This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users. Despite the emergence of new techniques to nucleic acids investigation such as next generation sequence and array chips, the Polymerase Chain Reaction (PCR) and its variations still prevail in clinical laboratories. The use of PCR has grown increasingly in different applications ranging from microorganisms detection to diagnosis of complex genetic diseases –3. The sThe secondary structure formed by a particular nucleic acid molecule influences their DNA melting profile. Many bioinformatic tools designed to predict melting curves of nucleic acids are available –17. SoftVienna RNA Package[RNAfold calculates the minimum free energy and predicts an optimal secondary structure using McCaskill’s algorithm [Vienna RNA Package also provides the unique tool to assess ssRNA melting curves, the RNAheat software, which reads RNA sequences and calculates their specific heat in a determined temperature range, from the partition function by numeric differentiation [Using carefully calculated thermodynamic parameters, algorithms can be used to predict the secondary structure of a RNA strand –33. One  Package. Among tlgorithm . Vienna ntiation –33.The identification of RNA secondary structures is particularly interesting when viral genomes are analyzed. Previous studies demonstrated that conserved stem loops, extensive long-range interactions and small palindromic stem–loops generate structures that are generally associated with viral packing capacity and regulate viral replication , 21, 34.RNAheat and MELTSIM, were used to generate melting curves of nucleic acid sequences from Totiviridae viruses. Melting curves generated were used to reconstruct groups determined by a traditional phylogenetic analysis, based on RdRp sequence alignment. Subsequently, ssRNA and dsDNA melting curves were compared for its potential to discriminate Trichomonas vaginalis virus isolates. Our results indicate that the information obtained by ssRNA denaturation may be used as a support to the development of more accurate methods to detect differences in nucleic acid sequences.In the present study we propose that the information extracted from a melting curve of a single stranded RNA molecule allows more precise detection of nucleotide variations than the traditional HRMA. To demonstrate our hypothesis, two softwares, et al. classification [Giardiavirus and ScV-like comprises viruses of the genus Totivirus. The genus Victorivirus includes two gropus, MoV-like and GaRV-like. The genera Leishmaniavirus and Trichomonasvirus include groups LRV-like and TVV-like respectively. IMNV-like group appears as less derived group near to GLV-like and does is not classified by ICTV. The Zygosaccharomyces bailii virus (ZbV-Z) and two other related viruses isolated from plants and fungus clustered together to form a ZbV-Z-like clade, on a basal branch of the phylogenetic tree . It is clear that the similarity between sequences increases the chance of finding regions with conserved RNA structure. In agreement with phylogenetic trees showed in Figure In HRMA, nucleotide variations between two PCR products are detected comparing their melting curves. Although this approach has been successfully used to identify sequence polymorphisms –8, and tRNAfold. RNA fragments that show conserved RNA secondary structures in IMNV-like group are indicated in Figure in silico melting curve analysis using softwares RNAheat and MELTSIM, in order to obtain ssRNA melting curves .To confirm that information extracted from a ssRNA melting curve is more detailed than its correspondent dsDNA melting curve, clustering analyses were performed using melting curves from each ssRNA and dsDNA sequences of groups IMNV-like, GaRV-like and GLV-like. The curves were compared per group and clustered using R . The ress Figure  were comRNAz. Due to this fact the Totiviridae family seems a perfect model. During all replication steps the genetic material of Totiviridae remains as RNA and the formation of RNA secondary structures occur only when RNA is being replicated. This factor can be decisive for the perfect reconstruction of phylogenetic groups comparing secondary structures of RNA.It is already known that the formation of secondary structures in DNA can exerts significant influence in the molecule functions during DNA replication, transcription or translation. These secondary structures may vary within the molecule or when DNA is transcribed to RNA in according to cellular context involved. Considering this fact, is perfectly plausible that a given nucleic acid sequence may suffer different selective pressures in according with variations of it conformation in different stages of its “life cyle”. In single stranded RNA viruses, the secondary structure of RNA could be selected by a large number of factors acting at the same time, including the compactation of the genetic material into capsid. Therefore, we opted to eliminate any noise that could compromise the analysis of RNA conserved secondary structures and ensure that natural selection would be acting