Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels.Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in  Neuronal gap junction proteins mainly function at electrical synapses, where they form intercellular channels for ions and small molecules, such as second messengers, to flow bi-directionally, and sometimes directionally, allowing the coupled cells to synchronize their activities. In the nervous system, gap junction protein expression is tightly controlled throughout development -4. TransVertebrate and invertebrate gap junctions exhibit similar structures and conserved functions. A gap junction is composed of two tightly associated, apposing hemichannel complexes between two neighbouring cells. A hemichannel is a hexamer complex composed of proteins with four transmembrane domains ,16. WhilSome connexins participate in non-junctional forms of neuronal communication . For exaGap junction hemichannels can be involved in another form of neuronal communication, referred to as ephapses ,35. ThisC. elegans genome encodes 25 innexins, 19 of which are reported to be present in at least subsets of neurons , ZM3306 nca(lf); unc-7(e5); hpEx1218[pJH1300+Podr-1-GFP], ZM3645 nca(lf); unc-7(e5); hpEx1383[pJH1380 +Podr-1-GFP]; 2) For aldicarb-response experiments, ZM1917 unc-7(hp121); hpEx428[pJH641+ Podr-1-GFP], ZM5215 unc-7(hp121); hpEx2204[pJH1300+Podr-1-GFP], ZM4376 unc-7(hp121); hpEx1677[pJH1380+Podr-1-GFP], ZM1912 unc-7(hp121); hpEx433[pJH1608 +Podr-1-GFP], ZM4705 unc-7(e5); hpEx1577[pJH1556+Podr-1-GFP]; 3) For active zone morphology analysis, ZM3164 unc-7(e5); hpIs61; hpEx1135[pJH641+Podr-1-GFP], ZM4949 unc-7(e5); hpIs61; hpEx1996[pJH1380+Podr-1-GFP], ZM4918 unc-7(e5); hpIs61; hpEx1975[pJH1300+Podr-1-GFP].Extrachromosomal arrays : 130 CsCl, 2 MgCl2, 10 Hepes, 4 Na2ATP, 10 glucose, adjusted to pH7.4 with KOH and to ~330 mOsm with sucrose. Adjacent cells both with bright GFP-fluorescence were clamped at the same voltage (-80 mV), and alternately applied 2s step voltages to +90 mV. Coupling was assessed by the occurrence of a slow, poorly voltage-dependent, non-inactivating current in one cell, in response to a step voltage applied to the other cell. Hemichannel currents were detected by step voltage stimulation from -120 to +100 mV in increment of 20 mV for 100 ms.Neuro2A cells were assessed for electrical coupling on the same setup. After the transfection (see below), cells were bathed in an extracellular solution containing (in mM): 140 NaCl, 10 glucose, 5 Hepes, 4 KCl, 2 CaCl2 and saturated humidity, and grown in MEM high glucose media supplemented with 10% Fetal Bovine Serum (Invitrogen), 1X Non-essential Amino Acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 100U Penicillin, and 100 ug/mL Streptomycin (Invitrogen). When reaching 70-80% confluence, cells were dissociated with 0.25% trypsin (Invitrogen) in 1X PBS, and replated at 15% confluence for maintenance and 70% confluence for transfections. 4 μg of DNA per well was transfected into Neuro2A cells with Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol.Neuro2A cells were cultured in 60 mm culture dishes for maintenance and electrophysiology analyses, and on glass coverslips for immunofluorescent staining. They were kept at 37°C, 5% COunc-7 and unc-7 cys-less cDNA were subcloned into a modified pTracer-CMV to give rise to pJH1874 and pJH1879, respectively. In both constructs, the CMV promoter drove the expression of UNC-7 (wild type or Cys-less form), while the EF1 promoter drove the expression of GFP for identification of transfected cells.Mammalian expression plasmids are pTracer-CMV (Invitrogen) based constructs. 2+/Ca2+), fixed with 4% PFA for 20 minutes, washed and kept in PBS at 4°C until staining. They were permeabilized in 0.2% Triton X-100 for 5 minutes, washed in PBS, followed by blocking with 1% Bovine Serum Albumin for 15 minutes. Cells were then incubated overnight with rabbit anti-UNC-7 antibody [For immunofluorescent staining, 24 h, 48 h and 72 h after each transfection, Neuro2A cells were washed in PBS(+Mgantibody  at 1/300hpIs61 (Punc-25-GFP::UNC-10) in intact C. elegans was performed by visual examination and counting of fluorescent puncta along the entire dorsal nerve cord in L4 stage animals as previously described [Quantification of the fluorescent marker escribed . All scoAldicarb plates were prepared as described previously . At t = unc-7 loss of function mutations, such as uncoordinated locomotion  animals. As both e5 and hp121 alleles harbor early stop codons, are protein-null alleles for UNC-7, and exhibit identical phenotypes [unc-7 null mutants in this study.Defects associated with the enotypes ,45, theyunc-7 mutants under our experimental conditions. In the absence of stimulation, the amplitude and frequency of the body wall muscle miniature postsynaptic currents (mPSCs) were identical between wild type and unc-7 animals. The evoked postsynaptic currents (EPSCs) in response to electrical stimulation of the motoneurons (electric-EPSCs) were also comparable in kinetics and amplitude between wild type animals and unc-7 mutants  or Gamma-Aminobutyric-Acid (GABA) to induce muscle contraction or relaxation, respectively [unc-7 animals ), or the auxiliary subunits of the channel complex, UNC-79 or UNC-80, result in a 'fainter' phenotype, where mutant animals are capable of normal locomotion when stimulated, but fail to sustain the activity and succumb to muscle relaxation shortly after [nca(lf); unc-7 triple loss of function mutants. They no longer faint but exhibit strong, sustained jerky locomotion characteristic of unc-7 mutants  in regards to locomotory behavior.To determine the role of UNC-7 at the neuromuscular system, we further examined its possible functional interactions with the NCA cation channel, first alluded to by their reported genetic interactions in volatile anaesthetic sensitivities ,60. In Cly after . Consistly after . We genehpIs61 (Punc-25-GFP::UNC-10) marker; Figure hpIs61 is normal in nca(lf) mutants, with bright, even GFP::UNC-10 fluorescent puncta regularly spaced along the dorsal nerve cord . In nca(lf); unc-7 animals, the active zone puncta appeared even in brightness, size and distribution, reflected by an increased total number of bright puncta that was similar to that of wild type (p > 0.05) or nca(lf) (p > 0.05) animals  rescuing the NMJ morphology defect of unc-7 mutants.Do NCA and UNC-7 also exhibit genetic interactions for the NMJ morphology? We analyzed the active zone morphology and distribution at GABAergic NMJs using the d Figure , whereasnalyses  was also reflected at a functional level, we examined the spontaneous and electrically evoked synaptic activity at the NMJs of nca(lf); unc-7 animals ; unc-7 mutants  was similar to that of nca(lf) animals . The mPSC frequencies for both nca(lf); unc-7 and nca(lf) were significantly decreased when compared to both wild type animals , and to unc-7  ; unc-7, n = 10; 21.7 ± 3.4pA for nca(lf), n = 7; 25.1 ± 1.9 pA for unc-7, n = 7) as compared to wild type   and nca(lf); unc-7 mutants thus suggests a functional dominance by the NCA channel at NMJs at rest.To assess whether the genetic interaction between ) Figure . The ave) Figure . The simunc-7 animals  was identical to that of wild type animals . While the response was reduced by approximately 60%  in nca(lf) mutants ([nca(lf); unc-7 animals  was identical to that of wild type (p > 0.05) and unc-7 mutants (p > 0.05). Therefore, upon stimulation, the effect of the UNC-7 channel dominates that of NCA, which is opposite to the situation at rest.When the nerve cord was stimulated, the electric-EPSC amplitude of mutants  and unc-7 mutants are complex, as their epistatic relationships are phenotype-dependent. These interactions do, however, confirm the antagonistic interactions between these two channels, and further indicate a cooperative modulation of the activity and development of the neuromuscular system.Overall, the genetic interactions between unc-7 mutations [unc-7 mutants is of neuronal, but not of muscular origin [unc-7 mutants and the active zone defects, while the expression of UNC-7 in muscles rescued neither phenotypes [The NCA channel is expressed and required in the nervous system. UNC-7, on the other hand, is expressed not only in neurons, but also in body wall muscles . Studies to date indicate that UNC-7 is either inactive or functionally redundant in muscles: the electrical coupling between body wall muscle cells is not affected by utations , and ther origin ,45. Pan-enotypes .nca(lf) mutant background, we compared the effect of restoring UNC-7 expression in either neurons, or muscles in nca(lf); unc-7 mutants. The pan-neuronal expression of UNC-7 in nca(lf); unc-7 mutants led to a complete behavioral reversion to the fainter phenotype, identical to that of nca(lf) mutants ; unc-7 mutants carrying a transgenic array expressing a pan-neuronal promoter-driven UNC-7 was also reduced by about 65% when compared to nca(lf); unc-7 animals not expressing the transgene  mutants, indicating that neuronal UNC-7 mediates such an interaction with NCA. These results further indicate that neuronal UNC-7 channel functions together with the NCA channel to modulate neuronal activities.To test if UNC-7 functions in neurons to modify the NMJ function in the Pannexins are now proposed to function mainly as hemichannels, rather than gap junctions . To examunc-7 or nca(lf); unc-7 mutants. Their subcellular localizations were examined subsequently by immunofluorescent staining with UNC-7 antibodies. Strains expressing the pan-neuronal wild type UNC-7 displayed prominent staining along both ventral and dorsal nerve cord neurites, with both large patches (on average ~0.71 ± 0.38 μm in length) that likely correspond to gap junctions described by ultrastructural reconstruction of the C. elegans nervous system, and numerous, tiny puncta of unknown correlating structures ; unc-7 mutants to the characteristic nca(lf)-like 'fainter' locomotion ; unc-7 mutants. Consistently, the genetic and functional interactions between the NCA and UNC-7 channels are also partially independent on UNC-7's ability to form gap junctions. Although expressing pan-neuronal C191A or Cys-less UNC-7 in nca(lf); unc-7 mutants did not alter their locomotion, it did revert the rescuing effect of the unc-7 mutation on the evoked postsynaptic currents in body wall muscles: the electric-EPSCs in these transgenic animals ('+') were reduced by >40%, when compared to their respective controls (their non-transgenic siblings '-') ; unc-7 mutants led to a statistically similar degree of decrease in the EPSC amplitude, though the decrease mediated by wild type UNC-7 showed a tendency to be slightly larger than that by C191A or Cys-less UNC-7 , unc-7 or nca(lf); unc-7 did not alter mPSC frequency or amplitude  mutants, suggesting an antagonistic interaction between the NCA cation leak channel and the UNC-7 innexin channel in neuronal activity modulation. We further demonstrate in vivo and in vitro that neuronal UNC-7 exerts these functions