification, suggesting that the infectivity of virions was preserved. Indeed, for MS2 bacteriophage, we estimated the resulting loss of viral titer after the process to be -0.86 log10 after the sucrose gradient step and -0.80 after the CsCl gradient step; for CoxB3, the resulting loss of viral titer was estimated at -1.12 log10 after the sucrose gradient step and -1.00 after the CsCl gradient step.For non-enveloped viruses , suggesting a degradation of nucleic acids during the extraction process.Three nucleic acid extractions processed on an EMEM sample spiked with the reference viruses were evaluated according to the extraction yield and the degradation of RNA: Trizol LS, QIAmp viral RNA mini kit from Qiagen, and High Pure viral nucleic acid kit from Roche. Although no difference was observed in the extraction yield between Qiagen and Trizol LS , Roche extraction did not reach the same yield . The RNA profile of the 3 extracts presented the same small RNAs as presented in 10 (3 Ct) between Qiagen (the best), Trizol LS, and Roche, Trizol LS extraction was chosen because of its capacity to extract large sample volumes (> 1 mL), as recovered after the sucrose gradient ultracentrifugation step. Moreover, the Trizol LS extraction allowed for separate extraction of DNA and RNA, while Qiagen and Roche both extracted total nucleic acids and therefore required a DNAse post-extraction treatment for the RNA virome preparation.Although real-time PCR targeting the DNA and RNA reference viruses conducted on these 3 extracts revealed an average difference of one logHereafter, the 3 metagenomes are designated as “Froussard”, “Wang”, or “Victoria”, according to the sequence-independent reverse transcription and amplification methods used to generate them.Total RNA was processed in three different random reverse transcriptions (RT), as previously described by Froussard , Wang 3, and VicFor RNA sequence-independent amplification, dsDNA generated by random RT-Klenow reactions was used in random PCR. To minimize the amplification step and the potential resulting bias of sequence representation in the metagenome, three cycling conditions were tested for each random PCR: 10, 20, and 40 cycles. No difference in dsDNA size profile was observed between 20 and 40 PCR cycles, but difference was observed in the yield of amplification. Ten cycles of amplification failed to reach the minimum amount of dsDNA material required by Illumina MiSeq whereas 20 and 40 cycles of amplification produced a sufficient quantity. For example, a cDNA sample quantified at 0.23 ng/μL was amplified at the yield of 0.27 ng/μL, 0.42 ng/μL, and 84.06 ng/μL after 10, 20 and 40 cycles of Froussard random amplification, respectively. Although only 20 cycles were enough to obtain a sufficient quantity of dsDNA for several samples , we decided to use 40 cycles in all cases to ensure enough sequencing material and to ensure similar sample treatments. Finally, we compared the effect of the use of different random PCR on the amplicon size profile for a given reference virus. For each tested reference virus, Froussard, Wang or Victoria amplification methods generated different amplicon sizes , revealiin vitro with human blood supplemented with YF, CPX, CoxB3, H3N2, MS2, and T4 viruses, and then further processed with the methodology for RNA virome preparation and analysis described above. T4 DNA bacteriophage was added to the RNA viruses’ panel in order to assess the residual DNA contamination of the RNA fraction. Twenty non-engorged body lice were concomitantly used as a positive control, i.e. they were spiked with the same amount of viruses as that used for engorged lice, and further processed and sequenced as previously described. Viral concentrations applied to either fed- or spiked-lice followed a Gaussian distribution that mimics “natural” conditions. This range of concentrations was also used to evaluate the sensitivity of the virome protocol.To evaluate the protocol on a complex sample, twenty body lice were fed 10 was observed for YF (ΔCt = 2.1) and CoxB3 (ΔCt = 2.6), more than one log10 for H3N2 (ΔCt = 4.06), and no difference for MS2 before and after the purification process, suggesting that the process did not affect the viral load of the reference viruses. For T4 DNA phage, a difference of less than one log10 (ΔCt = 1.2) was observed before and after the purification steps. CPX was negative, both before and after the purification process, probably due to a low viral load and a high limit of detection of the qRT-PCR system. We then purified this artificial positive control in the same way as the engorged lice, as described hereafter.We first evaluated by qRT-PCR and qPCR the loss of viral particles during the purification process of the positive control. A difference of less than one logAfter homogenization of the engorged lice, clarification and filtration through a 0.45 μm filter, the clarified homogenate was treated with a cocktail of DNA and RNA nucleases to remove the majority of host DNA/RNA contaminants. To purify viral particles from their complex environment, the supernatant was ultracentrifuged on a 66%-30% sucrose gradient, and total RNA were extracted from the resulting interface using the Trizol LS® extraction method. To control the recovery of viral particles and the efficiency of viral purification, fluorescence microscopy was conducted on an aliquot of the purified viral fraction . No partQuantification estimated 1.3 ng/μL of total RNA in the engorged lice extract. To evaluate the remaining host DNA and RNA contamination, real-time 18S PCR and RT-PCR were conducted on RNA extracts, resulting in a Ct = 28.85 for the DNA 18S PCR and a negative result for the RNA 18S RT-PCR. These results highlight the presence of a residual host DNA contamination in the RNA fraction. Positive qRT-PCR signals were detected for MS2 (Ct = 15.10) and CoxB3 (Ct = 23.02) viruses, but not for YF, CPX, and H3N2 viruses amplified with published primers.RNA extracted from engorged and spiked lice was further processed with the Froussard, Wang, or Victoria random reverse transcription-Klenow reactions described above. The resulting dsDNA was quantified using the Quanti-it Picogreen, but quantifications were negative. The low amount of nucleic acid material required the use of random PCR. Only 40 cycles of random PCR achieved the quantity of DNA compatible with Illumina MiSeq requirements.2 cluster density with 12,380,000 passed filter paired reads . Within this pooled run, the index representation for the 6 cDNA samples ranged from 4.8% to 7.8%, corresponding to 590,972 to 969,911 passed filter paired reads.i.e. spiked lice) RNA metagenome are presented in 5 PFU of H3N2 in the blood meal. Although a negative qRT-PCR result was obtained for H3N2 in the lice extract using primers from the literature, amplification using primers specifically designed on H3N2 reads recovered after sequencing was positive (data not shnown), confirming the presence of H3N2 viruses in the lice. The discrepancies observed between PCR results obtained with published or virome-based primers are likely due to differences in primers sensitivities. Additionally, no reads were obtained for YF and CPX, which were also negative after qRT-PCR (both with published and virome-based primers), most likely due to the low amount of viral particles ingested by the lice during their blood meal (less than 1.0 x 103 PFU of YF and 1.19 x 102 PFU of CPX added to the blood). Indeed, as described by Cheval et al. in 2011  and 102 PFU [as for CPX]).Sequencing of the spiked lice resulted in the same pattern of viral relative abundances and coverage as the one observed for engorged lice , S4 Fig,i.e. with low viral loads), only the Froussard SIA seemed to be able to randomly amplify each segment of segmented viruses , while the Froussard method seemed to randomly amplify all segments . Interes viruses .There was no notable difference in the taxonomic assignment of reads when comparing the 3 engorged lice metagenomes . FroussaViral metagenomics has been used worldwide to describe viral communities in various complex environments –17 or toThe RNA metagenome preparation protocol is based primarily on the “particle-associated nucleic acid amplification” strategy . That isTo eliminate the majority of contaminating host DNAs and RNAs, various strategies are used, such as soft cell lysis, which leaves nuclei intact , but theTM) density gradient medium. The main disadvantage of CsCl ultracentrifugation is that when analyzing the viral communities of an environment, the lack of prior knowledge of the viruses constituting these communities prevents the harvesting of specific fractions where viruses are located, resulting in recovery of all fractions and thereby diluting the viruses. Iodixanol gradient ultracentrifugation is used either in continuous or in isopycnic gradient to purify viral particles, resulting, as for CsCl, in the necessity to harvest all viral fractions and further diluting the viruses, unless the density of a specific virus is known [The next purification step was ultracentrifugation on a discontinuous 66%-30% sucrose gradient. A broad range of methods has been described to purify and concentrate viral particles. These include tangential flow filtration (TFF) ,44,45, Pis known . Furtheret al. [Recently, a protocol for virome preparation has been published by Kohl et al.  in whichet al. concluded that the quality and integrity of the nucleic acids isolated were compromised using these kits [One key step of metagenome preparation concerns total nucleic acid extraction. Indeed, the assessment of the description of viral diversity of an environment, as well as the genome coverage of viruses, depends on the quality and quantity of extracted nucleic acids. Usually, column-based extraction kits allow for preferential extraction of either DNA or RNA, or both in the same tube, which leads to the splitting in two of the extracts to generate separate DNA and RNA viromes. Recently, column-based kits allowing