mainly on the structure detected by Trichomonas vaginalis virus, revealed four five sub-groups called TVV2, TVV3, TVV4 and TVV1, all belonging to the group TVV-like and to genus Trichomonasvirus. The sub-group TVV1 was selected to generate melting curves in silico. An alignment of RdRp RNA sequences was used for RNAz screening. This analysis revealed one region with conserved RNA structure shared by all viruses of this group in the second half or RdRp RNA sequence. Then, two regions were selected, a non-conserved region and the conserved region detected by RNAz  was used as outgroup due to its higher proximity and similarity to the family Totiviridae. The RdRP sequences were then aligned at the amino acid level, using the program MAFFT v. 6.85 [6 generations, sampled every 100 generations, and a consensus tree was estimated by using a burning in of 1,000,000 trees. The convergence of the simultaneous runs was assessed using the Tracer tool v. 1.5 [The nucleotide and amino acid sequences from Totiviridae viruses were retrieved from public repositories such as GenBank [gorithms  using degorithms . ORFs an v. 6.85 , 49 with v. 6.85 . Then, t v. 6.85 . Afterwa v. 6.85  and the  v. 6.85 , 54 and  v. 6.85 . All indl v. 1.5 , in ordel v. 1.5 .RNAz software, provided by Vienna RNA Web Services[z-score is calculated from the relation z = (m -μ )/σ, where μ and σ are the mean and standard deviations, respectively, of the MFEs of the random samples [z-scores indicate that a sequence is more stable than expected by chance. The structural conservation is predicted using the RNAalifold approach [RNAz output using RNAfold software provided by Vienna RNA Package[RNAfold reads RNA sequences, calculates their MFE structure and free energy. The -p option was used to compute the partition function (PF) and base pairing probability matrix, as well as the overall free energy of the thermodynamic ensemble. RNAfold produces PostScript files with plots of the resulting secondary structure graph and a dot plot of the base pairing matrix. Default parameters were used to generate the interactive RNA structure plots.Conserved RNA secondary structures were detected from TCOFFEE multiple alignments of RdRp RNA sequences Tables  and 4, uServices. This to samples . Negativapproach . The sec Package. RNAfoldMELTSIM software, which generates derivative profiles. In the model used by this software, proposed by Blake et al.[RNAheat software [The dsDNA melting curves were estimated using the e et al., the looe et al.–62. By dsoftware . This prsoftware  for the software . The ressoftware .MEGA5[MUSCLE algorithm. Each alignment was used in a neighbor-joining grouping analysis, using Maximum-composite likelihood distance and 500 bootstrap replications. The obtained dendograms were visually compared to the ones from hierarchical cluster analyses, based on the single and double strand DNA melting denaturation cores.Based on the melting denaturation scores, the melting curves were clustered using a hierarchical cluster analysis, using R . This teMEGA5 using thAll supporting data are included in Additional files.RNAz software, from the alignment of ORF2 RNA sequences of GLV-like group members. (B) Secondary structure calculated using RNAfold, corresponding to each conserved region identified by RNAz. (C) Melting curves calculated from the conserved region, using the software RNAheat which considers ssRNA denaturation. (D) Melting curves calculated from the conserved region, using the software MELTSIM which considers dsDNA denaturation. (PPT 270 KB)Additional file 1: Figure S1: Regions with conserved RNA secondary structures identified in GLV-like group and their respective melting curves. (A) Regions with secondary structures identified using RNAz software, from the alignment of ORF2 RNA sequences of GaRV-like group members. (B) Secondary structure calculated using RNAfold, corresponding to each conserved region identified by RNAz. (C) Melting curves calculated from the conserved region, using the software RNAheat which considers ssRNA denaturation. (D) Melting curves calculated from the conserved region, using the software MELTSIM which considers dsDNA denaturation. (PPT 302 KB)Additional file 2: Figure S2: Regions with conserved RNA secondary structures identified in GaRV-like group and their respective melting curves. (A) Regions with secondary structures identified using in silico. Columns (B) and (C) shows the dendograms calculated from the curves generated by the programs RNAheat and MELSTSIM for the members of GLV group. (PPT 355 KB)Additional file 3: Figure S3: Cluster analysis and dendogram of GLV-like group. The curves generated for each sequence were compared and clustered using a statistical inference. The proximity between individuals of groups indicated in the column (A) is due exclusively to the similarity between the melting curves generated