in part through non-junctional activities. UNC-7 may function as both gap junctions and hemichannels to regulate different aspects of nervous system development and function.In the present study, we showed that, although C. elegans mutants that display superficially normal locomotion, but altered synaptic transmission at NMJs, the opposite example, mutants with strongly uncoordinated locomotion, of a neuronal origin and with altered synaptic morphology, but no detectable synaptic transmission defects at NMJs, such as the case for unc-7, are rare. Moreover, although nca(lf); unc-7 animals expressing Cys-less UNC-7 did not affect their locomotion, or they did show a full reversion of unc-7's modifying effect on synaptic transmission at NMJs.While there have been reports of several unc-7 mutants result mainly from defects in neurons that send inputs to motoneurons, either in the activation and/or a coordination of motoneurons, which we would also bypass or mask through the direct motoneuron stimulation in the current electrophysiological analyses. It has indeed been shown that the major locomotory defects associated with unc-7 mutants could be rescued by restoring UNC-7 expression in a small set of command interneurons [unc-7 mutants.Why do mutations in UNC-7 result in locomotory and pharmacological defects that are often associated with altered cholinergic synaptic transmission, but have no detectable synaptic transmission alteration by electrophysiology analyses? One possibility is that UNC-7 does affect the motoneuron activity, but the effect was obscured when applying strong stimulations to these neurons as required for the current electrophysiological analyses. Another possibility is that the locomotory defects of rneurons . Given iunc-7 mutants and synaptic activity of unc-7 and nca(lf); unc-7 mutants implicated that, in addition to its role in electrical coupling, UNC-7 is also involved in roles independent of gap junctions to affect activity at NMJs. Below we propose two potential mechanisms through which non-junctional UNC-7 may affect neuronal activity. Both were based on studies of Pannexins, the closest mammalian homologues of innexins, which were proposed to primarily function independently of gap junctions [The strong rescuing ability of Cys-less UNC-7 proteins in the altered pharmacological response of unctions .Mouse taste receptor cells released ATP through Pannexin-1 hemichannels, which further triggered serotonin release from adjacent taste receptor cells ,69. PannC. elegans neurons (data not shown), but the resolution limit of C. elegans neurites prevented ruling out this possibility completely.Another intriguing possibility is suggested by the study where pannexins were shown to play a role in calcium signalling and homeostasis at the ER . In heteunc-7 mutants on their own did not exhibit obvious synaptic transmission defects at NMJs indicates either a functional redundancy between UNC-7 and other innexins, or that UNC-7 functions with/through other channels to modulate neuronal activity in either the same or different sets of neurons. Its antagonistic genetic interaction with the NCA channel strongly supports the latter possibility.The fact that the C. elegans, the NCA complex regulates NMJ activities and affects the propagation of activity-related calcium signals along neurites [The NCA channel family is involved in setting the activation threshold, and therefore, the activity of neurons. In neurites ,54. Its neurites . NALCN wneurites .nca(lf) and unc-7 innexin mutants are of an antagonistic nature, suggesting that UNC-7 innexin regulates neuronal activity cooperatively with the NCA leak channel. Pannexins are proposed to either directly or indirectly modulate intracellular calcium. It is widely accepted that they form large, poorly selective channels in the plasma as well as intracellular membranes [The genetic interactions between the embranes -75. Togenca(lf) and unc-7, with the nca(lf) phenotype being dominant at rest, and unc-7 being dominant when neurons were stimulated, may be explained by these channels being activated under different membrane/intracellular conditions. For example, it is possible that at rest, the UNC-7 hemichannel is closed, and this contributes to a membrane/intracellular property that potentiate the opening of the NCA channel to modulate the basal neuronal activity. nca(lf); unc-7 therefore resembles the phenotype of nca(lf) at rest. Upon strong stimulation, however, UNC-7 hemichannels are also activated, and NCA-dependent neuronal activity, such as the increase of intracellular calcium concentration or membrane potential, may be required to inhibit the UNC-7 hemichannel activity to prevent a further increase or prolongation of neuronal activity. Deleting UNC-7 in nca(lf) would therefore lead to an increase of evoked currents.The complex, antagonistic genetic interactions between As previously reported, both NCA and UNC-7 are expressed in punctate patterns along neurites, and both appear excluded from synapses. However, our whole-mount co-immunofluorescent staining with antibodies against UNC-7 and NCA-1 did not show obvious co-localization (data not shown). Though not definitively conclusive due to the limited resolution afforded by the neurite, this further implies that the nature of the NCA and UNC-7 genetic interaction probably reflects a functional, rather than a physical interaction. A better understanding of the properties of the NCA leak and UNC-7 hemichannels, and a detailed examination of their functional requirements in specific neurons for different phenotypes, should help address the mechanistic details of such a functional relationship.The authors declare that they have no competing interests.MB and MZ designed the study and drafted the manuscript. MB and SBG performed electrophysiology experiments; MP and HL performed molecular biology experiments; MB performed all other experiments; WH, JG and JR offered advice and help with imaging and electrophysiology setup; MP, WH and JG edited the manuscript. All authors read and approved the final manuscript.Supplemental Movie 1. wild type (N2) animalClick here for fileSupplemental Movie 2. unc-7 (e5) animalClick here for fileSupplemental tables.Click here for fileSupplemental Movie 3. nca(lf) animalClick here for fileSupplemental Movie 4. nca(lf); unc-7 (e5) animalClick here for fileSupplemental Movie 5. nca(lf); unc-7 (e5) animal expressing UNC-7 by a pan-neuronal promoterClick here for fileSupplemental Movie 6. unc-7 (e5) animal expressing UNC-7 by a pan-neuronal promoterClick here for fileSupplemental Movie 7. unc-7 (e5) animal expressing Cys-less UNC-7 by a pan-neuronal promoterClick here for fileSupplemental Movie 8. unc-7 (e5) animal expressing C191A UNC-7 by a pan-neuronal promoterClick here for fileSupplemental Movie 9. nca(lf); unc-7 (e5) animal expressing Cys-less UNC-7 by a pan-neuronal promoterClick here for fileSupplemental Movie 10. nca(lf); unc-7 (e5) animal expressing C191A UNC-7 by a pan-neuronal promoterClick here for file


Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus replication.A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the “field”. Clinical samples from suspect cases of foot-and-mouth disease (FMD) were obtained from within Pakistan and Afghanistan. The samples were treated to preserve the RNA and then transported to National Veterinary Institute, Lindholm, Denmark. Following RNA extraction, FMDV RNA was quantified by real-time RT-PCR and samples containing significant levels of FMDV RNA were introduced into susceptible cells using electroporation. Progeny viruses were amplified in primary bovine thyroid cells and characterized using antigen ELISA and also by RT-PCR plus sequencing. FMD viruses of three different serotypes and multiple lineages have been successfully rescued from the RNA samples. Two of the rescued viruses (of serotype O and Asia 1) were inoculated into bull calves under high containment conditions. Acute clinical disease was observed in each case which spread rapidly from the inoculated calves to in-contact animals. Thus the rescued viruses were highly pathogenic. The availability of the rescued viruses enabled serotyping by antigen ELISA and facilitated genome sequencing.The procedure described here should improve the characterization of FMDVs circulating in countries where the disease is endemic and thus enhance disease control globally. Aphthovirus within the family Picornaviridae. FMDV particles comprise a single copy of the positive-sense RNA genome (ca. 8300 nt in length) within a near spherical protein capsid of ca. 28 nm which contains 60 copies of 4 different structural proteins, 1A (VP4), 1B (VP2), 1C (VP3) and 1D (VP1)  is one of the most economically important diseases of farm animals FMD can be controlled successfully by vaccination. However, there are seven distinct serotypes  and many subtypes have also been described In countries lacking the infrastructure to identify rapidly the serotype of FMDV in samples, these can be transported to reference laboratories, e.g. the World Reference Laboratory at Pirbright, U.K., for characterization. However, this currently involves the transportation of samples containing infectious FMDV which represents a significant bio-security hazard and also incurs significant costs. Moreover, the virus samples have to be collected and stored under appropriate conditions, until submission, to allow virus to be isolated successfully in the receiving laboratories. In practice, this can be difficult when ambient temperatures are high and distances from points of collection to national laboratories are large and the infrastructure is poor. It is therefore desirable to have a system where samples from suspect clinical cases of FMD can be treated at the point of collection to inactivate the virus but preserve the RNA in a manner which allows the subsequent recovery of infectious virus within a contained laboratory environment.The availability of commercial reagents which preserve the viral RNA, in conjunction with systems for the efficient rescue of picornaviruses from RNA .An aliquot of bovine tongue epithelium suspension, stored at -70°C,  from a bull calf infected with FMDV (serotype O-UKG 34/2001) was used as a source of FMDV RNA obtained from clinical material. Nucleic acid was isolated using both robotic  and manual (QIAamp®RNA Blood Mini Kit) protocols as described by the manufacturers and the RNA samples were eluted in 50 or 60 µl of water respectively.5 ( = 32)-fold decrease in the level of FMDV RNA.FMDV RNA, within these RNA preparations, was quantified using two different real time RT-PCR assays, that target either the 5′ untranslated region or the 3D coding region, as described previously RNAlater (Ambion) in a laboratory prior to transportation to DTU Vet, Lindholm. From these preserved samples, RNA was isolated and the level of FMDV RNA was quantified by real time RT-PCR as above. Samples prepared from epithelium samples typically had the highest amounts of FMDV RNA.Clinical samples (over 1500) were collected in Pakistan and Afghanistan as part of an FAO Regional Project (GTFS/INT/907/ITA). Mouth swab samples were placed directly into RLT buffer (Qiagen) while epithelium tissue samples, from clinically diseased animals, were initially collected in phosphate buffered saline (PBS) and then transferred to 6 cells/ml) in PBS were mixed with 7 µl of RNA in a cuvette (0.4 cm) and subjected to a single, square wave, electrical pulse (25 ms) of 240V using a BioRad Gene Pulser Xcell electroporation system. The treated cells were added to a monolayer of primary bovine thyroid (BTY) cells (in a 6 well plate) and incubated at 37°C for 2 days prior to harvesting by freezing at −70°C. An aliquot (200 µl) of the harvest was added to fresh BTY cells and incubated for a further 1–2 days prior to harvesting as above. Cells were examined on a daily basis to assess the generation of CPE. Stocks of the rescued viruses were grown in BTY cells in 25 cm2 flasks and were titrated as described below.Trypsinized BHK cells (800 µl of 2×1050The titre of the rescued virus samples was determined from 5 replicate dilution series in BTY cells (96 well plates), CPE was scored after both 1 and 2 days and the titre determined at 3 days post infection as TCIDA previously described FMDV antigen ELISA www.ncbi.nlm.nih.gov) and the comparison of VP1 amino acid sequences was assessed using MEGA version 4 Samples of the virus harvest were also used to generate RNA samples as described above and used in RT-PCR assays using appropriate primer sets to obtain overlapping cDNA fragments corresponding either to selected regions of the genome or, in some cases, to the near complete genome (lacking the extreme 5′ untranslated region on the 5′ side of the poly(C) tract). DNA sequences of the amplicons were generated by Agowa (Germany). Sequence assembly was achieved using SeqMan Pro, sequence comparisons were performed using BLAST  of FMDV. Each inoculated animal was kept in contact with two other similar calves in pens within high containment animal accommodation and were monitored on a daily basis for signs of clinical disease  for a period of 10 days (unless euthanized prior to this time). Serum and mouth swab samples were collected before and daily after inoculation of the animals until euthanasia. RNA was then extracted from these samples and assayed for the presence of FMDV RNA by real time RT-PCR as above. The level of RNA detected in serum samples was converted to genomes per/µl by reference to a standard curve of reference RNA samples assayed in parallel . The serum samples were also screened for the presence of anti-FMDV antibodies (using a 1∶10 dilution of serum) using serotype specific antibody ELISA systems (for O and Asia 1 as appropriate) and positive samples were then titrated to quantify the antibody content Two similar experiments were performed to determine the pathogenicity of two distinct rescued FMD viruses of different serotypes (O and Asia 1). In each case, two male calves (ca. 200 kg) were inoculated into the tongue, at two sites, with ca.10To optimize the procedure for the rescue of infectious FMDV from RNA preparations derived from clinical samples, initially RNA was isolated from bovine tongue epithelium suspension which was generated from cattle infected with serotype O FMDV (O-UKG 34/2001). The RNA was prepared both manually and by robot, then the FMDV RNA was quantified in each preparation using a standard diagnostic real-time RT-PCR assay It should be noted that on day 1 following electroporation, it is difficult to score low levels of CPE due to the large number of dead and floating cells, however, when high levels of viral RNA were present in the samples, then CPE could be observed on day 2. Furthermore, even when the lowest level of RNA was used, the generation of CPE in the second passage was evident on day 2.nd passage. This assay has the feature that it has an absolute requirement for virus replication in order to generate the virus protein which is detected since only viral RNA was applied to the cells. The results are shown in To determine that the CPE resulted from the rescue of virus from the electroporated FMDV RNA, a serotype specific antigen ELISA for FMDV was employed using the material harvested after the 2RNAlater to preserve the RNA and inactivate the virus. Following transportation to DTU Vet, Lindholm, total RNA was extracted from these samples and the presence of FMDV RNA was established and quantified using real time RT-PCR assays. With samples shown to contain FMDV RNA, conventional RT-PCR assays were used to amplify cDNA corresponding to the entire VP1 coding sequence and the amplicons (ca. 900 bp) were sequenced in order to identify the serotype of FMDV present within the clinical samples.Clinical samples (over 1500) were collected from cattle and buffalo from within Pakistan and Afghanistan. These materials included samples of bovine tongue epithelium from clinically diseased animals which were collected in PBS, transported back to the National Veterinary Laboratory in Pakistan or Afghanistan and then transferred to In an initial experiment, a collection of nine different RNA samples prepared from clinical samples obtained in Afghanistan and Pakistan and shown to contain high levels of FMDV RNA  were selected to attempt virus rescue. From comparisons with other FMDV sequences (using BLAST) with the VP1 coding sequences of amplicons generated in conventional PCRs, it was known that these clinical samples contained FMDV RNA derived from either serotype A or Asia 1 viruses see . FollowiAFG-07 and A IRN05BAR-08 . As with the Asia 1 viruses, VP1 coding sequences determined from the initial RNA preparations and from the rescued serotype O and A viruses were identical except for a single nucleotide substitution. Thus, in total, some 850 bp have been sequenced from amplicons, including the complete VP1 coding sequence derived from the six RNA samples generated both before and after virus rescue, and only a single nucleotide discrepancy  in one of the six rescued viruses has been detected.Subsequently, four other viruses have been rescued from epithelium samples collected in Pakistan and Afghanistan, each successful rescue was from RNA samples which had a Ct value of <20 see . One of Thus from 16 clinical samples, containing high levels of FMDV RNA  that were tested in the virus rescue system, some 6 different viruses have been rescued which are representatives of 3 different serotypes .The availability of the rescued viruses greatly facilitated the process of generating near complete genome sequences. To obtain these sequences (ca. 8000 nt) for the rescued serotype O (O/ISL/PAK/L1573/2009) and Asia 1 (Asia-1/BAM/AFG/L590/2009) viruses, some 15 separate RT-PCR products were generated to provide overlapping cDNA fragments corresponding to all the genome sequence on the 3′ side of the poly(C) tract. These amplicons were sequenced in both directions and the sequences were then assembled and shown to include a single large open reading frame as expected. These sequences have been submitted to Genbank (accession numbers HQ113232 and HQ113233 for these type O and Asia 1 viruses respectively).The Asia-1/BAM/AFG/L590/2009 virus is most closely related (96–98% nt identity in VP1 coding sequences) to viruses causing disease outbreaks in 2003 in Tajikistan, Uzbekistan and Afghanistan, in 2004 in Kyrgyzstan and Pakistan and in 2005 in Hong Kong and this represents a reappearance of this serotype in this region. The serotype O virus (O/ISD/PAK/L1573/2009) is related to the Pan Asia II strains that have circulated widely in recent years. However the VP1 coding sequence is only about 94% identical to the representative PanAsia II viruses [e.g. O/Pak4/2006 (accession no. EF494501.1) and O/Pak6/2006 (accession no. EF494500.1)] and thus represents a new sub-lineage. This has been tentatively assigned as a PanAsia III lineage but this will probably be dependent on the survival and spread of this strain. Analysis of the near complete genome sequences of this virus and other more typical PanAsia II strains (which are still in circulation in Pakistan (e.g. O/NWF/PAK/L1417/2009)) indicated a sequence identity of about 95% across the entire genome. However, it is interesting to note that the deduced amino acid sequences of the VP1 proteins from the O/ISL/PAK/L1573/2009 virus and some other samples collected in Pakistan during 2009 differ from the Pan Asia II serotype O viruses which are still currently circulating there and include changes flanking the receptor binding motif (RGDLXXL) within residues 140–160 (G-H loop) which is an important antigenic site see . A fulleTo test the pathogenicity of the rescued viruses, two experiments were performed using the serotype Asia 1 and serotype O viruses. In each case, two bull calves were inoculated into the tongue and each of these was then kept with two in-contact calves and all six animals were closely monitored over a period of 10 days during which time serum and mouth swab samples were collected. The presence of FMDV RNA in these samples was determined and the generation of anti-FMDV antibodies within the sera was assayed.In the experiment using the rescued serotype Asia 1 virus (Asia-1/BAM/AFG/L590/2009), the two inoculated cattle exhibited an elevated temperature at 1 dpi and this remained high for another 2 days . VesicleSera collected from the animals were tested for antibodies against FMDV in a competition ELISA, initially using a single dilution of serum . Both thSimilarly, in the experiment using the rescued serotype O virus (O/ISL/PAK/L1573/2009), the two inoculated cattle exhibited a markedly elevated temperature at 1 dpi which gradually subsided over the next 3 days . VesicleBoth the inoculated cattle (2C1 and 2C5) seroconverted to FMDV at 5 dpi and the three surviving in-contact animals seroconverted on day 8 see . Not sur9 genomes/ml serum on days 3 and 4 which was about 30-times higher than observed in any other animal  was detected with the serotype O virus in an in-contact animal which showed particularly severe clinical signs of disease (and was therefore euthanized at an early stage of the experiment). Due to the high particle to pfu ratio (ca. 100) known for FMDV, and other picornaviruses, it is likely this level of viræmia is roughly equivalent to the high level (ca. 107.8 TCID50/ml) described for the serotype C Noville strain of FMDV in cattle (see Both the FMDV Asia 1 strain from Afghanistan and the serotype O strain (tentatively assigned as belonging to a new PanAsia III sub-lineage) from Pakistan obtained through this virus rescue procedure proved to be capable of initiating severe clinical disease when inoculated into the tongues of calves kept within high containment animal accommodation. Furthermore, the initial infections rapidly spread to in-contact animals and classical symptoms of FMD became apparent in each of the animals with fever and multiple lesions in the mouth and on the feet. The profile of viræmia (lasting 3–4 days) and