the extraction of separate DNAs and RNAs from a single sample tube have emerged, but a recent study conducted by Mathieson ese kits . For thiet al. concluded that SIA introduces a strong amplification bias, consisting of inhomogeneous genome coverage and sequence depth [Usually, extraction yields of DNA and RNA obtained after all the viral purification steps are not sufficient for direct sequencing, and they often require a sequence-independent amplification. DNA SIA is biased ,51 but qce depth . These pce depth . In factce depth , Wang [3ce depth , and Vicce depth  random aWhen characterizing the virome of a biological sample, especially when metagenomics is used in diagnostic virology to determine the etiology of pathology, it is important to isolate the virus. Doing so allows for a clear determination of whether the cultivated virus is able to cause disease in healthy individuals, as described in Koch’s postulate in 1876 for bacteria  and reviThe resulting protocol for host-associated infectious RNA virome preparation is therefore composed of (1) a nuclease digestion of homogenized samples with Turbo DNAse, RNAse A, Benzonase, and Exonuclease I (2) a purification of viral particles on a discontinuous 66%-30% sucrose gradient ultracentrifugation at 130 000 g for 2 hours (3) a Trizol LS® RNA extraction followed by a Turbo DNAse digestion and (4) Froussard-based random RT and PCR.The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins, with no loss of viral infectivity and an important elimination of contaminating host DNA and RNA after the process. Moreover, the pipeline described here allows for further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses.S1 FigA. Cytotoxicity of sucrose and CsCl on Vero cells infected with non-purified, sucrose-purified and CsCl purified CPX . B. Cytopathic effects on Vero cells infected with non-purified, sucrose-purified and CsCl purified CPX .(TIF)Click here for additional data file.S2 FigA: amplification profile using Froussard method with different amount of random primers (red: 0.05 μg/μL / blue: 0.10 μg/μL / green: 0.15 μg/μL). B: amplification profile using Wang method with different amount of random primers (red: 20 pmol / blue: 40 pmol / green: 60 pmol). C: amplification profile using Victoria method with different amount of random primers (red: 50 pmol / blue: 100 pmol / green: 150 pmol). D: amplification profile using Froussard method with different amount of random primers (red: 0.10 μg/μL / blue: 0.01 μg/μL / green: 0.001 μg/μL). E: amplification profile using Wang method with different amount of random primers (red: 40 pmol / blue: 4 pmol / green: 0.4 pmol). F: amplification profile using Victoria method with different amount of random primers (red: 100 pmol / blue: 10 pmol / green: 1 pmol). G: amplification profile with 20 (red) or 40 (blue) cycles of random PCR. H: amplification profile using Froussard method with different elongation durations (red: 1 min / blue: 3 min). I to L: amplification profile of RNA viruses according to Froussard (red), Wang (blue) and Victoria (green) random PCR.(TIF)Click here for additional data file.S3 FigAll images were acquired with a Leica SP5 inverted confocal microscope with 4 lasers, a 100X objective and a numerical aperture of 1.4. Scale bar means 30 μm.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


In conclusion, our study reveals that the HBV 2.2 kb singly-spliced RNA encodes a 21.5 kDa viral protein that significantly interferes with the assembly of nucleocapsids during HBV nucleocapsid formation. These findings provide a possible strategy for elimination of HBV particles inside cells.Hepatitis B virus (HBV) is the smallest DNA virus and the major cause of acute and chronic hepatitis. The 3.2 kb HBV viral genome generates four major species of unspliced viral transcript as well as several alternatively spliced RNAs. A 2.2 kb singly-spliced RNA is the most abundant spliced RNA and is widely expressed among all HBV genotypes. The expression of the singly-spliced RNA, as well as that of its encoded protein HBSP, is strongly associated with hepatopathology during HBV infection. Here, we report a novel inhibitory role of a p21.5 protein, which is encoded by a 2.2 kb singly-spliced RNA, in the modulation of HBV replication. We show that overexpression of the singly-spliced RNA is able to efficiently inhibit HBV replication. Furthermore, a mutation in the ATG start codon of the precore region completely abolishes the inhibitory effect of the singly-spliced RNA, indicating that a viral protein (p21.5) derived from the singly-spliced RNA is the mediator of the inhibition. Furthermore, p21.5 is able to form a homodimer that interacts with core dimers forming hybrid viral assembly components. Sucrose gradient fractionation revealed that co-expression of p21.5 resulted in a spread distribution pattern of core proteins ranging from low to high sucrose densities. When compared with p22, p21.5 is almost ten times more efficient at destabilizing HBV nucleocapsid assembly in Huh7 cells overexpressing either p21.5 or p22 protein. Moreover,  Hepatitis B virus (HBV), a member of the hepadnaviridae family, is the smallest DNA virus and the major cause of acute and chronic hepatitis. More than 350 million people worldwide are living with chronic hepatitis B (CHB), which causes progressive liver damage and leads to the development of cirrhosis and hepatocellular carcinoma (HCC) , 2. The in vivo and in vitro [In HBV-producing hepatoma cells, a 2.2 kb singly-spliced RNA that lacks intron 2447/489 is the most abundant spliced viral RNA and represents between 20% and 30% of the spliced viral transcripts , 7. Althin vitro , 7–11; fin vitro .The 2.2 kb singly-spliced RNA has been reported to encode an HBV splice-generated protein (HBSP) . Severalin vitro and in vivo. The inhibitory effect by which this singly-spliced RNA modulates HBV capsid formation is explored in the present study.In this study, we report that a splice-generated protein (p21.5), derived from the one-amino acid shorter precore/core protein, exerts an inhibitory effect on HBV capsid formation. Overexpression of the 2.2 kb singly-spliced RNA was found to increase the expression level of p21.5 and to reduce significantly the amount of nucleocapsids in HBV-expressing hepatoma cells. In addition, p21.5 was found to form a homodimer that interacts with the core dimer; this interaction interferes with nucleocapsid formation both This study was conducted under the approval of Institutional Animal Care and Use Committee (IACUC) of National Health Research Institutes (Protocol No. NHRI-IACUC-102002-A).2, and 100% relative humidity in an incubator.The human hepatoma cell lines HepG2 and Huh7, and human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 μM non-essential amino acids. They were grown at 37°C in 5% CO-/R was constructed by cloning a fragment of the cDNA sequence of singly-spliced RNA with a single mutant of nucleotide A to T (nt 1919) into the pShuttle/R vector. This mutant is able to express singly-spliced RNAs but produces core protein that is only 6 amino acids in length. pShSS_prestop mutant/R was constructed by cloning a fragment of the cDNA sequence of singly-spliced RNA with a single mutant of nucleotide C to T  into the pShuttle/R vector to generate a prestop codon at the 7th amino acid upstream of the authentic stop codon of p21.5. pShPC-1/R was constructed by cloning the sequence of the precore/core ORF of the singly-spliced RNA into the pShuttle/R vector. pShSS_PCmut/R and pShSS_Cmut/R were derived from pShPC-1/R by mutating the ATG start codon of the precore ORF and core ORF to GTG. pShSSFlag/R was also derived from pShPC-1/R and had amino acids 78–81 (DPAS) of the core ORF replaced by the FLAG sequence (DYKDDDDK) as previously described [p1.3HBcl/Hyg is an HBV-expressing plasmid that contains 1.3 copies of the entire HBV genome and is able to support HBV replication . pShuttlescribed . pSh22/Rescribed .6 cells were plated in 10 ml of the Dulbecco’s modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) per 10-cm dish one day before transfection. After culturing overnight, the medium was replaced with 5 ml of 10% DMEM medium before transfection. For each dish, 10 μg of DNA was diluted into 250 μl 150 mM NaCl. In parallel, 20 μl of jetPEI reagent was diluted into 250 μl 150 mM NaCl. The diluted jetPEI reagent was combined with the diluted DNA and incubated for 15 minutes at room temperature. The DNA-jetPEI complexes were then added to the dishes and mixed gently by rocking the plate back and forth. Finally, the dishes were incubated at 37°C in a CO2 incubator.DNA transfection was performed using jetPEI polymer-based DNA transfection reagent (Polypus-Transfection). In brief, 3×102 incubator to allow the virus to infect the cells. After 2 hours, 5 ml fresh complete growth medium was added. The cells were then incubated at 37°C for 3 or 5 days.Recombinant adenoviruses  were prepared according to the BD Adeno-X Expression System 1 User Manual (Clontech). In brief, a gene fragment was cloned from the pShuttle vector into the pAdeno-X vector, and pAdeno-X was transfected into HEK-293T cells. The recombinant adenoviruses carrying the EGFP (AdIE) gene, singly-spliced RNA (AdSS), or 1.3 copies HBV (Ad1.3HBV) were collected from the cells. To infect HepG2 cells with adenovirus, cells were plated on a 10 cm-dish 24 h before infection. The next day, the growth medium was removed and 5 ml of virus (diluted to achieve the desired MOI) was then added to the dish. The plates were tipped to spread the viruses evenly and the dish was incubated at 37°C in a CO32P-labeled HBc DNA fragment.For HBV RNA analysis, total RNA was extracted following the TRIzol RNA Isolation Reagent (Invitrogen) protocol. Equal amounts of total RNA were then separated by gel electrophoresis and analyzed by northern blot hybridization using a Cells were lysed in NET buffer  in the presence of protease inhibitor cocktails. Equal amounts of total proteins