The present study was designed to evaluate the antibacterial activities of synthetic The broth microdilution method was used to determine the minimal inhibitory concentrations (MIC).p-Chloro-benzyliden-selenosemicarbazide, 4-methyl-2-[(4-chloro-benzyliden)-hydrazinyl]-1,3-selenazole, p-chloro-benzoyl-selenosemicarbazide and 4-chloromethyl-2-[(4-chlorobenziliden)-N-acetyl-hydrazinyl]-1,3-selenazole were more active than the choramphenicol on Klebsiella pneumoniae KP63. Tested alone, the lowest MIC value of 16 mg/L was obtained with p-methoxy-benzyliden-selenosemicarbazide against Enterobacter aerogenes ATCC13048, K. pneumoniae ATCC112296 and KP55. Tested in the presence of an efflux pump inhibitor, phenylalanine arginine β-naphthylamide (PAβN), the activity of p-chloro-benzyliden-selenosemicarbazide, 4-methyl-2-[(4-chloro-benzyliden)-hydrazinyl]-1,3-selenazole, p-chloro-benzoyl-selenosemicarbazide and p-methoxy-benzyliden-selenosemicarbazide significantly increased with MIC values below 10 mg/L obtained respectively on 43.8 %, 31.3 %, 62.5 % and 100 % of the 16 tested bacterial strains. The lowest MIC value of 0.5 mg/L in the presence of PAβN was recorded with p-chloro-benzoyl-selenosemicarbazide against Escherichia coli ATCC8739 and KP55 as well as p-methoxy-benzyliden-selenosemicarbazide against E. aerogenes KP55. p-Chloro-benzyliden-selenosemicarbazide and p-chloro-benzoyl-selenosemicarbazide contained the same pharmacophore as p-methoxy-benzyliden-selenosemicarbazide.The results demonstrated that the best activities were obtained with hydrazinoselenazoles. p-chloro-benzyliden-selenosemicarbazide, p-chloro-benzoyl-selenosemicarbazide and p-methoxy-benzyliden-selenosemicarbazide could be explored more to develop novel antimicrobial drugs to fight MDR bacterial infections.This study indicates that  Enterobacteriaceae or MexAB-OprM in Pseudomonas aeruginosa, are involved in multidrug resistance of pathogenic Gram-negative bacteria -thiazole, C15H18N3O4S2 ; 5-acetyl-4-methyl-2-[2-(benzenesulfonyl)-hydrazino]-thiazole, C13H18N3O4S2 ; 5-acetyl-4-methyl-2--thiazole, C17H20N3O5S2 ; 4-Phenyl-2--thiazole, C22H24N3O2S2, ; 5-bromo-4-Chloromethyl-2--thiazole, C17H20BrClN3O2S2 ; p-toluenesulfonylthiosemicarbazide, C10H18N3O2S2 ; 4-methyl-2--thiazole, C16H20N3O4S2 ; 4-chloromethyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole, C12H15ClN3O2S2 ; 4-phenyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole, C17H18N3O2S2 ; 5-acetyl-4-methyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole, C14H18N3O3S2 ; 5-acetyl-4-methyl-2--thiazole, C18H22N3O5S2  and hydrazinoselenazoles: p-chloro-benzyliden-selenosemicarbazide, C8H8ClN3Se ; 4-methyl-2-[(4-chloro-benzyliden)-hydrazinyl]-1,3-selenazole, C11H10ClN3Se ; p-chloro-benzoyl-selenosemicarbazide C8H8ClN3OSe ; 4-chloromethyl-2-[(4-chlorobenziliden)-N-acetyl-hydrazinyl]-1,3-selenazole, C13H11Cl2N3OSe ; p-methoxy-benzyliden-selenosemicarbazide, C9H11N3OSe ; 4-phenyl-[2-(4-metoxybenzyliden)-hydrazinyl]-1,3-selenazole, C17H15N3OSe ; 4-chloromethyl-[2--hydrazinyl]-1,3-selenazole C16H19ClN4SSe ; and 4-methyl-5-acetyl-[2--hydrazinyl]-1,3-selenazole, C18H22N4OSSe . The synthesis of p-toluenesulfonyl-hydrazinothiazoles (1–11)  can be considered as the phamacophoric moiety. Within this moiety, the presence of a free selenoamide is primordial for the antibacterial activity, as compounds 12 and 14 (bearing a free selenoamide) were also very active, especially when they were combined with EPI  were much lower than that of 16 (with -OCH3 substituent).When regarding the structure-activity relationship, it appears that amongst the tested compounds, P. aeruginosa tested in the present study, play a key role in multidrug resistance of pathogenic Gram-negative bacteria [p-Toluenesulfonyl-hydrazinothiazoles and hydrazinoselenazoles are synthetic compounds that previously displayed good anticancer, analgesic and anti-inflammatory activities [p-toluenesulfonyl-hydrazinothiazoles. However, all the tested chemicals were substrates of bacterial efflux pumps. Interestingly, when efflux pumps were blocked by EPI, significant increase of the activity of three compounds 12, 14 and 16 was observed. More importantly, the obtained activity was better than that of the reference drug chloramphenicol in many cases (Table 12, 14 and 16 on 43.8 %, 62.5 % and 100 % tested bacterial strains when they were tested in the presence of EPI, highlighting their possible use in combination with PAβN in the control of MDR bacterial infections.Bacterial multidrug resistance represents a major problem in the treatment of infectious diseases. In the present study, we tested a panel of bacterial strains including both reference ATCC strains and MDR phenotypes expressing active efflux pumps , 5, 22. bacteria , 24. p-Ttivities –11. HerePseudomonas aeruginosa is an important nosocomial pathogen highly resistant to clinically used antibiotics, causing a wide spectrum of infections and leading to substantial morbidity and mortality [K. pneumoniae, E. aerogenes, P. stuartii and E. coli, have also been classified as antimicrobial-resistant organisms of concern in healthcare facilities [Regarding the involvement of MDR bacteria in treatment failures and the re-emergence of infectious diseases , 27, 28,ortality  and was cilities .12, 14 and 16 are promising