the induction of an anti-FMDV immune response detectable from 4–5 days post inoculation were fairly typical see . It was ttle see .A feature of the virus rescue system described here is that virus amplification is achieved by infection of primary bovine cells (BTY). This system can be expected to maintain the “field” characteristics of the viruses and not introduce specific adaptations to cell culture within the capsid region which are frequently seen in BHK cells for example. The initial introduction of the viral RNA into BHK cells by electroporation was required due to the large number of cells used for this process and is not expected to induce adaptations, at least within the capsid region, since no capsid functions were required to initiate the infection cycle within these cells. The marked pathogenicity of the rescued viruses studied here clearly shows that this characteristic of these viruses was not significantly diminished in this procedure. There has been a recent description Using RNA samples generated directly from clinical material within our laboratory, it has been possible to rescue infectious FMDV from samples containing quite low levels of viral RNA . However, using RNA samples derived from clinical samples collected in Pakistan and Afghanistan and then transported to Denmark, we have found that only samples containing a much higher level of FMDV RNA  have been successfully rescued, this represents about a1000-fold higher level of viral RNA. It should be noted that even a single break in the RNA strand (ca. 8300 nt) is sufficient to destroy the infectivity of the RNA whereas it will have rather little effect on the apparent level of FMDV RNA detected in the real time RT-PCR assays as these target small regions of the genome (ca. 100–150 nt). Furthermore, the epithelium samples used here were transported to regional laboratories before the RNA preservation was performed. Direct placement of such samples into an RNA preservative at the point of collection can be expected to improve the efficiency of virus rescue. Clearly, if virus can be rescued from swab samples collected directly into an RNA preservative then this will be beneficial, on the basis of simplicity, and thus attempts to achieve this are in progress.When outbreaks of FMDV occur, it is important for national authorities to be informed of the characteristics of the strain to allow selection of appropriate vaccines. To obtain detailed information about the properties of an outbreak strain, analysis of the live virus is required. The identification of serotype can be achieved by antigen ELISA if virus is available; however the sensitivity of these assays is relatively low so the ability to grow the virus can be important. Serotype identification can also be achieved by sequencing of amplicons corresponding to the VP1 (1D) coding sequence. Such assays can be performed directly on RNA obtained from clinical material but the analyses are greatly facilitated by the ability to grow the virus and hence obtain RNA preparations containing high levels of FMDV RNA. The pathway of infections within an outbreak can be traced using full-length genome sequence analysis


Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron. How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk  Normal aging is characterized by progressive deterioration of several functions -3. What  Aging also differentially alters components of neural circuitry. In a study that measured long-term potentiation (LTP) in middle aged and young rats, Rex et al showed that LTP at basal dendrites induced by theta burst stimulation decayed rapidly in older rats, whereas LTP induced at apical dendrites did not show any significant difference . Aging impacts brain regions differentially. For example, in rats, the cerebellum showed earlier senescence in the hippocampus. Stereological studies of rats at different ages have shown significant loss of cerebellar Purkinje neurons, whereas hippocampal pyramidal neurons were stable across age groups . MoreoveAplysia. Neurons of Aplysia are larger and easily tractable from animal to animal  in order to facilitate the dissection of the connective tissue sheath overlying cells. Importantly bursting activities of R15 neurons were not affected by the protease treatment [ Both mature and old animals were anaesthetized, injecting 30-35% of their body weight of a 380 mM MgClreatment . We alsoreatment ,30.  R15 was impaled with single intracellular sharp microelectrode (10-15 MΩ), filled with a 3M KCl solution for measuring membrane potential (voltage) and bioelectrical activity, which were recorded (10 kHz band pass) through an intracellular recording system BRAMP-01R ver. 4.2 , digitally converted through the Instrutech ITC-18 , and then processed by Axograph X  and analyzed using a pCLAMP 10.0 . Only R15 neurons from mature and old animals with a rhythmic stable bursting activity were included in the analysis. Once R15 rhythmic bursting patterns were observed, the baseline activity was recorded for 30 minutes or more in order to check the stable activity before drug application. ACh 1 M (volvolume 0.3 uL) was directly applied into the bath using micropipette to obtain a final 1 mM concentration inside the recording chamber. Following the ACh application, R15 activity was observed for ≅ 8 minutes. Bath ACh application leads R15 to a depolarization. The depolarization rapidly reaches a plateau, indicating the point at which the drug is most effective. Any effect of ACh on the R15 membrane potential or bursting activity shown was reversible upon washing. Due to the volume of the chamber and the ASW flow rate, a complete washing is accomplished 2 minutes after ACh application.  To study the effect of ACh on R15 membrane potential, the following parameters were analyzed: (i) membrane potential at reference condition; (ii) amplitude of the membrane potential shift (from the reference condition to the plateau); and (iii) latency of effect (time elapsed from the beginning of the shift to the plateau). To identify age-related changes in R15 membrane potential and ACh-responsiveness, the values obtained from mature animals were compared to those obtained from older animals using a Student’s t test. In order to study ACh-related changes in the bursting activity of each recorded R15 neuron, both burst duration and the number of burst APs, before and after ACh, were separately evaluated, and the value was calculated with a duration of 90 seconds. The effect of the ACh on the basal activity was evaluated starting from the beginning of the shift of the membrane potential. Furthermore, burst duration and the number of burst APs of the first burst following neurotransmitter application were also evaluated, and the value was calculated separately.  The baseline values were measured during the period preceding the neurotransmitter application. For each of these analyses, we calculated the number of action potentials (before and after), and duration of each burst measured 90 seconds before and 90 seconds after ACh treatment, and averaged for one animal. Then we combined all of the data for statistical analysis. First burst action potentials and burst duration was calculated by comparing the number of action potentials in the first burst after ACh application and compared to burst immediately before ACh exposure. Off-line analyses of the following waveform parameters of bursts APs were studied: (i) Depolarization amplitude, (ii) Depolarization Duration, (iii) Repolarization amplitude and (iv) Repolarization duration. Baseline AP waveform parameters values were obtained from baseline bursts collected during the 90-second period preceding the neurotransmitter application. To evaluate ACh-induced changes in the bursts APs, waveform parameters were calculated from those bursts collected during a window time (with a duration of 90 seconds) starting from the beginning of the shift of the membrane potential. As in the case of measurements of bursting described above, we analyzed all of the APs produced in bursts 90 seconds before and 90 seconds after ACh treatment for each animal, and data was combined for statistical analysis (One way ANOVA followed by Tukey’s HSD post hoc test). All the data shown in the figures are included in the supplementary section . RNA from single neurons were subjected to two rounds of linear T7 RNA Polymerase–driven transcription using MessageAmp™ II aRNA Amplification Kit from Ambion before cDNA synthesis. cDNA was generated by reverse transcription from 1 μg of RNA from abdominal ganglia and single neurons using Quanta cDNA supermix (Quanta Biosciences) according to the manufacturer’s instructions. The expression profiles of eight AChR genes in the abdominal ganglia were first studied by qPCR using SYBR green PCR master mix . All of the qPCR amplifications were performed in a total volume of 10 μl containing 2 μl of H2O, 2 μl of cDNA, 5 μl of 2X Master Mix, 1.0 μl of 10 μM (each) forward and reverse primer (see Table S5 in Tables S1) designed based on the AChR sequences  available at NCBI (http://www.ncbi.nlm.nih.gov/). The qPCR reaction was carried out in a 7900HT Fast Real-Time PCR System  under the following conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 s, 60°C for 1 minute. Quantification of the target transcripts was normalized to the Aplysia 18S rRNA reference gene using the Pfaffl method [Aplysia. Tukey’s multiple comparison test was used for post hoc analysis. Gene transcripts were considered differentially expressed when there was at least a 2-fold change (up or down), and the p value was set at <0.05. Total RNA was isolated from Figure S1Resting membrane potential analysis of all the mature and old R15 neurons . Statistical analysis suggests that resting membrane potentials of mature and old R15 neurons are not significantly different .(TIF)Click here for additional data file.Figure S2Analysis of rhythmic bursting R15 neurons. A representative trace, baseline, ACh treatment, first burst analysis, shift and calibration graph are shown.(TIF)Click here for additional data file.Figure S3Comparison of amino acid sequences of AChRs expressed in R15 neuron.AChRs sequences obtained from NCBI were translated and the open reading frames (ORFs) were compared using multalin . Amino acids in red indicate identity, and blue indicate similarity among the five AChRs.(TIF)Click here for additional data file.File S1Table S1: Intracellular measurements of R15 neurons from mature and old Aplysia. Table S2: Membrane potential measurements in old and mature R15 neurons. Table S3: Analysis of ACh induced changes in rhythmic bursting in mature and old R15 neurons. Table S4: Single spike analysis of bursting. Measurements of amplitude and duration of depolarization and repolarization of AP waveform. Table S5: Sequences of qPCR primers used in the gene expression analysis. Table S6: AChR gene expression data obtained from single R15 neuron qPCR experiments.All supporting tables are contained File S1: (XLSX)Click here for additional data file.