were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membrane . Each membrane was then blocked with 5% nonfat milk in 1× PBS, which was followed by incubation with anti-HBV core antibody , anti-GFP antibody (Clontech), or anti-beta-actin antibody (Sigma). The immunoblot signals were examined using enhanced chemiluminescence reagent (PerkinElmer Life Sciences).in situ by denaturing the membranes with 0.2 N NaOH/1.5 M NaCl followed by neutralizing with 0.2 N Tris-HCl/1.5 M NaCl. Finally, the membranes were hybridized with 32P-labeled HBx DNA fragment.Analysis of intracellular HBV core particles was performed as previously described . In brieEqual amounts of total proteins were ultracentrifuged at 500,000 × g through 2 ml of a 20% sucrose–NET  (wt/wt) cushion for 1 h at 4°C using a TLA 100 rotor . Under these conditions, viral core particles are pelleted, whereas free core protein and soluble HBeAg remain in the supernatant as has been described previously . The pel32P-labeled HBx-specific probe.HBV replication following transient transfection of cell lines was assessed by Southern blot analysis as described previously with some modifications . The isoCells were lysed in NET buffer  in the presence of protease inhibitor cocktail. Equal amounts of total proteins were then layered onto 10 ml of a 10 to 60% (wt/wt) sucrose–NET  mixture. Gradients were established by ultracentrifugation at 30,000 rpm with a SW41 rotor for 17 h at 4°C. Fourteen 0.75-ml aliquots were collected from the gradient and equal volumes were then analyzed by western blot to detect the proteins that were present.BALB/c mice  were anesthetized with isoflurane. Fifteen micrograms of plasmid DNA was injected into the tail vein of each mouse in a volume of PBS equivalent to 8% of the mouse body weight. The total volume was delivered within 5 seconds. Liver samples were then collected for particle blot analysis on day 3 after injection.The HBsAg in mice serum was measured with an enzyme-linked immunosorbent assay (ELISA) Kit purchased from General Biology Corp. The amount of formazan dye formed correlated with the amount of HBsAg, which was determined quantitatively using a scanning multi-well spectrophotometer (ELISA reader) at an absorbance of 450 nm. By using standards of HBsAg with known concentrations, the amounts of the secreted HBsAg were estimated.In order to investigate the effect of the singly-spliced 2.2 kb RNA on HBV replication, Huh7 cells were cotransfected with plasmids expressing HBV (p1.3HBcl/Hyg) together with the singly-spliced RNA (pShSS/R) or a control plasmid pShuttle/R (Mock); then the replication capacities of HBV in the presence or absence of singly-spliced RNA expression were analyzed. A dramatic decrease in the amount of nucleocapsid was observed when there was overexpression of the singly-spliced RNA  together with expression of either EGFP (AdIE) or singly-spliced RNA (AdSS); these were co-transduced into HepG2 cells. A similar inhibitory effect of the singly-spliced RNA on HBV replication was observed in HepG2 cells. Furthermore, while the expression level of pgRNA was also slightly reduced after adenovirus AdSS transduction . Interestingly, the formation of HBV capsids was inhibited by cotransfection with pShSS/R but not with pShSScore-/R  in hepatoma cells that are overexpressing singly-spliced RNA raised the possibility that this viral protein may act as a mediator in the inhibition of nucleocapsid assembly. To investigate this possibility, a nonsense mutation was made within the plasmid pShSS/R by introducing a premature stop codon at nt 1919 (numbering starting at the EcoRI site) in order to terminate the translation of both the precore and core proteins  and a high-density group (capsid particles) after 10%–60% sucrose gradient fractionation; in such circumstances the assembly intermediates are hardly detectable as they exist in very low amounts or for a very short time , 22. To It has been reported that p22, a nonsecreted HBeAg precursor protein is also able to exert an inhibitory effect on HBV replication through the formation of hybrid capsids with the HBV core protein . To compin vitro, we next explored whether singly-spliced RNA is able to exert its inhibitory effect in vivo. An HBV-expressing plasmid (p1.3HBcl/hyg), together with either singly-spliced RNA-expressing plasmid (pShSS/R) or mock control plasmid (pShuttle/R), were co-injected into the tail vein of BALB/c mice as described by Huang et al. [in vitro and in vivo.Based on the fact that our findings indicate that the singly-spliced RNA is able to interfere with the formation of intracellular nucleocapsids via the p21.5 protein g et al. . As showA variety of viruses generate spliced viral RNAs or splice-related proteins that act as functional molecules and are able to modulate host-virus interactions –28. Retr-/R) or a precore ATG mutation (pShSS_PCmut/R) of the singly-spliced RNA was found to completely abolish its inhibitory activity, which indicates that a viral protein derived from the precore protein is responsible for the inhibition of virus replication that is mediated by the singly-spliced RNA . Therefore, the predicted coding sequence of p21.5 is that of the intracellular HBeAg precursor, but being one-amino acid (Cys183) shorter than that of previously identified HBeAg precursor p22 . As showturation , 37. As turation . When th4 IU/ml [Although singly-spliced RNA has been known for over two decades and has been well characterized –9, the e4 IU/ml .in vitro and in vivo. This may provide insights into the development of potential therapeutic strategies for controlling HBV replication.Our current study suggests that expression of minute amounts of p21.5 is sufficient to interfere with nucleocapsid formation, raising the possibility that the elevation of singly-spliced RNA in CHB patients may play a functional role in the feedback modulation of HBV replication. In this case, the increased level of p21.5 translated from the singly-spliced RNA would seem to diminish the formation of nucleocapsid and thus contribute to the establishment of a viral persistent infection. Evolving such an inhibitory effect involving destabilization or disruption of viral nucleocapsids constitutes a common anti-viral strategy for controlling of HBV infection. For example, some antiviral cytokines, such as tumor necrosis factor alpha and various interferons, have been shown to destabilize pre-existing viral particles through unknown mechanisms , 42, 43.S1 FigHuh7 cells were co-transfected with pShCore/R together with either pShSS/R or a control plasmid (Mock), and total mRNA was harvested at day 3 post-transfection. The expression of core mRNA was confirmed by northern blot analysis via hybridization with an HBc-specific probe. The expression level of co-transfected GFP was used as a transfection control and 18s rRNA was used as a loading control.(TIFF)Click here for additional data file.S2 Fig(A) Schematic of the products of the HBV precore/core gene. pShPC/R was constructed by cloning the coding sequence of the HBV precore protein into the pShuttle/R vector. (B) Huh7 cells were co-transfected with p1.3HBcl/Hyg and pShPC/R in the ratio of 1:1, 1:0.5, 1:0.25, 1:0.125, 1:0.0625, or control plasmid (Mock) (lane 1–6) and the cells were harvested three days post-transfection for particle blot and western blot analyses. Top two panels, the expression levels of nucleocapsid were analyzed by particle blot analysis. The encapsidated nucleic acid was revealed by hybridizing with an HBx-specific probe. Lower two panels, the expression levels of HBV core protein and GFP protein were examined by western blot analysis.(TIFF)Click here for additional data file.S3 Fig(A) BALB/c mice were injected with p1.3HBcl/Hyg together with either pShSS/R (N = 6) or p1.3HBcl/Hyg and control plasmid (Mock) (N = 6) by tail vein injection. (B) BALB/c mice were injected with p1.3HBcl/Hyg together with either pShPC-1/R (N = 10) or p1.3HBcl/Hyg and control plasmid (Mock) (N = 10) by tail vein injection. The mice were sacrificed three days post-injection and the sera were harvested to determine the amount of secreted HBsAg. The cutoff values of the ELISA assay in S3A Fig. and S3B Fig. are 6.81 ng/ml and 5.62 ng/ml, respectively.(TIFF)Click here for additional data file.S4 Fig(A) Top panel, a schematic of the structure and open reading frames of the singly-spliced RNA. Lower panel, a schematic of the plasmid constructs. pShSS/R, a plasmid expressing full-length singly-spliced RNA. pShSS_prestop mutant/R, a plasmid expressing singly-spliced RNA with a prestop mutation at the position 2428 (numbering starting at the EcoRI site) within the precore/core ORF. (B) Huh7 cells were transfected with the indicated plasmids. Lane 1: p1.3HBcl/Hyg and pShSS/R; lane 2: p1.3HBcl/Hyg and pShSS_prestop mutant/R; lane 3: p1.3HBcl/Hyg and control plasmid (Mock). The cells were harvested three days post-transfection for particle blot and western blot analysis. Top two panels, the expression levels of nucleocapsid were analyzed by particle blot analysis. The encapsidated nucleic acid was revealed by hybridizing with an HBx-specific probe. Lower two panels, the expression levels of HBV core protein and GFP protein were examined by western blot analysis.(TIFF)Click here for additional data file.S5 Fig293T cells were transiently transfected with p1XHBV, pShSS/R or pSh22/R alone, or p1XHBV combined with pShSS/R or pSh22/R for three days and cell lysates were harvested for sucrose gradient centrifugation (10–60%). (A) The sucrose densities of fractioned samples were detected and shown. (B) Distribution patterns of core species after sucrose gradient fractionation were assessed by western blot analysis. Equal volumes of total protein were separated by electrophoresis on SDS-PAGE, and the distribution of core proteins species in each fraction was detected by western blot analysis using an anti-core antibody. The mobility of wild type core, p21.5 and p22 was not clearly resolved in the blot.(TIFF)Click here for additional data file.


Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.Ethephon, an ethylene releaser, is used to stimulate latex production in  The ethylene biosynthesis and signalling pathways have been well established in plant model species. Ethylene has a wide variety of applications in agriculture and horticulture. The effect of ethephon, an ethylene releaser, was tested as early as the 1970s on natural rubber production [ex cells ,3. Latexex cells . The mecex cells . Bark woex cells .Ethylene is also associated with negative effects, such as the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase located at the surface of lutoids , which generates reactive oxygen species (ROS) . Excessiin situ coagulation of rubber particles. Numerous studies have shown that TPD appears to be a physiological disorder resulting from excessive recurrent tapping and overstimulation by ethylene [The Tapping Panel Dryness (TPD) syndrome is characterized by the partial or, ultimately, total stoppage of latex flow upon tapping, due to ethylene ,8,10,11.ethylene ,13,14,15etc.) [TPD susceptibility depends on the planting material (genetic susceptibility and rootstock-scion interaction) and the environment  ,17. So tetc.) .cis-1,4-polyisoprene. A high sucrose content must be maintained to regenerate latex after latex flow and avoid any cell dysfunction that can lead to TPD [Latex diagnosis consists in measuring four main parameters: sucrose, inorganic phosphorus (Pi) and thiol contents, and the total solid content (TSC) of latex ,19. Sucrd to TPD . The inod to TPD ,21. For d to TPD ,22. By cd to TPD .Many ethylene-responsive genes have been related to these physiological changes. More recently, transcriptome analyses led to the identification of candidate genes and microRNAs related to TPD occurrence ,26,27,28Our objective was to provide a basis for understanding the molecular regulation of ethylene biosynthesis and signalling pathways, as well as the ROS-scavenging system during the TPD process. We used morphological, histological and biochemical analyses to classify three contrasting rubber tree clones according to their latex metabolism and TPD susceptibility. Latex production and the dry cut length were measured over a period of 3 years, revealing the higher TPD susceptibility of clone PB 260 compared to other tested clones. Data from latex diagnoses showed a gradient of metabolic typology between the three clones studied  600, and Sungei Putih (SP) 217). A decrease in some biochemical components revealed lower latex metabolism activity in TPD-affected trees than in healthy trees. At the same time, we characterized the regulation of genes related to ethylene biosynthesis and signalling pathways, and ROS-scavenging systems. This study provides insight into the transcriptional regulation of ethylene-related and ROS-scavenging genes in rubber-producing tissues. The comparison with model species led us to propose some candidate genes that could be used to identify molecular markers associated with TPD tolerance for breeding programmes.The metabolic typology of three rubber clones with a contrasting latex metabolism  was checked by a latex diagnosis analysis. Latex samples were analysed from trees tapped every two days and without ethephon stimulation after one year of tapping a,b. RubbThe sucrose content peaked at 1.6 mM in latex from clone PB 260 under the d1 ET 12/y treatment, whereas it could reach 5.79 and 9.84 mM for clones SP 217 and RRIM 600 under d1 ET 0/y and d2 ET 12/y, respectively a,d,g. ThIn order to predict the occurrence of TPD for each studied clone, the dry cut length was measured over a period of three years . Clone PThe interaction between clones, tapping and ethephon frequencies was analysed for the dry cut length . A cloneIn order to estimate yield production, dry rubber was calculated for each year during the observation . The higIn a previous analysis, susceptibility to TPD was shown in three contrasting clones in which clone PB 260 was the most susceptible compared with the other two clones. Regardless of tapping frequency and ethephon stimulation, a latex diagnosis was carried out three years after the first tapping on healthy and TPD-affected trees from this clone a–c. The A histo-cytological analysis was carried out on healthy a and twoHevea brasiliensis genes comparing TPD-affected and healthy trees was analysed in latex and bark of 6-year-old mature trees  and HbAP2-10. In latex, 3 genes  were induced by TPD as opposed to 4 inhibited genes  in non-stimulated trees. By contrast, 12 genes  were induced by TPD as opposed to 1 inhibited gene (HbACO1) in stimulated trees. In bark, 2 genes (HbACS3 and HbERF-Ib2) were induced and inhibited by TPD, respectively, in non-stimulated trees. Meanwhile, 9 genes  were induced by TPD in stimulated trees.The transcript abundance ratio of 70 re trees . These gThe TPD susceptibility of clones is related to their growth and latex metabolism . The ideSurprisingly, the occurrence of TPD did not result in a decrease in latex production over the duration of this study. Of course, such latex production was not likely to be sustainable, since a drop in dry rubber content began in the third year of production. The continued latex production when partial TPD occurred might be explained by the large latex-producing zone of the tapping panel involved in latex flow and only local oxidative stress in part of the tapping cut. Latex diagnosis is an efficient method for early detection of laticifer dysfunctioning. Typically, an increase in sucrose content and a decrease in thiols content were observed. When TPD symptoms occurred significantly, all the latex diagnosis parameters fell slightly but significantly, revealing a general decrease in metabolic activity in the laticifers.TPD occurrence increased regularly during the plantation cycle, and the frequency of its occurrence depended dramatically on the latex metabolism of the clones, and environmental and harvesting conditions. As TPD occurrence was spread over time, its quantification remained complicated and hampered any genetic analysis of TPD tolerance. Clones with a high latex metabolism showed earlier TPD occurrence than those with a low latex metabolism when using a harvesting system with a high tapping frequency (d1) and ethephon stimulation (ET 12/y). This suggests that dry cut length measurement could be used as a phenotyping parameter for trees subjected to this stressful harvesting system. Monitoring the dry cut length could be used to distinguish a population derived from a cross between TPD susceptible and tolerant clones. Quantitative Trait Loci (QTL) detection has been successfully carried out to understand the genetic determinism of South American Leaf Blight (SALB) in rubber trees ,35. Thisc-oxidoreductase from lutoids is particularly high [Rboh genes but also on ethylene-response genes. NADPH oxidase (RbohD) was needed for the ROS-responsive expression of Arabidopsis ERF6 via a calcium-dependent manner [Hevea, HbERF-VII was recently linked to potential hypoxia-responsive genes in laticifers [HbAPX2 in latex tissue  in bark was significantly induced by ethephon but not in the case of TPD. Besides this transcriptional regulation, post-transcriptional regulation is expected for several ERF groups. For example, the ubiquitination of protein degradation has been demonstrated for ERF-VII [Ethylene was differentially perceived and transduced in latex and in bark during ROS-TPD. In general, the relative transcript abundance of in latex . The HbEethylene . This di tissues . ERF-VII ERF-VII . Alterna ERF-VII ,51,52.HbERF-IIb2, HbERF-VIIIa14 and HbERF-IXb2 [HbERF-IIb2 is orthologous to OCTADECANOID RESPONSIVE ARABIDOPSIS AP2/ERF (ORA) 47, which controls jasmonate biosynthesis. Several expression marker genes were also related to the programmed cell death (PCD). ERF group VIII is renowned for Programmed Cell Death (PCD) induction during ectopic over-expression [HbERF-VIIIa14, a repressor-type ERF, potentially displayed PCD regulation. Recently, ERF-IXb was identified as an ERF subgroup involved in PCD [HbERF-IXb2 was not regulated by ethylene [HbAP2 genes were also significantly accumulated including HbAP2-6, an ortholog to WRI1 [HbAP2-10, which was an expression marker gene in both the bark and latex. This regulation of HbAP2 genes during TPD suggested metabolic and developmental regulation. All these expression marker genes represent good candidate genes for studying the allelic variability and development of genetic markers associated with susceptibility or tolerance of TPD.Of the 70 genes related to ROS-scavenging systems, and ethylene biosynthesis and signaling pathways, this study highlighted 27 TPD-expression marker genes in latex and bark. The expression marker genes differed between latex and bark except for two genes. This suggests tissue-specific regulation of genes involved in ROS-TPD occurrence. Regardless of the tissue, more expression marker genes of ROS-TPD were identified in ethephon-stimulated trees (22) than non-stimulated trees (9), revealing a complex regulation of ROS-TPD. Among the expression marker genes, several had a putative important function predicted by phylogenetic analysis, such as ERF-IXb2 . HbERF-Ipression . The repd in PCD ,54. The ethylene . Transcr to WRI1 , and HbAHevea clones  at the Sembawa Research Centre of the Indonesian Rubber Research Institute. The latex harvesting system was a half spiral tapping (S/2), with a tapping frequency of 1 (d1), 2 (d2) and 4 days (d4), respectively. Ethephon (Eth) was applied at 2.5% to the tapping panel of the bark at various frequencies (0 (0/y), 12 (12/y) and 24 (24/y) times a year). Three replicates of one tree each were observed for each combination (clone × tapping frequency × ethephon treatment). The length of dry cut was observed just after tapping before tissue sampling in order to measure visually the tapping panel dryness gradient. Samples of healthy