antibacterial agents to combat MDR phenotypes, but are also substrates of efflux pumps. They should therefore be combined with an EPI to combat MDR phenotypes. We also found that compound 16 skeleton was the pharmacophoric moiety of the tested hydrazinoselenazoles and that the presence of a free selenoamide scaffold is necessary for their antibacterial activity. However, more compounds should be further synthesized to fully investigate the structure-activity relationship for this series of hydrazinoselenazoles. Toxicological studies will also be performed to ensure the safety of the active hydrazinoselenazoles, especially compound 16.Regarding the medical importance of the studied microorganisms, the results obtained in this study demonstrated that hydrazinoselenazoles are strong source of inspiration in antibacterial drug discovery. Thus, the present data showed that hydrazinoselenazoles


A combination of 3D modeling and high-throughput sequencing may offer a faster way to determine the three-dimensional structures of RNA molecules. Related research article Cheng CY, Chou FC, Kladwang W, Tian S, Cordero P, Das R. 2015. Consistent global structures of complex RNA states through multidimensional chemical mapping. eLife4:e07600. doi: 10.7554/eLife.07600Image Multidimensional chemical mapping predicts the structures of RNAs based on interactions between nucleotidesThe structures of molecules often hold the key to understanding their roles in cells. Thus, when Watson and Crick proposed the double-helix structure for DNA, they immediately speculated on how DNA may replicate. Unfortunately, working out the structures of RNA molecules is challenging, and the techniques needed to do so—X-ray crystallography and NMR—are only available at a small number of locations worldwide. This has prevented many small laboratories from embracing structural work.eLife, Rhiju Das and co-workers—including Clarence Yu Cheng as first author—describe a new approach that promises to simplify the determination of RNA structures to nanometer resolution  structure. Previously, Das and others have combined protocols that predict the secondary structure of RNA with three-dimensional modelling algorithms  to generSeveral experimental approaches can fill this gap, but many require specialized reagents or equipment, which limits their use. Just as the growth of social media depended on developments in technological infrastructure, the current progress in multidimensional chemical mapping has been made possible by the widespread availability of instruments for deep sequencing DNA. In a wry twist of fate, sequencing has become a key tool for defining structure.The idea to use sequencing to determine RNA structures is not new  with deep sequencing and algorithms that predict the RNA secondary and tertiary structure . MOHCA uPreviously, gel electrophoresis has been used to show where the RNA molecule is cleaved during MOHCA, but combining MOHCA with deep sequencing provides much more detailed information and significantly increases throughput. Cheng et al. constructed maps showing the interactions between the nucleotides in the molecule and combined these with algorithms that predict secondary and tertiary structures to generate a well-defined three-dimensional model of the RNA. To demonstrate the accuracy and utility of this new approach, Cheng et al. applied it to five RNAs of known structure, and to one RNA whose structure had not been released at the time. Multidimensional chemical mapping successfully predicted the three-dimensional structures of all six RNA molecules to within nanometer resolution.However, RNA often interacts with cofactors, which remain largely undefined or otherwise make structure determination more challenging. Multidimensional chemical mapping works in solution and can help define structures in the absence of these cofactors. To test this, Cheng et al. used multidimensional chemical mapping to predict the structures of several RNAs without their cellular cofactors. Their prediction for the structure of the internal ribosomal entry site in human Hox messenger RNA will aid efforts to determine the structure of this mRNA in complex with the ribosome and other partner molecules using techniques such as cryo-electron microscopy. Cheng et al. also modeled ligand-free conformations of several riboswitches, which may guide the development of drugs that stabilize non-functional RNA conformations.But what challenges are still ahead of us? Currently, the size of the RNA limits the analysis. Refining the modeling algorithm and the way the samples are prepared for sequencing may help to improve the predictions. A more general concern is the presence of naturally occurring modifications to RNA molecules inside cells. In highly modified structures—like transfer RNAs—these chemical groups influence the folding of the molecule. Thus, when solving the structure of an unmodified RNA molecule produced in an artificial system, an important piece in the puzzle is missing. Even when they are present, how these chemical modifications affect the cleavage of RNA by the