Due to an error in the production process, Ming Liang Wang was not included in the author byline. He should be listed as the third author, and his affiliations are 1 Department of Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China, 2 Shanghai Institute of Digestive Surgery, Shanghai, People’s Republic of China, 3 Shanghai Minimally Invasive Surgery Center, Shanghai, People’s Republic of China. The contributions of this author are as follows: Conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, and wrote the manuscript.The correct citation is: Feng B, Dong TT, Wang ML, Wang LL, Zhou HM, et al. (2012) Colorectal Cancer Migration and Invasion Initiated by microRNA-106a. PLoS ONE 7(8): e43452. doi:10.1371/journal.pone.0043452


Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors. Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer in clinical applications and basic research. The most commonly encountered problem is that the virus titer is too low. Retroviral vectors produced by DNA transfection of producing cells usually contain a large amount of non-infectious virus. The ratio of infectious virions to the total MLV particles could be as low as 1/100 In vitro experiments suggested that host cells control the production of genomic RNA-deficient virus. The synthesis of such deficient virus was greatly different in different host cells For the wild type replication-competent MLV virus, the genome-length viral RNAs of MLV are believed to be separated into two non-equilibrating pools in virus producer cells, with one serving as mRNA for translation of Gag and Gag/Pol polyproteins and the other as genomic RNA for packaging c-myc mRNA from endonuclease cleavage and increases its stability Insulin-like growth factor II mRNA binding protein 1 (IMP1) is a member of the VICKZ family Based on the abilities of IMP1 to inhibit the translation and to increase the stability of some RNAs, we investigated the role of IMP1 in regulating the production of infectious MLV vectors. Our results demonstrated that overexpression of IMP1 increased the viral genomic RNA levels both in virus producer cells and virions and thereby the production of functional MLV vector.To test whether expression of IMP1 affected the production of MLV-luc, a retroviral vector carrying the firefly luciferase reporter To test whether IMP1 enhanced the production of infectious MLV-luc in a dose-dependent manner, different amounts of the IMP1-expressing plasmid was transfected with the MLV-luc producing constructs. Consistent with the above results, the expression levels of Gag protein in the producer cells were little changed , and theTo test whether IMP1 facilitated the incorporation of viral genomic RNA into the virions, the viral genomic RNA levels in the virion particles were measured. MLV-luc pseudovirus produced in the presence of empty vector, IMP1 or IMP3 were purified and concentrated. The same amounts of virion particles were used, as indicated by the amount of capsid (CA) protein in the virions . The virThe increased incorporation of MLV-luc RNA into the virion particles could be accounted for by increased abundance of the RNA in the producer cells or more efficient encapsidation of the RNA into the virions, or both. To test these possibilities, we first analyzed whether IMP1 bound to the viral genomic RNA in the producer cells. IMP1 was co-expressed with MLV-luc  in 293T To test whether expression of IMP1 increased the abundance of the viral genomic RNA in the producer cells, the viral genomic RNA levels in the producer cells were analyzed by Northern blotting. In the IMP1-expressing cells, the viral genomic RNA level was significantly higher compared with that in the control cells . When inWe then further analyzed whether IMP1 was incorporated into virion particles. APOBEC3G, which has been shown to be incorporated into virions To explore whether endogenous IMP1 regulated the production of retroviral vectors, the expression level of IMP1 was downregulated by RNA interference in the producer cells and the production of MLV-luc vector was assayed. The plasmids expressing shRNAs directed against hIMP1 were first confirmed for their ability to downregulate the expression of hIMP1 in 293 cells . MLV-lucTo examine whether IMP1 exerts the same effect on the production of wild-type MLV, the virus was produced in the presence of IMP1. Consistent with the above result that IMP1 inhibited the expression of luciferase from the viral genomic RNA , the proEfficient packaging of virial genomic RNA is critical for the production of high-titer retroviral vectors. In this report, we demonstrated that overexpression of IMP1 in the producer cells facilitated the packaging of viral genomic RNA and thereby improved the production of infectious MLV-luc vector in an IMP1 expression level-dependent manner , 2. FurtThe stimulatory effect of IMP1 on the production of infectious virion particles was specific only for retroviral vectors. In the retroviral vector producing cells, the viral proteins are expressed from constructs separate from that expressing the viral genomic RNA. Therefore, IMP1 increased the abundance of the viral genomic RNA in the pool for packaging without affecting the expression of the viral proteins. For wild-type MLV, the same unspliced viral RNA serves as both genomic RNA for packaging and mRNA for translation. IMP1 inhibited the expression of the viral proteins while increased the incorporation of viral genomic RNA into the virions . As a coIn contrast to the stimulatory effect of IMP1 on MLV vectors, it has been reported that IMP1 bound to HIV-1 RNA but inhibited viral genomic RNA packaging MLV producing plasmids pCMV-VSVG, pHIT60 and pMA-Luc have been previously described GAATTCGCCACCATGAACA AGCTTTACATCGG-3′5′-CCG;IMP1-SP: GCGGCCGCCCTTCCTCCGAGCCTGGGCCA -3′5′-AAGGAAAAAA;IMP1-AP: AAGCTT GCCACCATGAACAAATTGTACATCGGGAA-3′5′-CCC;IMP3-SP: GAATTCGCCTTCCGCCTTGACTGAGGTG-3′5′ -CCG;IMP3-AP: To generate the shRNAs directed against IMP1, oligonucleotides were designed and cloned into pSuper following the manufacturer's instruction (OligoEgine). The antisense sequence of the shRNAs directed against mIMP1 and hIMP1 are listed below:5′- TTATGAGCCAGGATCTTCAGGG-3′.mIMP1-26i: 5′- TCTTGCTCTACCTTCTTCA-3′;hIMP1-4i: 5′-TTTCACGATGACCTGGTCG-3′.hIMP1-9i: NIH 3T3 cells (ATCC CRL-1658), Rat2 cells (ATCC CRL-1764), 293 cells (ATCC CRL- 1573) and 293T cells (ATCC CRL-11268) were maintained in DMEM supplemented with 1% antibiotics and 10% fetal bovine serum (Invitrogen).To produce VSV-G pseudotyped MLV-luc, pCMV-VSV-G, pHIT60 and pMA-Luc were transfected into 293T cells. The reneilla expressing plasmid, pRL-TK (Promega), was included to serve as a control for transfection efficiency and sample handling. At 48 h post transfection, the supernatants were collected, centrifuged at 2,000 rpm for 5 min and filtered through 0.45 µm filters to remove cell debris. To purify and concentrate the virion particles, supernatants were first subjected to 25%/45% sucrose step gradient centrifugation at 25,000 rpm with Hitachi P40ST rotor for 2 h at 4°C and then concentrated by ultracentrifugation with 25% sucrose cushion for 2 h. The pellets were resuspended in TNE buffer  and stored at −80°C for future use.The luciferase activities were measured 48 h after transfection or infection by the dual luciferase assay system following the manufacturer's instruction (Promega).The mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology) was used to detect proteins with the myc epitope and the mouse monoclonal antibody M2 (Sigma) was used to detect proteins with the Flag epitope. The N-terminal domain of MLV-CA with a His-tag was bacterially expressed and used to immunize rabbits to generate polyclonal antiserum. The antibody was affinity purified using the recombinant CA protein. The rabbit polyclonal antiserum against MLV-RT was a generous gift from Dr. Stephen Goff of Columbia University, New York, USA.The homopolymer RT assay and the endogenous RT assay have been previously described The assays for detecting the minus strand strong stop DNA and nuclear circular DNA have been previously described 5′-TCACTGCCACCCAGAAGACTGTGG-3′) and GAPDH-RP (5′-GGTCCACCACCCTGTTGCT GTAGCC-3′). The PCR conditions for the MSS DNA and GAPDH were 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 21 cycles for MSS and 22 cycles for GAPDH. The same conditions and primers were used for realtime-PCR analysis.The virions were lysed in Trizol buffer (Invitrogen). To each sample, 10 µg of total RNA of 293 cells was added to help as well as to serve as a control for sample handling. The RNA was extracted, ethanol precipitated and re-suspended. To remove possible DNA contamination, the RNA was treated with the DNA Free kit (Ambion) followed by ethanol precipitation. The viral genomic RNA level was measured by reverse transcription followed by PCR or realtime-PCR using specific primers for the minus-strand strong stop (MSS) DNA To analyze the viral genomic RNA levels in the producer cells, the cells were trypsinized, washed and lysed in the Trizol buffer (Invitrogene). The total RNA was isolated following the manufacturer's instruction. The RNA levels were measured by Northern blotting analysis following the procedure described previously pMA-luc was cotransfected with pcDNA4-IMP1 or empty vector into 293T cells. At 48 h posttransfection, cells were washed extensively with diethyl pyrocarbonate (DEPC)-treated PBS. A quarter of the cell was lysed in the Trizol buffer (Invitrogene) to extract total RNA for analyzing the expression of pMA-luc and GAPDH RNAs. The rest cells were lysed in the lysis buffer . An aliquot of the cell lysate was used to examine IMP1 expression by Western blotting. The rest lysate was precleared by incubating with mouse anti-HA antibody and proteins G-sepharose (Pharmaicia) in the presence of RNasin (40 u/ml) at 4°C for 2 h, followed by brief centrifugation. The precleared cell lysate was incubated with new protein G-sepharose beads with or without 3 µl anti-myc antibody at 4°C for 2 h. The beads were washed with the washing buffer  for seven times. The RNA was extracted with the Trizol buffer, treated with DNase I (Ambion) and analyzed by RT-PCR to detect the viral RNA as described above.