and TPD-affected trees were then collected 24 h after ethephon treatment. Sample tissues were collected and immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction.Latex and bark tissues were collected for three years after the first tapping from 6-year-old mature rubber trees of three Histological samples of TPD-affected trees were collected 2 cm below the tapping cut using a punch without damaging the cambium. The samples were fixed as described by . After ii.e., sucrose (Suc), inorganic phosphorous (Pi), and thiol contents (R-SH) were measured from March to May after the rainy season and before leaf senescence in order to avoid any physiological variation. Fresh latex (0.5 mL) was supplemented with 2.5% trichloroacetic acid (TCA) with 4.5 mL of volume to induce protein precipitation, and then with 0.2% phosphotungsic acid (PTA) to coagulate rubber particles. This clear latex serum was then used for the measurement of sucrose, inorganic phosphorous and thiols using the Dische anthrone method [The gradient of tapping panel dryness was assessed by measuring the dry cut length (DCL). The DCL was measured by visual estimation of the percentage of half-spiral tapping cut without latex flow instantly after tapping. Latex diagnosis parameters e method , the Taue method , and thee method , respectg at 4 °C for 30 min. The supernatant was transferred to a new tube containing 8 mL of 5.7 M CsCl. Ultracentrifugation in a swinging bucket was carried out at 89,705× g at 20 °C for 20 h. The supernatant and caesium cushion were discarded whilst the RNA pellet was washed with 70% ethanol. After 30 min of air drying, the pellet was dissolved in 200 µL of sterile water. Although DNA could not cross the caesium cushion for this centrifugation condition, a DNAse treatment was performed using TurboDNAse . DNA contamination was then checked by PCR amplification using primers of the Actin gene including the intron sequence. RNAs were conserved at −80 °C.Total RNAs were isolated using the caesium chloride cushion method adapted from Sambrook  by Duan et al. [Real-time RT-PCR experiments followed recommendations specified by Udvardi et al. . Specifiet al. ,62,63,64® 480 SYBR Green I Master, Roche Applied Sciences, Basel, Switzerland) in a 6-µL volume. PCR cycling conditions comprised one denaturation cycle at 95 °C for 5 min, followed by 45 amplification cycles . Expression analysis was performed in a 384-well plate. Samples were loaded using an automation workstation .cDNAs were synthesized from 2 μg of total RNA to the final 20 μL reaction mixture using a RevertAidTM M-MuLV Reverse Transcriptase (RT) kit according to the manufacturer’s instructions . Full-length cDNA synthesis was checked on each cDNA sample by PCR amplification of the Actin cDNA using primers at the cDNA ends. Quantitative gene expression analysis was finally carried out by real-time RT-PCR using a Light Cycler 480 . Real-time PCR reaction mixtures consisted of 2 µL RT product cDNA, 0.6 µL of 5 µM of each primer, and 3 µL 2× SYBR green PCR master mix  (HbRH2b was selected as the best reference gene according to its stability in tissues from various treatments in mature trees and juvenile trees. The homogeneity of the HbRH2b gene Cp (crossing point-PCR-cycle) confirmed that it could be used as an internal reference gene. The HbRH2b gene was amplified in each reaction plate in parallel with target genes. The transcript abundance level for each gene was relatively quantified by normalization with the transcript abundance of the reference HbRH2b gene. Relative transcript abundance took into account primer efficiencies. All the normalized ratios corresponding to transcript accumulation were calculated automatically by Light Cycler Software version 1.5.0  provided by the manufacturer using the following calculation: Normalized Ratio = Efficiency (Cp target − Cp RH2b)−Δ.Real-time PCR was carried out for eleven housekeeping genes in order to select the most stable gene as the internal control for all types of sample used (HbActin) . HbRH2b F-test for latex diagnosis data. The values were considered as significantly different when p value ≤0.05. The level of interaction between clone, tapping, ethephon, the combination of two, i.e., clone × tapping, clone × ethephon, tapping × ethephon, and a combination of the three (clone × tapping × ethephon) was assessed for the DCL. The level of interaction between clones , tapping frequencies , and ethephon stimulation frequencies per year  was equally assessed.Dry cut length values and latex diagnosis physiological data  were the mean of three biological replicates. The statistical analysis was carried out on raw data using an ANOVA followed by a Bonferroni test for dry cut length and by Fisher’s F values for each interaction and the corresponding p values were noted as follows: <0.001 (***); <0.01 (**); <0.05 (*); <0.1 (°).Each relative transcript abundance value was the mean of six biological replicates. Statistical analysis was performed after logarithmic transformation of raw data. The analysis of relative transcript abundances of TPD-affected and healthy trees was carried out using an ANOVA followed by a Newman–Keuls test. The level of interaction between ethephon (Eth), tapping panel dryness (TPD) and a combination of both (Eth × TPD) was assessed for each tested gene. The analysis generated variance tables that included HbAP2/ERFs also suggested the involvement of other hormones and developmental processes such as jasmonate, and PCD and cambial activity, respectively. Beyond improving our knowledge of the TPD, this study contributes to a better understanding of the role of transcription factors and in particular ethylene signalling in perennial species. Unveiling the role of AP2/ERF transcription factors in the plant defence processes in Hevea, may help molecular breeders in improving tolerance to abiotic and harvesting stress.Environmental constraints are known to induce the production of ROS and ethylene. Monitoring TPD occurrence is a simple method to highlight ROS-related latex cell dysfunctions. The dramatic regulation of genes involved in ethylene perception and signalling during TPD reinforces the critical role of this hormone in plant defence. The putative functions of some


The current standard practice of lung lesion diagnosis often leads to inconclusive results, requiring additional diagnostic follow up procedures that are invasive and often unnecessary due to the high benign rate in such lesions . The Percepta bronchial genomic classifier was developed and clinically validated to provide more accurate classification of lung nodules and lesions that are inconclusive by bronchoscopy, using bronchial brushing specimens . The analytical performance of the Percepta test is reported here.Analytical performance studies were designed to characterize the stability of RNA in bronchial brushing specimens during collection and shipment; analytical sensitivity defined as input RNA mass; analytical specificity  as tested on blood and genomic DNA; and assay performance studies including intra-run, inter-run, and inter-laboratory reproducibility.RNA content within bronchial brushing specimens preserved in RNAprotect is stable for up to 20 days at 4 °C with no changes in RNA yield or integrity. Analytical sensitivity studies demonstrated tolerance to variation in RNA input (157 ng to 243 ng). Analytical specificity studies utilizing cancer positive and cancer negative samples mixed with either blood (up to 10 % input mass) or genomic DNA (up to 10 % input mass) demonstrated no assay interference. The test is reproducible from RNA extraction through to Percepta test result, including variation across operators, runs, reagent lots, and laboratories .Analytical sensitivity, analytical specificity and robustness of the Percepta test were successfully verified, supporting its suitability for clinical use.The online version of this article (doi:10.1186/s12885-016-2153-0) contains supplementary material, which is available to authorized users. Lung cancer has the highest mortality of all malignancies with approximately 160,000 deaths per year in the U.S. and a 5-year survival rate of only 17 % , with loIt has been shown that exposure to tobacco smoke alters gene expression in airway epithelial cells , 6, and Analytic validity, Clinical validity, Clinical utility and associated Ethical, legal and social implications) have defined parameters which should be used to evaluate analytical validity of novel genomic tests [While the clinical validity of the Percepta test has been demonstrated in independent studies , 10, it ic tests , 14. HerNormal appearing bronchial epithelial cells (BEC) were collected from the mainstem bronchus using standard cytology brushes during AEGIS 1 and AEGIS 2, two prospective, multicenter, observational studies (NCT01309087 and NCT00746759). The enrolled patients had already been referred for bronchoscopy examinations as part of their clinical care for suspicion of lung cancer. Following sample collection, brushes were immediately clipped and submerged in RNAprotect preservative solution  post-collection. Samples were stored at 2–8 °C before being shipped in a NanoCool shipper  with active cooling to 2–8 °C, and stored at 2–8 °C upon receipt prior to RNA extraction.Fresh peripheral blood samples were collected from healthy voluntary participants. Immediately after collection, the blood samples were mixed with the RNAprotect preservative at a 1:5 ratio as recommended by the manufacturer. Subsequently, the pure blood samples were tested following the Percepta molecular test lab procedure as outlined below from total RNA extraction to array results.Ethics approval was obtained prior to the initiation of the studies described in this report. To characterize analytical performances of the assay, we used total RNA samples  that were derived from human bronchial epithelial cell specimens. These total RNA