hydroxyl radicals still needs to be assessed.The field of structural biology is undergoing dramatic changes as improvements in technology—such as the development of free-electron lasers and improved detectors for electron microscopy—are making it possible to solve structures at atomic resolution, almost in a high-throughput manner. Will the combination of three-dimensional modeling and sequencing become a similar game changer in the field of RNA structures, being used and adapted by large numbers of researchers? Multidimensional chemical mapping clearly has the potential to transform structure determination by putting it into the hands of researchers who have experience in molecular biology and access to deep sequencing.Today, this is almost everybody: structures to the people!


Papp) of Caco-2 cells to marketed drugs. While in some cases there are some significant interlaboratory disparities, most are quite minor. Most drugs are not especially permeable through Caco-2 cells, with the median Papp value being some 16 ⋅ 10−6 cm s−1. This value is considerably lower than those  recently used in some simulations that purported to show that Papp values were too great to be transporter-mediated only. While these values are outliers, all values, and especially the comparatively low values normally observed, are entirely consistent with transporter-only mediated uptake, with no need to invoke phospholipid bilayer diffusion. The apparent permeability of Caco-2 cells to marketed drugs is poorly correlated with either simple biophysical properties, the extent of molecular similarity to endogenous metabolites (endogenites), or any specific substructural properties. In particular, the octanol:water partition coefficient, logP, shows negligible correlation with Caco-2 permeability. The data are best explained on the basis that most drugs enter (and exit) Caco-2 cells via a multiplicity of transporters of comparatively weak specificity.We bring together fifteen, nonredundant, tabulated collections (amounting to 696 separate measurements) of the apparent permeability ( Most pharmaceutical drugs, and all oral ones, must necessarily cross at least one cell membrane to act. Understanding how this transport is effected remains a major challenge . We havede facto standard in studies of pharmaceutical drug transport. They form a more or less (and otherwise) impermeable layer that is polarised, in the sense of having ‘apical’ and ‘basolateral’ faces in which transporters are differentially expressed. They express hundreds of transporters  they hasporters .Papp) rates for different drug molecules that Caco-2 cells can sustain. Although there are undoubtedly larger databases in-house in commercial and other enterprises, we have sought to bring together what we can of published data to determine the kinds of permeability values that Caco-2 cells can sustain, and what might determine that. We recognise that many factors can affect a specific measurement, e.g., the seeding density, age of the cells, pH and so on. An interlaboratory comparison , essentially as described  . Although there are older papers, we have started with the compilation of Hou and colleagues (We have selected a set of 15 studies (indicated in the legend to bank.ca) , we complleagues . Our metlleagues . We incolleagues , one froA → B direction. In the case of the interlaboratory comparison  are from a study  There is a slightly clearer relationship between Caco-2 permeability and a drug’s total polar surface area, but again the relationship is fairly weak ). It is also of interest that there is no significant relationship between total Polar Surface Area and SlogP (P.If unstirred layer effects and pure diffusion (as opposed to transporter-based enzyme kinetics) were significant in Caco-2 permeability (notwithstanding the evidence that they are not ), one mind SlogP . In partnd SlogP  and othend SlogP  find thar2 = 0.156, with the number of endogenites exceeding a Tanimoto similarity of 0.75, for the 21 molecules that had at least one, data not shown.) One interpretation of this is that while in some cases a rather small number of transporters are typically involved in drug uptake , and is best explained by the intermediacy of a very heterogeneous set of transporters.Finally, we wondered whether a standard machine learning approach (a random forest learner ) might bPapp for verapamil and propranolol, claimed that the apparent permeability values were such that they could not be supported by known (random) transporters at random expression level, mK and kcat values. It was stated , as most drugs exploit multiple but often unknown transporters with overlapping specificities. Which they are and how much each contributes to a given Caco-2 permeability must be determined by varying their activities as independent variables (A recent publication , using es stated  that sucs stated , it promariables , whetherariables  or genetariables . The avaariables  imply th10.7717/peerj.1405/supp-1Supplemental Information 1Drugs_Caco2_compilation_with_descriptors_2.xlsClick here for additional data file.