There has been increased interest in the study of molecular survival mechanisms expressed by foodborne pathogens present on food surfaces. Determining genomic responses of these pathogens to antimicrobials is of particular interest since this helps to understand antimicrobial effects at the molecular level. Assessment of bacterial gene expression by transcriptomic analysis in response to these antimicrobials would aid prediction of the phenotypic behavior of the bacteria in the presence of antimicrobials. However, before transcriptional profiling approaches can be implemented routinely, it is important to develop an optimal method to consistently recover pathogens from the food surface and ensure optimal quality RNA so that the corresponding gene expression analysis represents the current response of the organism. Another consideration is to confirm that there is no interference from the "background" food or meat matrix that could mask the bacterial response.Salmonella cells. First, we tested the optimum number of Salmonella cells required on the poultry meat in order to extract high quality RNA. This was analyzed by inoculating 10-fold dilutions of Salmonella on the chicken samples followed by RNA extraction. Secondly, we tested the effect of two different bacterial cell recovery solutions namely 0.1% peptone water and RNAprotect (Qiagen Inc.) on the RNA yield and purity. In addition, we compared the efficiency of sonication and bead beater methods to break the cells for RNA extraction. To check chicken nucleic acid interference on downstream Salmonella microarray experiments both chicken and Salmonella cDNA labeled with different fluorescent dyes were mixed together and hybridized on a single Salmonella array. Results of this experiment did not show any cross-hybridization signal from the chicken nucleic acids. In addition, we demonstrated the application of this method in a meat model transcriptional profiling experiment by studying the transcriptomic response of Salmonella inoculated on chicken meat and exposed to d-limonene. We successfully applied our method in this experiment to recover the bacterial cells from the meat matrix and to extract the RNA. We obtained high yield and pure RNA. Subsequently, the RNA was used for downstream transcriptional profiling studies using microarrays and over 600 differentially regulated genes were identified.Our study involved developing a food model system using chicken breast meat inoculated with mid-log Salmonella is ideal to obtain optimal RNA amount and purity. Our results demonstrated that RNAprotect yielded higher RNA amounts (approximately 10 to 30 fold) when compared to 0.1% peptone water. The differences between the RNAprotect and 0.1% peptone samples were significant at a p-value of 0.03 for the bead beater method and 0.0005 for the sonication method, respectively. The microarray experiment demonstrated that the chicken samples do not interfere with the hybridization of Salmonella cDNA on the array slide. Hence, the background chicken RNA will not interfere with the microarray analysis when poultry meat models are used. Finally, we successfully demonstrated the application of the poultry meat model proposed in this study by conducting transcriptional profiling analysis of Salmonella inoculated on the poultry. Results of this study proved that this method has the potential to be employed in other meat model studies.Our result showed that 8 log cfu/g of  Salmonella is the most common cause of foodborne illness among enteric pathogens [Salmonella due to fecal contamination of carcasses is a major issue for the poultry industry. Additionally, the USDA-FSIS report established a "Salmonella verification program with a goal of having 90% of the poultry houses meeting new standards" [Salmonella is one of the leading foodborne pathogens potentially present on the surface of poultry. Several hurdles are used by the poultry and meat industry to combat these foodborne pathogens and eliminate their presence on carcasses. Chemical washes such as chlorine, organic acids, cetylpyridinium chloride, and trisodium phosphate are commonly used in the United States but are prohibited in some countries belonging to the European Union (EU) [Disease caused by foodborne pathogens contributes to serious public health concerns . The Fooathogens . The Worathogens . Accordiandards" . Salmoneion (EU) -7.Staphylococcus aureus on the surface of pork products over time [It is important to understand the physiological status of the pathogens stressed by these antimicrobial treatments as well as their genomic and proteomic responses. This will enable researchers to identify the best possible combinations of antimicrobial treatment that does not lead to cross-protection responses by the pathogen . Studyinver time .Salmonella from the chicken meat surface and perform the subsequent RNA extractions. In addition, the effect of "the background meat matrix" cross-contamination in the downstream expression profiling study was evaluated by hybridizing the chicken tissue RNA and Salmonella RNA to the Salmonella genomic microarrays. Subsequently, to test this methodology we applied this method to study the transcriptional response of Salmonella inoculated on poultry meat and exposed to d-limonene antimicrobial treatment. In this experiment Salmonella cells were recovered from the poultry meat treated with d-limonene (test) and sterile water (control). The cell recovery and RNA extraction was carried out by the method described in this paper and the RNA was used for downstream transcriptomic analysis using microarrays.To study the gene expression of pathogens on the surface of chicken carcass, recovery of cells and their corresponding purity of RNA is crucial. Therefore, in this study we compared RNA extraction methods for pathogen-poultry surface transcriptome studies. In order to develop this standard method, two different bacterial cell recovery solutions (0.1% peptone water and RNAprotect solution) and two different cell lysing methods (sonication and bead beater), were used to recover the Salmonella enterica serovar Typhimurium ATCC 14028 from the culture collection of the Center for Food Safety, University of Arkansas was used for this study. Stock cultures of the strain were held in Luria Bertani broth (LB) and glycerol at -80°C. The cultures were grown overnight in LB at 37°C and transferred twice before use in the experiment.The citrus oil, d-limonene, was obtained from Firmenich Citrus Center, Safety Harbor, FL, and stored at 4°C as per the manufacturer's recommendation.The outline of the overall method is shown in Figure Chicken breast was purchased at retail stores, cut into pieces in a sterile environment, and stored in sterile stomacher bags. Five ml of RNAprotect solution was added directly to the stomacher bag and stomached manually. The liquid portion was recovered and centrifuged in polypropylene tubes. The pellets were stored at -80°C overnight and used for subsequent RNA isolations.600 approximately 0.4) Salmonella for 30 min. The pieces were placed in sterile stomacher bags and sprayed with either d-limonene (test) or sterile water (control) and incubated for 15 min. Five ml RNAprotect was added to the stomacher bags and stomached by hand for 30 sec. The liquid portion was transferred by pipette to 50 ml polypropylene tubes and centrifuged at 10,000 rpm for 10 minutes at 4°C . The supernatant was discarded and pellets were used for RNA isolation.The chicken samples were inoculated with mid-log (ODThe RNeasy mini kit (Qiagen Inc.) was used for RNA isolation with some modifications described in the next section. One ml of TRIzol  was added to the bacterial or chicken tissue sample and vortexed for 40 sec. The samples were either subjected to sonication or to a bead beater in order to break the cells. For the sonication method, the sample was transferred to a glass sonicator tube and sonicated three times at 1 min intervals . For the bead beater process, the samples were transferred to FastPrep blue tubes containing glass beads . The cells were broken in FastPrep for 40 sec at a bead beating frequency speed setting of number 6. Following this, 200 μl of chloroform was added and then centrifuged at 4°C for 15 min at 12,000 rpm . The upper phase containing nucleic acid was collected and transferred to an RNeasy mini kit column and further purification steps were conducted according to kit instructions. In addition, the RNA was subjected to DNase treatment (Qiagen Inc.), to remove any DNA residues that could affect the downstream reaction. The total RNA quantity and purity ratios were calculated using a Nanodrop 8000 . The RNA was stored at -80°C and used for downstream molecular analysis.Salmonella samples were annealed with random hexamers as primers (Invitrogen). The samples were placed in a heating block at 70°C for 5 min and then incubated in ice for 1 min. Following this, SuperScript III reverse transcriptase (Invitrogen) and 0.1 M dithiothreitol 12.5 mM dNTP/aminoallyl-UTP  mix was added and the samples were incubated at 42°C for 18 h. Residual RNA was removed and cDNA was purified with a QIAquick PCR purification kit (Qiagen Inc.). Purified aminoallyl-modified cDNA was subsequently recovered, labeled with Cy3 or Cy5 mono-functional NHS ester cyanogen dyes , and purified, using a PCR purification kit  following the manufacturer's instructions. Purified labeled cDNA were hybridized on S. Typhimurium microarray version 5 slides provided by the Pathogen Functional Genomics Resource Center (PFGRC). Hybridized slides were scanned using a GenePix 4000B microarray scanner .cDNA was synthesized from the purified RNA as described by Muthaiyan et al. (2008) . BrieflyStatistics were done to analyze differences between the two different methods using Microsoft Excel (Microsoft Office 2003). Type 2 t-tests statistics were performed to obtain the p-value and test significant differences among various groups.Salmonella. These results were further confirmed by the increased adhesion of heat stressed Salmonella to Caco-2 cells [Salmonella inoculated on a chicken matrix and performing downstream transcriptional profiling analysis.There is an increasing need to study the effect of antimicrobial treatments on the transcriptome of the pathogen. Results from these studies enable researchers to elucidate the molecular mechanisms by which foodborne pathogens tolerate and survive on complex food matrices that are subjected to a variety of antimicrobial treatments. Our previous microarray studies have demonstrated the induction of adhesion genes in heat stressed -2 cells . Only a -2 cells -14. Tran-2 cells . MicroarSalmonella cells were added to 25 g chicken breast samples. We observed that 8 log cfu/g resulted in a RNA yield of 92.7 ± 6.5 ng/μl with a purity ratio of 2.0 (260/280 and 260/230). The lower dilutions of 7, 6, and 5 log cfu/g mid-log Salmonella cells exhibited a lower concentration of RNA with poor purity. Results of this study showed that 8 log cfu/g of bacterial cells were required to obtain both high yield and pure RNA. The RNA yields obtained after inoculating 8 log cfu/g on chicken was more than sufficient for molecular analysis using quantitative real- time reverse transcriptase PCR (qRT-PCR) since the technique requires approximately 100 ng RNA per reaction [To test the minimum number of cells required for quantifiable bacterial RNA yield serial dilutions of 8, 7, 6, and 5 log cfu/g mid-log reaction . Howeverreaction . For thiSalmonella cells recovered with 0.1% peptone water and RNAprotect solution using two different methods for cell lysis was tested  was the dominant color and very few spots with Cy5 (cDNA made from chicken RNA) hybridization was observed  was not interfering with downstream molecular gene expression analysis we performed a microarray analysis using pure culture rganisms -28. Thisrganisms . In our n genome . Howevern sample . TherefoSalmonella inoculated on the poultry meat matrix. Previous studies in our laboratory have shown d-limonene, a natural citrus oil, to be inhibitory against several strains of Salmonella [600 approximately 0.4) Salmonella on poultry samples for 30 min as described in the previous sections. The poultry samples were sprayed with d-limonene and Salmonella was recovered and used for RNA extractions as described in the previous sections. We obtained 1000 ng/μl of RNA yield with high level purity (260/280 and 260/230 ratios approximately 2). This ratio is generally recommended for downstream molecular analysis [Salmonella exposed to d-limonene. We observed an optimum Cy3 and Cy5 dye labeling of the cDNA synthesized from the RNA extracted from the Salmonella cells recovered from the meat matrix. Hybridization signals on the microarray did not display any background signal interference from the chicken RNA. Further analysis of the hybridization signal revealed a total of 359 upregulated and 313 downregulated Salmonella genes in response to the d-limonene treatment  following the application of antimicrobials .In order to examine the practical application of this optimized method we designed an experiment to test the effect of an antimicrobial on the transcriptome of lmonella . Transcranalysis . In ordeSalmonella inoculated on chicken pieces. From a practical standpoint, the methodology demonstrated in this study can be used to recover bacterial cells from the different meat matrices and used in further molecular studies such as microarray and qRT-PCR. The method described in this study could be useful to devise microarray approaches as laboratory tools to screen genes that may be differentially expressed after antimicrobial addition where a large number of bacterial cells would need to be inoculated onto the poultry matrix.Gene expression analysis of pathogens inoculated on food matrix is crucial to understand the state of the pathogen on a molecular level. This type of analysis is complicated because of background food matrix contamination. Results of our study demonstrated that the meat matrix does not interfere with the microarray analysis of The authors declare that they have no competing interests.SAS designed and performed the experiments and drafted the manuscript. AM participated in the experimental design and manuscript preparation. SCR participated in the experimental design and production of the draft of the manuscript. All authors have read and approved the final manuscript.Salmonella Typhimurium genes in response to d-limoneneSupplementary table S1. Differentially regulated . Fold changes of the genes induced and repressed due the effect of d-limonene on the genome of S. Typhimurium is demonstrated in this table.Click here for file


Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a ‘lure and lock’ model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA–protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding. RNA recognition motifs (RRMs) are the most commonly found RNA-binding domain in eukaryotes and are implicated in many critical RNA–protein interactions in the cell . RRM-conRRMs are characterized by a β-α-β-β-α-β secondary structure that results in the formation of an antiparallel β-sheet comprising the RNA-binding platform . The mosStructural analyses of RRM-containing proteins have indicated that regions C-terminal to the RRM domain can be important for RNA-binding affinity and specificity by increasing the number of contacts between the protein and its RNA target . The RRMTo interact with U1hpII, RNP-1 must be exposed. This occurs through a shift in the position of helix C relative to the RNA-binding region by >90° to an ‘open’ protein conformation C 20,21),2120,21)Biochemical, structural and computational analyses of the N-terminal region of U1A containing RRM1  suggest that helix C is dynamic and may be stable in both the ‘closed’ and ‘open’ conformations independent of interactions with RNA targets . HoweverHere, we use a surface plasmon resonance-based biosensor (Biacore) to analyze the role of helix C in the interaction with U1hpII. Biacore allows the collection of high-quality kinetic measurements that facilitate the dissection of features important for complex formation (through measurements of association rates) versus complex stability (through measurements of dissociation rates). Previous kinetic analyses of the U1A–U1hpII interaction have led to the development of a ‘lure’ and ‘lock’ model in which well-placed positively charged amino acids bring U1A into proximity with U1hpII, and then close-range interactions lock the complex into place ,32,33. IU1A mutagenesis, production and quantification were as previously described with the following modifications ,33. To f−1 yeast tRNA  and 62.5 µg ml−1 acetylated bovine serum albumin . To provide an optimal comparison of the results obtained from all different U1A mutants, we prepared two surface densities on the sensor chip, an intermediate density RNA surface (100–125 resonance units) that would yield sufficient signal, even when proteins with lower affinities were used, and a low-density RNA surface (35–50 resonance units). Proteins were serially diluted in running buffer to the concentrations indicated in t-test; equal and unequal variance for the samples was determined using the F-test.Binding experiments were performed on a Biacore 2000 instrument as previously described ,33. To iMolecular dynamics simulations were performed in AMBER10 with solvation of the protein–RNA complex in a periodic box of TIP3P water molecules at 298 K with a time step of 1 fs and a vdW cutoff of 10 Å. Chains B and Q of the U1A-U1hpII radiographic structure  were usSupplementary Figure S1) indicate that the fold of RRM1 is maintained in U1A90 compared with U1A and that the reduced affinity of U1A90 for U1hpII is not caused by perturbation of the RRM structure owing to complete removal of helix C.The role of helix C in the interaction of U1A A with U1Kinetic analysis of U1A95 showed a modest but statistically significant increase in the association rate (1.7-fold increase), which was offset by a ∼35-fold loss in complex stability  and 3. TSupplementary Figure S2). Therefore, the large loss in complex stability we observe in the Ile94Pro mutant is most likely due to disruption of the conformation of helix C.To examine the effect of disrupting helix C, rather than removing it, we introduced a proline at position 94 (Ile94Pro). Ile94 makes a backbone hydrogen bond to Ser91, forming the first turn of the helix, and its mutation to proline should disrupt the short helix C, as predicted by a helix propensity scale for globular proteins . The mutThe much greater loss in stability of the U1A90–U1hpII complex relative to that of U1A95 or the Ile94Pro mutant points to a major role for amino acids 91–94 in stabilizing the protein–RNA complex. Amino acids in this region form multiple types of intra- and intermolecular interactions, including the quadruple stack contributed by Asp92, the hydrogen bond from Asp92 to C7 and the hydrogen bond between Ser91 and Thr11. We undertook site-directed mutagenesis to examine the contributions of these amino acid residues in more detail.We investigated the kinetic contribution of the quadruple stacking interaction in which Asp92 is connected to RNP-1 residue Phe56 via bases A6 and C7 of the U1hpII RNA loop E 21). M. M21). Mka of 2.5- to 4-fold. . The loss of Thr11 OH in the Thr11Ala mutant eliminated the water bridge in all except five models (Supplementary Table S2). Water bridges were retained in 24 of the 43 models for the Ser91Ala mutant, mainly due to bridging between Thr11 OH and Ala91 NH (Supplementary Table S3). The decrease in water bridges between residues 11 and 91 was significant in the two mutants . An illustration of the solvent calculation is shown in Supplementary Figure S3. The bridging water molecule in the wild-type protein  is lost in the mutated proteins . Loss of this water bridge and further disruption of the solvation shell between helix C and the RRM may result in easier movement of helix C, as suggested by the increased ka values for interaction of U1hpII with the mutated proteins.To test whether these mutations affect helix C positioning before interacting with U1hpII, we undertook computational analyses to examine solvation of the mutants. We used an algorithm (WATGEN) for interface solvation that was developed in our laboratory ,38. The The N-terminal region of U1A, containing RRM1, and its interaction with its RNA target in the U1snRNA, U1hpII, has served as a powerful model for defining the structure and mechanism of RRM-containing proteins interacting with their RNA targets. The U1A–U1hpII interaction is among the strongest non-covalent interactions described, and determining its molecular basis is of interest with respect to both understanding the U1A–U1snRNP complex as well as understanding how tight biological interactions can be formed and maintained.kd. In the Thr11Ala mutant complexed with U1hpII, only the Thr11 to Ser91 hydrogen bond is lost, but in the Ser91Ala mutant, both the hydrogen bonds to Thr11 and to N1 of base A6 are lost. The merely 2-fold faster dissociation of Ser91Ala than Thr11Ala might suggest that the hydrogen bond to A6 plays a modest role in complex stability. However, experiments in which the N1 hydrogen donor of nucleotide A6 was mutated showed a 50-fold loss in affinity between U1A and the mutated RNA target . This helix is thought to participate both in complex formation and stability. Here, we dissected its two roles. We find that helix C is of key importance in stabilizing the RNA–protein complex; the dissociation rate increases by three orders of magnitude with helix C removal. The biggest contribution of helix C to complex stability was not, as expected, the quadruple stack terminated by Asp92. We found that the role of Asp92 in this interaction is largely dispensable for a stable U1A–U1hpII complex. Mutations of Asp92 to Glu, Ala or Phe exhibited minor defects in complex stability (∼2-fold), but showed more substantial increases in the rate of complex formation (2.5- to 4-fold), so that net affinity was unaffected or even slightly improved. Our previous kinetic analysis of Phe56 mutants has indicated that loss of this aromatic residue results in a 6500-fold reduction in complex stability . Thus, iA target , indicatIn other RRM proteins, adjacent helices also assist in mediating stable protein–RNA complexes. Numerous other proteins that lack a C-terminal helix in the free protein increase the number of contacts between their RRM domain and their RNA target by forming a C-terminal helix on interaction with RNA  ,42–46. TBesides negatively affecting complex stability, helix C removal or disruption positively affects the association of U1A with U1hpII. The contradictory consequences of helix C perturbations mask its key role in stabilizing the RNA–protein complex when equilibrium measures are used to assess binding; they reduce the net effect of helix C perturbations on the affinity. This emphasizes the importance of kinetic studies when dissecting the role of structural elements in binding interactions. The largest increase in the rate of complex formation was observed when the helix was completely removed (U1A90), providing experimental confirmation that this helix occludes the RNA-binding surface. However, improved association was even observed in more subtle mutants. In both the Thr11Ala and Ser91Ala mutants, an association rate increase mitigated the effect of the loss of the intraprotein bond these residues provide in the complex, so that the net loss in affinity is <10-fold. Computational examination of the solvent network between helix C and the RRM indicated loss of bridging water molecules between Thr11 and Ser91 in both mutated proteins. This suggests that in these mutants, the helix is not packed as tightly against the RRM, which may allow easier association with U1hpII. These data indicate that solvation can influence the rate of association, while previous work has shown that ionic strength can affect the dissociation rate . These oC-terminal helices in other RRM-containing proteins appear to play diverse roles that contribute significantly to the function of these proteins. In U1A and proteins including hnRNP F, p14, U2AF, La protein and CstF-64, a preexisting C-terminal helix covers the RRM surface of the unbound protein, forming a hydrophobic core involving RNP amino acids. In hnRNP F, p14 and La protein, the helix remains in the ‘closed’ position because the RRM interacts with RNA via a non-canonical mechanism, without using the typical RNA-binding platform ,49. MainIn the above examples, a short alpha helix plays a role in inhibiting RNA contacts with the RNA-binding surface of the RRM. Accessory domains could lie at either side of RRMs and could come in many different forms. Examples of RRM autoinhibition exist in which a fully formed downstream RRM domain initially occludes the RNA binding surface, such as in HuD and U2AF65 ,56. HuD In addition to modulating access of U1hpII to the RNA-binding surface, the snug fit of U1A helix C onto the unbound RNA-binding platform might also improve the stability of the free protein. For example, in the analogous spliceosomal protein 65 K, a region N-terminal to the RNA-binding RRM forms two alpha helices in the unbound protein ,58. RemoWhile the study of the U1A–U1hpII complex is of great interest owing to its remarkable stability, it should be noted that in nature, U1A does not bind to U1hpII in isolation; both are sections of larger molecules. The full-length U1A protein and the complete U1snRNA are components of the U1snRNP, which contains numerous other proteins . Thus, rSupplementary Data are available at NAR Online: Supplementary Tables 1–3 and Supplementary Figures 1–3.National Science Foundation [MCB-0131782 to I.A.L.-O.]; National Institutes of Health [T32 GM067587 to M.J.L. and P.P.A.]; National Cancer Institute [P30CA014089]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation, the National Cancer Institute or the National Institutes of Health. Funding for open access charge: I.A.L.-O. donor funds.Conflict of interest statement. None declared.