samples were already available from previously registered clinical trials which were published as clinical validation studies , 10. AddThe Percepta molecular test lab procedure starts with the extraction of total RNA from bronchial brushing specimens using the miRNeasy Kit . Yield was measured using NanoDrop 8000 instruments  and quality was measured by the RNA Integrity Number (RIN) generated by the BioAnalyzer System . Samples with concentration <21 ng/μL and/or RIN <4 were stopped from further processing per pre-specified QC criteria. Positive (lung tissue lysate) and negative (water) controls were developed and used as applicable with pre-defined RNA yield and quality acceptance criteria to ensure the reliability of the procedure. For each sample, 200 ng of total RNA were amplified using the Ambion WT Expression Kit , fragmented and labeled using the Affymetrix WT Labeling and Controls Kit , followed by overnight hybridization of 2.75 μg biotin-labeled cDNA to a Gene 1.0 ST microarray (Affymetrix). The arrays were then washed, stained, and scanned on a GeneChip System GCS3000 or DXv2 (Affymetrix) following manufacturer’s protocols. Cancer positive and cancer negative total RNA controls were included in each sample batch starting from the amplification step. Pre-defined specifications for yield, quality, and Percepta classification of these control samples were used as batch acceptance criteria.To evaluate the genomic DNA amount present in the total RNA samples, the Quantifast SYBR Green PCR kit was used following manufacturer’s protocol , using a normalized 50 ng total RNA input for the test samples.All data analysis was done in R version 3.1.2 . To obtap-value had to be >0.05 under all attempted models, and the lowest p-value was reported so that lack of significance was ensured for all models fit. Transcript signal intensity Pearson correlations between arrays from different test sample groups were calculated from the genes used in the algorithm. All 95 % confidence intervals for standard deviations (SD) were obtained by bootstrap where the residuals of a linear mixed-effects model controlling for sample and other sources of variation (depending on the type of SD reported) were sampled with replacement to create a bootstrap sample. The distribution of blood contamination level in the total RNA from the AEGIS clinical samples was simulated based on 1) the observed blood contamination levels based on a predefined color scale, 2) the mass variation of pure fresh blood derived total RNA, and 3) the mass variation of total RNA from the clinical samples, assuming these three factors contribute to the blood contamination level independently. The simulation to assess the maximum tolerable level of variation in Percepta scores was performed by making multiple random draws from a normal distribution for each AEGIS sample, with the mean defined as the Percepta score obtained during the clinical validation [Brushing sample stability was established using an ANOVA test of means of yield and failure rates of RIN over time. Linear mixed effect models were used to evaluate the effects of RNA input amount, blood interference, and genomic DNA interference at the 5 % significance level. To claim that a variable was not statistically significant, its lidation  and the Multiple lots of lung tissue lysate were manufactured (at Veracyte) and used as process controls during RNA extraction. Three different lots of controls were tested over several weeks of independent runs with 7 replicates of each lot per run, by two different operators. Testing of three lots is standard practice to verify the reproducibility of a manufacturing or laboratory process. Lung lysate controls consistently produced the expected quantity and quality of total RNA, resulting in within-lot coefficients of variation (CV) ranging from 4 to 6 % for yield and 2–4 % for RIN (data not shown).Similarly, multiple lots of cancer positive and cancer negative total RNA were manufactured (at Veracyte) and tested for their use as process controls for amplification and hybridization steps. The reproducible Percepta results obtained from these controls enabled concurrent monitoring of assay performance for each run. All Percepta tests and studies outlined below included at least one cancer positive and one cancer negative total RNA controls.p = 0.148, Fig. p = 0.955, Fig. To demonstrate the cumulative stability of the RNA content within the preserved BEC samples under the typical collection and storage conditions, comprehensive sample tracking data were collected from AEGIS samples and the stability was evaluated using metrics of RNA yield and integrity. The length of time from sample collection till RNA extraction was accounted for. With the pre-specified sample quality criteria, no statistically significant difference was observed among samples with a cumulative 2–8 °C storage time of up to 20 days based on RIN failure rate . The transcript signal intensities of each sample were equally highly correlated within each single group of RNA input  and between the test input groups and the baseline 200 ng condition . Overall, this study demonstrated highly robust analytical sensitivity of the Percepta test to RNA input quantity variation within the tested range.While the standard total RNA input quantity to the Percepta assay is fixed (200 ng), concentration measurement (by NanoDrop) and pipetting (using Rainin LTS pipets) variability around this nominal input amount are expected in routine practice. Therefore, a study was performed to characterize the transcript array signal intensities and Percepta results relative to variability in total RNA input quantity. Based on the manufacturers’ specifications in concentration measurement and pipetting, the standard deviation (SD) of such variation translates to 15.2 ng, given the intended 200 ng input. Therefore, the titration levels were designed to be 200 ng ± 30 ng  and 200 ng ± 43 ng . Total RNA from a cancer positive and a cancer negative bronchial brushing sample were processed in triplicate through the Percepta test at the designed total RNA input levels . As shown in Fig. p-value = 0.515), supporting that the Percepta test is robust against blood contamination.Occasionally, bronchial brushing samples may contain small amounts of blood due to variation in the collection procedure or to individual patients. This was confirmed by the visual inspection of samples collected during the AEGIS trials, which showed that greater than 80 % of the samples have no visible blood contamination. The procedure to collect BEC specimens limits the volume of contaminating blood using standard sheathed cytology brushes  . Thus, assay testing was designed for up to 10 % genomic DNA contamination as a worst-case scenario (10 times above baseline). One cancer positive and one cancer negative brushing sample that were tested to have < 1 % genomic DNA were spiked with 1, 5 and 10 % additional genomic DNA and tested via Percepta in triplicate per condition. There was no significant difference in the Percepta score between samples with up to 10 % genomic DNA and samples with no additional genomic DNA spiked in (20) Fig. . This stin silico to the original Percepta scores obtained from the validation samples [In order to assess the maximum tolerable level of variation in Percepta scores, a simulation study was performed by adding increasing levels of random variation  samples . The res2 coefficients of 0.985 (range 0.945 to 0.998).The within-run and inter-run reproducibility of the Percepta test were evaluated using total RNA from 10 bronchial brushing samples with high, medium and low scores, and 6 control samples, processed in triplicate in three experimental runs (144 Percepta results), varying reagent lots, operators, and days (spanning three weeks). The pooled within-run SD of Percepta scores was estimated to be 0.222 . Thus, the Percepta test demonstrated sufficiently high reproducibility across reagent lots, operators, and processing runs. In contrast, the inter-class score SD was estimated to be 1.180 (95 % CI 1.115–1.246), which includes biological variation between cancer and non-cancer samples from the AEGIS 1 and 2 clinical studies.Additionally, the Percepta scores were estimated to have an inter-run pooled SD of 0.259 , demonstrating inter-laboratory reproducibility and accuracy of Percepta results. Inter-laboratory pooled standard deviation of Percepta scores was estimated to be 0.276 (95 % CI 0.172–0.389), which is in agreement with the SD of 0.259 calculated for within-lab inter-assay reproducibility. Similarly, transcript signal intensities were highly correlated between laboratories across all samples , consistent with expectations for inter-assay results.Total RNA from 22 different patient bronchial brushing samples was processed using the Percepta test in the laboratory where the test was developed (Veracyte Research & Development Laboratory). A second aliquot of RNA from the same samples was later tested in a different, CLIA-certified reference laboratory using different operators, reagent lots, and equipment . The Percepta calls for all samples were 100 % concordant between the two laboratories. Further, the Percepta scores of the 22 samples between the two laboratories are highly correlated , and up to 10 % genomic DNA spiked into the starting RNA sample have no detectable impact to the Percepta scores.Analytical reproducibility was evaluated following technical assessment criteria outlined by EGAPP and ACCE, using clinical samples with Percepta scores covering the entire range and concentrated around the decision boundary of the assay . It has The robustness of the Percepta test to induced variables, including those that may be encountered in clinical samples, supports that routine testing of bronchial brushing specimens can be achieved at high confidence from the standpoint of analytical performance and reproducibility.


Tripneustes gratilla, to evaluate the efficacy of their use as biocontrol agents in the efforts to reduce alien invasive algae. In flow-through tanks, we measured urchin growth rates, feeding rates and feeding preferences among diets of the most common invasive algae found in Kāneʻohe Bay, Hawaiʻi: Acanthophora spicifera, Gracilaria salicornia, Eucheuma denticulatum and Kappaphycus clade B. Post-transport survivorship of outplanted urchins was measured in paired open and closed cages in three different reef environments  for a month. Survivorship in closed cages was highest on the reef flat (∼75%), and intermediate in the lagoon and reef slope (∼50%). In contrast, open cages showed similar survivorship on the reef flat and in the lagoon, but only 20% of juvenile urchins survived in open cages placed on the reef slope. Urchins grew significantly faster on diets of G. salicornia (1.58 mm/week ± 0.14 SE) and Kappaphycus clade B (1.69 ± 0.14 mm/wk) than on E. denticulatum (0.97 ± 0.14 mm/wk), with intermediate growth when fed on A. spicifera (1.23 ± 0.11 mm/wk). Interestingly, urchins display size-specific feeding preferences. In non-choice feeding trials, small urchins (17.5–22.5 mm test diameter) consumed G. salicornia fastest (6.08 g/day ± 0.19 SE), with A. spicifera (4.25 ± 0.02 g/day) and Kappaphycus clade B (3.83 ± 0.02 g/day) intermediate, and E. denticulatum was clearly the least consumed (2.32 ± 0.37 g/day). Medium-sized (29.8–43.8 mm) urchins likewise preferentially consumed G. salicornia (12.60 ± 0.08 g/day), with less clear differences among the other species in which E. denticulatum was still consumed least (9.35 ± 0.90 g/day). In contrast, large urchins (45.0–65.0 mm) showed no significant preferences among the different algae species at all (12.43–15.24 g/day). Overall consumption rates in non-choice trials were roughly equal to those in the choice trials, but differences among feeding rates on each species were not predictive of feeding preferences when urchins were presented all four species simultaneously. In the choice feeding trials, both small and medium urchins clearly preferred A. spicifera over all other algae (roughly twice as much consumed as any other species). Again, however, differences were less pronounced among adult urchins, with adults showing a significant preference for A. spicifera and Kappaphycus clade B compared to the other two algal species. These findings indicate that outplanted urchins are surviving on the reef flats and eating a variety of alien invasive algae as intended. Although juvenile urchins show stronger feeding preferences, these animals grow quickly, and adult urchins are more generalist herbivores that consume all four alien invasive algae.We investigate the survivorship, growth and diet preferences of hatchery-raised juvenile urchins,  Kappaphycus clade B, Eucheuma denticulatum, Gracilaria salicornia, and Acanthophora spicifera. The species level taxonomy of Kappaphycus remains a subject of contention , henceforth it should and will be referred to as Kappaphycus clade B in this study .Within the last 70 years, Kāneʻohe Bay has become home to many introduced and invasive algal species, whose aggressive growth has smothered corals and overgrown many patch reefs across the bay . Some ofntention . Due to K. clade B and G. salicornia both spread rapidly across Kāneʻohe Bay after their introduction, and are now found at high abundance throughout the Bay  and showed that feeding preferences were generally unchanged after 5 months on monospecific diets, except urchins that were maintained on Padina sanctae-crucis and showed enhanced preference in subsequent choice trials, whereas those maintained on G. salicornia tended to avoid it when offered five species from which to choose at the end of the trial. Further, T. gratilla being outplanted in Kāneʻohe Bay. Together, these studies will aid both State and conservation group efforts to control alien algal overgrowth of corals on reefs in Kāneʻohe Bay and across Hawaiʻi.Currently, thousands of cultured urchins are outplanted at 20–25 mm test diameter, but comparatively few were observed in subsequent surveys of urchin density on the reef . A major motivation of this research was that it was unknown at that time whether the missing urchins were dying from transplant stress, starvation, being eaten by predators after outplanting, or simply moving into cryptic habitats at small sizes such that they were missing in subsequent surveys. Tripneustes gratilla received its common name from the habit of gathering fragments of coral rubble, rocks, or algae from the benthic environment as camouflage while it forages. Its body is predominantly black but often possesses a pentaradial bluish or reddish hue when its tube feet are retracted close to its body. Its spines are typically black, white or cream. This echinoid is relatively common in shallow waters (0–15 m) across the Hawaiian archipelago , medium (29.8–43.8 mm), and large (45.1–65.1 mm) that were then used for each of the experimental trials outlined below. Prior to the experiments, medium and large urchin size classes were raised on diets of all four alien algae. Small urchins were not fed before trials, but instead were placed directly into experiments within a few days of arriving from the hatchery. Therefore, urchins were starved 3–5 days prior to each experiment.Acanthophora spicifera, Gracilaria salicornia, Eucheuma denticulatum, and Kappaphycus clade B. Kappaphycus clade B  and Eucheuma denticulatum were intentionally introduced from the Philippines to Kāneʻohe Bay September 1974 by researchers from the University of Hawaiʻi for scientific research . In order to monitor individual growth rates of urchins without marking the animals, each tank held a single urchin of each size class: small, medium, and large. For each treatment, algae were provided ad libitum to reduce any resource competition, and all aquariums were cleaned twice a week during which freshly collected algae were provided to each tank. Urchin test diameter was measured using Vernier calipers (VWR) to the nearest tenth of a millimeter each week for a month. An analysis of covariance (ANCOVA) was employed to analyze growth, with initial urchin test size used as the covariate. Tukey’s HSD post-hoc comparison was then performed to determine significance of pairwise differences in average growth rates of urchins on each algae diet . Algae were blotted on paper towels to remove excess water and weighed before being placed in each tank. Urchins were allowed to graze for ∼5 days and the amount of remaining algae was weighed as before to calculate the amount of each species consumed per urchin per day. In a few cases, we stopped the experiment after the 4th day because we did not want the urchins to consume more than half of the algae offered in any trial. Consumption rates  were then compared by analysis of variance (ANOVA). Tukey’s HSD post-hoc comparison was used to identify significant differences between each of the algae . The data from the medium cohort from the no-choice trial was also normally distributed . However, the data from the large urchins of the no-choice feeding trial were only marginally non-normal . To test the homogeneity of variance among our feeding trial data, the Levene’s test was employed. The null hypothesis for the Levene’s test was that the variances are homogenous. For the urchins in the no-choice feeding trials, the data passed the homogeneity test  = 0.56, p = 0.64).No-choice feeding trials provided juvenile he algae . The assThe choice feeding experiment provided all four species of algae, in equivalent amounts, simultaneously to urchins. As with no-choice experiments above, each algal species was blotted and weighed before being introduced to the experimental tanks. For this assay, larger tanks  were used to allow room to separate algae into the four quadrants of the experimental tank. Four urchins were then introduced to the middle of the tank and allowed to graze at will for ∼7 days. Again, if any species of alga became low relative to the others , we stopped the experiment a day early to avoid biasing results. At the end of each experiment, algae were removed, blotted and weighed as previously to calculate the amount of algae consumed per urchin per day for each species.iA and fA were the initial and final blotted masses of algae subject to grazing by urchins; while iAC and fAC were the initial and final masses of the algae in the no-urchin control tanks. This equation was used to account for growth of algae over the course of the experiment, but can also account for any unexpected decline in algal biomass unrelated to the grazing trial . However, the data for the medium and large sized urchins of the choice feeding trials both passed the normality test . For the small urchins of the choice feeding trial, the data passed the homogeneity test  = 2.13, p = 0.13). The data from the medium urchins in the choice feeding trial fail to reject the null hypothesis  = 0.32, p = 0.81). The data from the cohort of large urchins in the choice feeding trials also passed the test for equality of variances  = 1.98, p = 0.13).For each choice and no-choice feeding trials, small urchins were provided with ∼100 g of algae initially, whereas medium and large urchins were offered a starting biomass of ∼150 g of algae. To account for any growth or decline of the algae not attributed to urchin grazing during the experiment, both choice and no-choice experiment tanks had a divider such that one half of the tank housed experimental algae and urchins whereas the other side housed only equivalent amounts of algae to serve as a no urchin control. The consumption rates of algae were then calculated as: ng trial . Becauseng trial  and rank2 galvanized chicken wire mesh. We constructed both open and closed cages. For closed cages, the mesh extended across all sides including the tops to prevent urchins from being able to crawl out and prevent access by fishes on the reef. In contrast, the sides of the open cages end with back-folded edges and no top to minimize escape of the juvenile urchins from the cage, but still allow open access of predatory fishes. Initial trials with urchins caged in seawater tables indicated that this back-folded edge design (approximating an upside down U) was the most effective for open cages, but urchins still escaped the cages at the rate of 1–2 animals per week. Urchins ranged from 18–22 mm at the start of the experiment. Cages were filled with G. salicornia to provide the juvenile urchins with food and a place to hide, because our initial aquarium trials revealed that urchins were far more likely to escape the open cages in the absence of hiding spots and food in the cage. In the absence of any cover or food, open cages were frequently empty within 24 h in our water table trials (data not shown).Cages measuring roughly 50 × 50× 75 cm were constructed from 1 cmCages were placed at 6 sites across three habitats, with four cages, three open and one closed control, at each site. 251 urchins were used during these caging experiments. Three habitats surrounding Coconut Island map in  were selT. gratilla, measured as maximum test diameter (mm), were significantly affected by fixed algal diets  = 10.80, p < 0.001, G. salicornia or K. clade B grew at significantly  higher rates (1.58 ± 0.14 and 1.69 ± 0.14 mm/week TD (Test Diameter), respectively) than those urchins that fed on a diet of E. denticulatum. Urchins that fed on a diet of E. denticulatum had the lowest growth rates (0.97 ± 0.14 mm/week TD) out of the four assays, though not significantly lower to urchins on a diet of A. spicifera (1.23 ± 0.11 mm/week TD).Growth rates of G. salicornia at the highest rate of all algal species offered (15.24 ± 0.001 g day−1), and K. clade B at the lowest rate (12.43 ± 1.51 g day−1) although these trends were not significant  = 1.94, p = 1.78, F = 8.49, p < 0.05, Gracilaria salicornia was eaten at a significantly higher rate (12.60 ± 0.08 g day−1) than either A. spicifera (10.33 ± 0.36 g day−1) or E. denticulatum (9.35 ± 0.90 g day−1) ; K. clade B was also eaten at a higher rate (11.87 ± 0.27 g day−1) than E. denticulatum , but not A. spicifera . Among the small collector urchins, feeding rate patterns were comperable to those of the medium urchins, but with more significant disparities among algal species,  = 51.30, p < 0.001, G. salicornia had a significantly higher mean consumption rate (6.08 ± 0.19 g day−1) than any other algal assay in the non-choice feeding trial . Small urchins likewise consumed Eucheuma denticulatum at the lowest rate (2.32 ± 0.39 g day−1), which was significantly lower than both A. spicifera and K. clade B .When presented with no choice, urchins consumed different species of algae at different rates, but the effect varied by urchin size . On averA. spicifera than any of the other three available algae species . Likewise medium urchins showed a significant preference , as it was consumed at the highest rate (3.69 ± 0.21 g day−1), whereas G. salicornia and E. denticulatum were consumed at intermediate rates . In contrast, medium urchins consumed K. clade B at the lowest rate (0.51 ± 0.10 g day−1). Unlike the small and medium urchins, which avoided K. clade B in the choice trials, large urchins exhibited significant preferences for both A. spicifera and K. clade B  in the choice feeding trials . Kappaphycus clade B was also significantly preferred to G. salicornia and E. denticulatum . Whereas G. salicornia and E. denticulatum were consumed at intermediate rates by small and medium urchins, these species tend to be avoided by the large urchins  when given a choice of algae on which to feed  when fed only A. spicifera relative to any other algal diet . The orange stars represent are caging sites along the reef flat . And the yellow stars mark caging areas along the reef slope . These three sites were used to assess post transport survival of juvenile urchins which are deployed in the Bay.Click here for additional data file.10.7717/peerj.1235/supp-2Supplemental Information 2K. clade B.Average growth rates of small, medium and large urchins while on a no-choice diet of either A. spicifera, G. salicornia, E. denticulatum or Click here for additional data file.10.7717/peerj.1235/supp-3Supplemental Information 3tab 1: Urchin growth data; tab 2: No choice feeding data on 4 different algal diets; tab 3: Choice feeding data with all 4 algal species; tab 4: Urchin caging survivorship data.Click here for additional data file.


Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way. However, fixing the cells before hybridization followed by extensive washes of the unbound probes prevent monitoring the critical dynamic regulation aspects of RNA. Such steps were circumvented with the development of molecular beacons where a quencher and a fluorophore, positioned at both 5′-and 3′-ends RNA extremities of a stem-loop hairpin structure, would become fluorescent once bound to its cognate complementary RNA strand  and its derivatives have been central to these efforts by tagging onto endogenously coding genes of proteins to map the expression, interaction, localization, and role of proteins in the cell and beyond  to enhance the fluorescence of a compound otherwise practically non-fluorescent , so that radiative decay pathways  can predominate, leading to an increase in fluorescence  is the low fluorescent background  in the absence of the RNA aptamer. The strong resurgence of the aptamer generation and selection field brought new tools to synthesize novel type of aptamers that would improve the area of e Figure .These light-up aptamers allow the tagging of RNAs, thus providing a simple approach for a simple protein-free live-cell imaging of RNAs by fluorescence. Being composed of RNA, light-up aptamers have a huge advantage over GFP. RNA is highly modular  purification, targeted RNA detection via strand displacement, ribozyme cleavage monitoring, and development of demethylase inhibitors.in situ  aptamer was developed to confine the formation of radicals on RNA upon laser activation, leading to the rapid degradation of such RNA -2-- 1H,1′H-2,5′-bibenzo[d]imidazol-2′-yl)phenoxy)acetamide] fluoresced only in the presence of a 71 nucleotide-long aptamer which were minimized and optimized to a 29 nucleotide-long RNA blue fluorescent aptamer  and dimethyl indole red (DIR) were synthetized and showed little nonspecific fluorescence activation from cell lysate  indicated that a stem-loop can be shortened , guanine, and guanosine triphosphate (GTP). Under certain conditions, these sensors produced ~20-fold increases in fluorescence upon metabolite binding. Moreover, SAM synthesis that was monitored in individual E. coli cells was highly variable from cell-to-cell, once again pointing the importance of population averaging versus single cell measurements.Using the S-adenosylmethionine (SAM) aptamer connected to Spinach Tm) of 34°C was central in the poor folding of Spinach where an estimated 32% of the aptamer was folded at 25°C while only 13% was at 37°C. To increase Spinach thermostability and brightness, systematic mutagenesis was performed on the aptamer, resulting in a more stable light-up aptamer appropriately named Spinach2 , where the excitation of a single spinach RNA attached to a slide in presence of 5 μM of DFHBI, the bright fluorescence of the Spinach-DFHBI complex showed fast decay  after only 2 s of excitation at ~100 W/cmIn terms of microscopy, a drawback of the Spinach-DFHBI fluorescence is that its spectral properties are different from current optical instrumentations that are optimized for GFP or fluorescein isothiocyanate (FITC) with a bandpass excitation filter transmitting light at 480 nm, a 505 nm dichroic mirror and an emission filter transmitting light at 535 nm. With a maximal emission wavelength slightly below 500 for the Spinach-DFHBI complex, most of the fluorescent light is not collected by the dichroic mirror or is blocked by the emission filter. During the initial development of HBI derivatives , the Vibrio proteolyticus 5S rRNA scaffold  integrated into Spinach to improve the brightness were still questionable for the detection of mRNA expressed at low-level (transcribed by the RNA polymerase II). To improve on the fluorescence signal, an array of Spinach aptamer repeats was created. Arrays of up to 64 Spinach repeats increased by 17-fold the fluorescence signal which allowed for imaging mRNAs that would otherwise be invisible with a single aptamer tag  of the fluorogen and the aptamer. Very short incubation times and stringent conditions during the selection of an aptamer against a thiazole orange derivative that was acetylated (TO1) then biotinylated (TO1-Biotin) allowed the development of the high-affinity light-up aptamer RNA Mango  but also on the dissociation constant . This 49 nucleotide-long RNA, named Broccoli  and it has a reduced magnesium dependence, which contributes to a nearly 2-fold brightness increase in E. coli. Additionally, the secondary structure of Broccoli, containing a hairpin stem-loop, was engineered to form a dimer by substituting the terminal loop with a second Broccoli aptamer resulting in a 70% increase in fluorescence.After a few “regular” rounds of SELEX, light-up aptamers were selected both for binding and fluorescence activation by using fluorescence-activated cell sorting (FACS) where the libraries were transformed in i Figure , activati Figure . For exavia the fusion of a metabolite-binding aptamer to Spinach, is a powerful application that seems to be more popular than the actual imaging of RNA trafficking in vivo. Light-up aptamers have been used to monitor, in vitro, and/or in live E. coli, the following metabolites: adenosine, ADP, guanine, SAM, guanine, and GTP  in solution showed affinity in the nanomolar range. For example, detection of expression of streptavidin in E. coli resulted in a 10-fold increase of the fluorescence signal when the streptavidin light-up aptamer was co-expressed. An immediate concern might be about the robustness of the quantitative protein concentration determination from such light-up aptamers. To alleviate uncertainty, a linear correlation between the fluorescence generated by the MCP light-up aptamer and the MCP protein levels (as measured by western blot) indicated that the MCP light-up aptamer is as reliable as direct readout of protein levels, without the need of additional antibody chemistry. The MCP sensor was able to show cell-to-cell MCP synthesis variability, indicating the use of this technology in single-cell interactions within population studies and may also provide a new approach to study molecular crowding.In addition to only RNA molecules, the light-up aptamers have been used to monitor proteins: thrombin, streptavidin, and the MS2 phage coat protein  was not detected in mammalian Mv1Lu cells nor in yeast methyl]amino]- 6- - 8- methyl-pyridopyrimidin-7(8H)-one]. This system has been named IMAGEtag , where the fluorophore becomes activated when the quencher binds to an endogenously produced aptamer at the 3′-end of an RNA of interest, offers the possibility of monitoring many types of mRNA in vivo purpose is much harder than in vitro. There are multiple reasons, such as having a controlled environment instead of a dynamic organism, having non-specific binding with cellular components, unknowingly activating pathways, variable permeability of membrane(s), etc. More specifically with light-up aptamers, the in vitro assays allows for excellent sensors as the background fluorescence is very low, leading to a strong signal-to-noise ratio. Moreover, those aptamers simply require in vitro transcription to generate the needed protein-free sensor. Several in vitro molecular biology applications (described below) have been developed with these aptamers, such as in-gel imaging (similar to SYBR Gold), radioactivity-free kinetic activity measurements of ribozymes, affinity purification of RNA, and ribonucleoprotein complexes, identification of the ligands from orphan riboswitches and also, development of a high-throughput in vitro screening to identify the new drugs binding to RNA-modifying enzymes.Although still in development, there are no doubts that developing light-up aptamers for E. coli and mammalian cells . Modified light-up aptamers have been developed for non-isotopic detection of the hammerhead ribozyme self-cleavage in real-time by fluorescence. Extra sequences are added to the hammerhead ribozyme to bind one half of the sequence of Spinach. Once the ribozyme self-cleaves, the half-Spinach sequence is released and binds its cognate Spinach second half to reconstitute a bi-molecular Spinach aptamer to induce fluorescence activation upon DFHBI binding . It should however be noted that under certain conditions, a single Spinach fluorescence may be barely above background . Moreover, Mango RNA aptamer selectively activates a thiazole orange derivative with high affinity. We are now entering in a new field of protein-free fluorescence detection of endogenously produced molecules. The in vitro and in bacteria will surely continue to increase over the next few years, several in vivo challenges must be prioritized for the light-up aptamers. Among others, low-level Pol II expressed mRNA must be easily monitored, transfection controls, and wide field view of raw images must be constantly be present in publication to identify the cell-to-cell fluorescence variation, which is crucial for the background subtraction. In the meantime, it is undeniably a great tool to consider for protein-free in vitro fluorescence applications.While the number  of applications JO was the sole writer of this review.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.