Renilla Luciferase, in which the Tat-peptide was inserted within the Luciferase, in the presence of the specific ligand and triggered the “Lighting-UP” switch of the engineered Luciferase.RNA and protein are potential molecules that can be used to construct functional nanobiomaterials. Recent findings on riboswitches emphasize on the dominative function of RNAs in regulating protein functions through allosteric interactions between RNA and protein. In this study, we demonstrate a simple strategy to obtain RNAs that have a switching ability with respect to protein function in response to specific target molecules. RNA aptamers specific for small ligands and a trans-activation-responsive (TAR)-RNA were connected by random RNA sequences. RNAs that were allosterically bound to a trans-activator of transcription (Tat)-peptide in response to ligands were selected by repeated negative and positive selection in the absence and presence of the ligands, respectively. The selected RNAs interacted with artificially engineered  Various biological functions are accomplished through allosterically-regulated interactions between biomolecules. Proteins are assumed to take the initiative in allosteric interactions with their targets, such as interactions with other proteins or the promoter regions of genomic DNAs, through structural changes in response to ligand binding or chemical modification. However, recent studies have revealed that RNA molecules also change structures to control allosteric interactions with proteins Riboswitches are RNA elements located in the untranslated region of mRNA, which can regulate gene expression through RNA conformational transition in response to specific metabolites Renilla Luciferase Renilla Luciferase, the catalytic activity was restored to approximately 20% of the wild-type Luciferase activity and enabled intracellular detection of RNAs As described previously, we constructed an artificially engineered Luciferases, in which the trans-activator of transcription (Tat)-peptide derived from bovine immunodeficiency virus (BIV) was inserted into the firefly or In this study, we demonstrate the strategy for constructing the Lighting-UP switches with a selection of RNAs that allosterically interact with the BIV Tat-peptide in the presence of specific target molecules. RNA libraries containing a randomized linker sequence between the aptamers known to bind certain ligands and the TAR-RNA sequence were synthesized and selected for their ability to bind the Tat-peptide only in the presence of a target molecule. This would be a simple strategy for constructing luminometric biosensors for various target molecules because aptamers specific for various targets have already been selected. In addition, the selection was performed only for the short linker sequences.in vitro transcription using ScriptMAX Thermo T7 Transcription Kit (Toyobo) and purified with a denaturing polyacrylamide gel. The th5N-temp DNA template had the sequence 5′-CTAGGATCCAGCTCGCTGCCAAGGGCNNNNNGGAGCTAATGA GCTACACNNNNNGCTGGTATCGAGCTAACCAGAGAGACCCTTAAGCC-3′, and the tc5N-temp was 5′-GGATCCGGC CTAGGTGGTCGT ATTCTTCACCTCTCCNNNNNGGAGCTAATGAGCTACACNNNNNGGTATGTTTTAGGCCAACCAGAGAGACCCTTAAGC-3′. th5N-temp was amplified using the 5N-PCR primer (5′-CTTAATACGACTCACTA TAGGCTTAAGGGTCTCTCTGGTT-3′) and the th5N-RT primer (5′-C TAGGATCCAGCTCGCTGCC-3′). tc5N-temp was amplified using the 5N-PCR primer and the tc5N-RT primer (5′-CTAGGATCCGGCCTAGGTGGT-3′).th5N-G0 and tc5N-G0 libraries were prepared by 2, 20 ng/ µL tRNA, 50 ng/ µL glycogen, and 0.001% (v/v) Tween 20) at final volume of 200 µL and was incubated at 37°C for 30 min. Before addition of biotin-labeled Tat-peptide, the RNA library was refolded by incubating at 70°C for 10 min and cooling to 37°C at 1°C min−1. Streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin: Invitrogen) (25 µL bed volume) initialized by the selection buffer was added to the reaction mixture and incubated at 37°C for 30 min. RNAs that did not bind to the Tat-peptide in the absence of trigger molecules remained in the supernatant and were ethanol precipitated for use in positive selection.RNA library (20 nM) was mixed with biotin-labeled Tat-peptide (100 nM), which was purchased from Scrum Inc., in a selection buffer (20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgCl−1. Biotin-labeled Tat-peptide  was added and the solution was incubated at 37°C for 30 min. Streptavidin-coated magnetic beads (2.5 µL bed volume) initialized by the selection buffer was added and samples were incubated at 37°C for 30 min. The supernatant was removed, and the magnetic beads were washed 3 times in selection buffer containing the trigger molecule. RNAs were eluted from the beads in 1 M NaCl solution and ethanol precipitated.RNAs remaining after the negative selection were dissolved in the selection buffer  containing the trigger molecules (3 mM theophylline for th5N library and 100 µM tetracycline for tc5N library). The RNA library was refolded by incubating at 70°C for 10 min and cooling to 37°C at 1°C minin vitro using ScriptMAX Thermo T7 Transcription Kit.After the negative and positive selection, complementary DNAs of th5N and tc5N libraries were synthesized by reverse transcriptase (Rever Tra Ace: Toyobo) using th5N-RT and tc5N-RT primers, respectively. Double-stranded DNA templates for transcription of RNA libraries were synthesized by PCR using 5N-PCR primer and appropriate RT-primer, and RNAs were transcribed 2, 20 ng/ µL tRNA, and 0.005% (v/v) Tween 20 in the absence or presence of the trigger molecules. Fluorescence intensities were measured at 37°C using a microwell plate reader  at 535 nm excitation and 590 nm emission after 60-min incubation at 37°C.Tetramethyl-rhodamine (TAMRA)-labelled BIV Tat-peptide (TMR-Tat) (2.5 nM) was mixed with varying concentrations of RNA libraries in a buffer containing 20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgClKobs) between the TMR-Tat and RNA libraries were calculated from the fluorescence increase as a function of RNA library concentration by statistically fitting an equation assuming an equilibrium of a simple 1∶1 binding reaction The observed association constants . A 0.04 µL aliquot of the translated product was mixed with varying concentrations of RNA libraries and trigger molecules in a buffer containing 20 mM phosphate (pH 7.4), 100 mM NaCl, 1 mM MgCl2, 100 ng/ µL tRNA, and 0.005% (v/v) Tween 20 in a total reaction volume of 100 µL. The reaction mixture was incubated at 37°C for 30 min, and luminescence signals of the reaction mixture were evaluated using a multispectro microwell plate reader . An equal volume of 10 µM coelenterazine (Promega) diluted in reaction mixture buffer was added prior to data collection.Plasmid vector for To create Lighting-UP switches that accommodate various target molecules, artificial aptamers selected by the synthetic evolution of ligands by exponential enrichment (SELEX) As shown in G25 is approximately 11–12 kcal mol−1) Renilla Luciferase in the absence of the target molecules. Thus, the random linker sequences on either side of the TAR-RNA region were designed to be 5 nucleotides in length because the −ΔG25 of the RNA duplex is predicted to be greater than 12 kcal mol−1 if the random regions hybridized with regions neighboring to the opposed random regions and formed a 10-mer RNA duplex 6) can be easily covered in a small reaction volume.The selected RNAs were not expected to adopt the TAR-RNA structure in the absence of the target molecule. The target molecule should induce an allosteric conformational transition allowing RNAs to interact with the Tat-peptide. The interaction between the TAR-RNA and the Tat-peptide is one of the strongest known RNA–peptide interactions with a dissociation constant in the low nanomolar range (−ΔKobs 37) were calculated from the fluorescence signals using the equation in the Experimental Section  and tc5N-G0 (0.51×108 M−1), respectively, were slightly higher compared with those in their absence, although the ratios of Kobs 37 in the presence and absence of the trigger molecules were less than double . The results indicated that some RNAs in the G0 libraries allosterically interacted with the Tat-peptide in response to the target molecule because the Kobs 37 of the wild-type TAR-RNA was almost the same regardless of the presence of the target molecules  with the Tat-peptide was evaluated on the basis of the signal caused by binding of the TMR-Tat  Section . The Komolecule . Thus, iolecules .Kobs 37 of th5N-G6 and tc5N-G6 in the presence of the target molecules were 2.53×108 M−1 and 1.46×108 M−1, respectively. They were significantly higher than those of the G0 libraries and almost the same as that of the wild-type TAR-RNA. In contrast, the Kobs 37 of th5N-G6 and tc5N-G6 in the absence of the target molecules were 0.22×108 M−1 and 0.14×108 M−1, respectively. They were slightly lower than those of the G0 libraries. These results indicated successful enrichment of RNAs that allosterically interact with the Tat-peptide. Our strategy first eliminated RNAs that interacted with the Tat-peptide by negative selection; only those RNAs that interacted with the Tat-peptide in response to the target molecules were subsequently selected by positive selection. The ratios of Kobs 37 in the presence and absence of the specific target molecules were 11.7 and 10.1 for th5N-G6 and tc5N-G6, respectively.Negative and positive selections were repeated in the absence and presence of the target molecules . The results of binding of the TMR-Tat to the th5N-G6 and tc5N-G6 libraries obtained after six rounds of selection are shown in Renilla Luciferase in response to the target molecules. The engineered Renilla Luciferase was prepared by in vitro translation, and 0.04 µL of the crude lysate was mixed with th5N-G6 or tc5N-G6 in the presence or absence of the target molecules. The luminescence signals from the engineered Renilla Luciferase were analyzed at 37°C -based detection of theophylline using theophylline-aptamer conjugated RRE-RNA and fluorescent protein consisting of Rev-peptide inserted between EYFP and ECFP In this study, we constructed “Lighting-UP” switches on the basis of allosteric interaction of selected RNA libraries and the engineered Figure S1The interaction of wild-type TAR-RNA with the Tat-peptide. (A) Sequence of wild-type TAR-RNA. (B) Fluorescence intensities (FI) of TMR-Tat at 590 nm mixed with wild-type TAR-RNA in buffer containing 20 mM phosphate (pH = 7.4), 100 mM NaCl, 1 mM MgCl2, 20 ng/ µL tRNA, and 0.005% (v/v) Tween 20 at 37°C. TMR-Tat was mixed with varying concentrations of wild-type TAR-RNA in the absence (blue) or presence of the target molecules, 3 mM theophylline (red) or 100 µM tetracycline (green). (C) The observed association constant (Kobs) for wild-type TAR-RNA and TMR-Tat at 37°C in the absence and presence of the target molecules.(TIF)Click here for additional data file.