Escherichia coli. We find that nucleotides that participate in RNA–RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA–RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell.Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure–function relationship of two RNA mechanisms that regulate translation in  Non-coding RNAs (ncRNAs) have diverse functions, ranging from regulatory roles in transcription, translation and messenger stability in prokaryotes ,2 to genThis effort has recently accelerated with the advent of high-throughput RNA structure characterization technologies that combine chemical probing with next-generation sequencing –24. In oThe use of next-generation sequencing has allowed these methods to be highly multiplexed, which has offered some of the first ‘transcriptome-wide’ glimpses of RNA structure –21,23,24To address these issues for researchers interested in studying the structure–function relationship of select RNAs in depth, we have developed in-cell SHAPE-Seq. In-cell SHAPE-Seq combines in-cell probing of RNA structure with SHAPE-Seq  and a meE. coli: the synthetic RNA riboregulator translational activator system developed by Isaacs et al. .E. coli. Specific primer designs and detailed cloning procedures to construct the plasmids used in this work, or plasmids for other RNA regulatory systems, can be found in Supplementary Methods. The cis-repressed sense RNA (crRNA) and trans-activating RNA (taRNA) plasmids were generated by introducing the riboregulator sequences from Isaacs et al. , was used to maintain a consistent cellular load to properly compare fluorescence levels with or without the antisense RNA present. Transformed cells were plated on LB+Agar media with 100 μg/ml carbenicillin, 34 μg/ml chloramphenicol or both and incubated overnight at 37°C. The next day, individual colonies were picked and grown in 1 ml of the appropriate LB+antibiotic in a 2 ml 96-well block (Costar) and grown approximately 17 h overnight at 37°C at 1000 rpm in a VorTemp 56 (Labnet) benchtop shaker. Twenty-four microliters of this overnight culture was then used to subculture into 1.2 ml of LB+antibiotic. E. coli TG1 cells without plasmids were prepared in the same way without antibiotic for probing endogenously expressed RNAs. The subculture was grown under the same conditions as the overnight culture for at least 3 h before measuring fluorescence  and performing structure probing. See Supplementary Methods (steps 17–21) for more details.Each sense and antisense plasmid was transformed separately, or in combination, into chemically competent In vitro transcription templates for crR12, taR12, RNA-IN S3 C24A A25C and RNA-OUT A3 were prepared using PCR with Taq polymerase (NEB), replacing the E. coli promoter with the T7 promoter (TAATACGACTCACTATAGG), followed by ethanol precipitation. The in vitro transcription reaction contained 5 μg of template, 40 mM tris-HCl pH 8.0, 20 mM MgCl2, 10 mM DTT, 20 mM spermidine, 0.01% Triton X-100, 5 mM NTPs, 60 U of SUPERase-In, 20 μl of purified T7 RNA polymerase, brought to a total of 1 ml with MilliQ H2O. The shorter RNAs (taR12 and RNA-OUT A3) were gel purified and passively eluted before ethanol precipitation. The longer RNAs containing the SFGFP sequence (crR12 and RNA-IN S3 C24A A25C) were purified from the transcription reaction using AMPure XP RNA beads according to the manufacturer's instructions.600), which typically ranged from 0.1 to 0.5. Fluorescence and OD600 were first corrected by subtracting values measured for a media blank. Relative fluorescence levels of each culture (except those used for endogenous RNA characterization) were determined by normalizing the fluorescence readout by optical density (FL/OD) and subtracting the FL/OD of the antisense plasmid containing cultures (which did not contain SFGFP) to correct for cell autofluorescence.Fluorescence was measured after at least 3 h of growth by pelleting 150 μl of each subculture and resuspending it in 200 μl PBS buffer with 100 μg/ml kanamycin (to prevent further translation) and assaying for fluorescence (485/520 nm) and optical density (ODFor RNA modification with 1-methyl-7-nitroisatoic anhydride (1M7), 500 μl of each 3 h subculture was modified in the 96-well block with 13.3 μl 250 mM 1M7 in DMSO  (+) or 13.3 μl DMSO (−) for 3 min before RNA extraction. For the DMS modification, the 1M7 was replaced with 27.75 μl of 13% DMS in ethanol and the DMSO replaced with 27.75 μl ethanol. After 3 min of incubation with DMS, the reaction was quenched with 240 μl 2-mercaptoethanol. See Supplementary Methods (steps 22–32) for a more detailed in-cell modification protocol.in vitro transcribed RNAs, 10 pmol of RNA total  were diluted in 12 μl H2O total before denaturing at 95°C for 2 min. The RNAs were than snap-cooled on ice for 1 min before adding 6 μl 3.3X Folding Buffer  and incubated at 37°C for 20 min. The resulting 18 μl were split and added to 1 μl 65 mM 1M7  or 1 μl DMSO and incubated at 37°C for 1 min. The modified in vitro RNAs were then ethanol precipitated and dissolved in 10 μl H2O before reverse transcription.For For in-cell probing experiments, both modified (+) and control (−) samples were pelleted, then resuspended in 100 μl of hot Max Bacterial Enhancement Reagent (Life Technologies) before extraction with TRIzol Reagent (Life Technologies) according to the manufacturer's protocol. Extracted RNA was dissolved in 10 μl of water. See Supplementary Methods (steps 33–39) for more details.btuB riboswitch samples to which 3 μl of 50 nM oligonucleotide were added instead. Sense RNAs were extended with an RT primer for SFGFP, antisense RNAs were extended with a primer for the ECK120051404 terminator, and endogenously expressed RNAs were extended with an RNA-specific primer (Supplementary Table S3). For samples containing sense-antisense pairs, 1.5 μl of each primer were mixed together. All RNAs were denatured at 95°C for 2 min, then 65°C for 5 min. After denaturing, each RNA sample was then snap-cooled on ice for 1 min before extension with Superscript III (Life Technologies) at 52°C for 25 min. After RT the RNA was hydrolyzed with 1 μl 10 M NaOH. The solution was then partially neutralized with 5 μl of 1 M hydrochloric acid and ethanol precipitated. See Supplementary Methods (steps 40–51) for more details.For each RNA sample, 3 μl of 0.5 μM oligonucleotide were added for reverse transcription (RT), except for the The cDNA from each RT reaction was separately ligated to a ssDNA adapter for Illumina sequencing with CircLigase I ssDNA ligase (Epicentre). Each ligation reaction was incubated at 60°C for 2 h, followed by deactivation at 80°C for 10 min. The ligated cDNA was then ethanol precipitated and dissolved in 20 μl water. Unligated oligonucleotides were removed by purification with 36 μl of Agencourt AMPure XP beads (Beckman Coulter) according to manufacturer's protocol. See Supplementary Methods (steps 52–57) for details.btuB riboswitch) were amplified for 15 cycles without the complete forward Illumina adapter primer first, which was then added for a second set of 15 cycles. Libraries that were derived from cultures that contained both sense and antisense plasmids were amplified separately with one selection primer to visually separate the library qualities of the independent priming locations. The fluorescently tagged amplifications were run on an ABI 3730xl Analyzer with GeneScan 500 LIZ standard (Life Technologies) and checked for the correct full-length product (indicating good RT and PCR) and minimal side product formation. See Supplementary Methods (steps 58–67) for further details.Each single-stranded cDNA library from a highly expressed RNA was PCR amplified with Phusion polymerase (NEB) for 15 cycles with two forward primers, a selection primer (containing an RNA-specific sequence and part of the forward Illumina adapter) and a longer primer containing all of the forward Illumina adapter, and a fluorescent reverse primer that binds to the reverse Illumina adapter sequence as part of the ligated ssDNA adapter ). Moderate to weakly expressed RNAs (RNase P and the btuB riboswitch) were amplified for 15 cycles without the complete forward Illumina adapter primer first, before it was added for a second set of 15 cycles. Excess primer was removed with ExoI (NEB) before purification with 90 μl of Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer's protocol. See Supplementary Methods (steps 68–75) for more details.Highly expressed RNA libraries passing quality analysis were PCR amplified with Phusion polymerase (NEB) for 15 cycles using three primers: a forward primer that contained an Illumina adapter, another RNA-specific forward selection primer, and a reverse primer that contained the other Illumina adapter and one of 24 TruSeq indexes . Moderate to weakly expressed RNAs . All libraries were mixed to have the same final molar concentration and sequenced with the Illumina MiSeq v3 kit or HiSeq 2500 rapid run using 2×35 bp paired end reads. Adapter trimming was turned off during Illumina post-sequencing processing.http://hannonlab.cshl.edu/fastx_toolkit/], then aligned to the target RNA sequences with Bowtie 0.12.8 (http://rmdb.stanford.edu/repository/)   using thRNA secondary structure predictions were performed using the RNAStructure web server . In-celltrans. In these systems, translation is controlled by specific RNA structures in the 5′ untranslated region (5′ UTR) of a ‘sense’ target mRNA. Interaction with a trans-acting complementary ‘antisense’ RNA sequence causes structural rearrangements to occur, turning downstream gene expression ON in the case of riboregulators, or OFF in the case of the IS10 system.One goal of the in-cell SHAPE-Seq platform is to characterize cellular structural and functional states of regulatory RNAs simultaneously Figure . Often, E. coli   ,9. In thgure S1)  on a medin vitro pools of purified RNAs, using the ability of certain SHAPE reagents to penetrate living cells   ,40,41. Te Figure  producede Figure  to improe Figure ,22,39.While successful, initial versions of our protocol suffered from an excess of RT primer-sequencing adapter ligation dimers, making it difficult to accumulate enough sequencing reads with our libraries for computational reactivity analysis ,30. In scis-repressed RNA (crRNA) is thus OFF in the basal state. To activate translation, a trans-activating antisense RNA (taRNA) is expressed that base pairs with the crRNA, causing structural rearrangements that expose the RBS and allow translation (ON state).We first used in-cell SHAPE-Seq to examine a synthetic riboregulator system that activates translation in bacteria . In the in silico using computational models of RNA folding . The first region is the hairpin stem, which is predicted to contain multiple sets of inner loops. Low reactivities in inner loops are not uncommon with SHAPE reactivities ,31 and cTo corroborate our findings, we also examined the taR10/crR10 riboregulator variant, which has a similar overall design and was the second best riboregulator pair in terms of fold activation . We repeAdditionally, we compared our in-cell SHAPE-Seq results to an equivalent in-cell ‘DMS-Seq-like’ approach , where tE. coli cells expressing both the crR12 sense construct and the taR12 antisense construct. We observed distinct in-cell SHAPE-Seq reactivity changes in several specific regions of crR12 caused by the addition of taR12 that lead to the observed 7.3-fold increase in gene expression . How. HowE. cSimilar features were observed when the structure–function relationship of the taR10/crR10 interaction was characterized with in-cell SHAPE-Seq . One difference, however, was a change in the specific nucleotides that were observed to decrease in reactivity as a result of taR10 binding. Overall, more of the 5′ end of crR10 appeared to bind to the taR10 sequence relative to taR12/crR12, though there is a seven nucleotide region from positions 17–23 on crR10 which does not appear to bind as strongly, if at all. One possible explanation for the difference in the interacting structures of these variants is the relative stabilities of the terminal stem loops in taR12 (nt 19–61) and taR10 (nt 22–62). The taR12 hairpin is more stable  than the taR10 hairpin  as predicted by RNAStructure . TherefoUnlike crR12 and crR10, no major reactivity changes were observed for either taR12 or taR10 when expressed with their corresponding crRNA targets . Since these RNAs are expressed in excess of their targets, our in-cell SHAPE-Seq experiment is likely capturing a majority of non-interacting taRNA states, as they take up a large portion of the cellular population. We also note that we did not observe significant reactivity changes in either crRNA's CDS when the corresponding taRNA was present.Because the riboregulator mechanism is thought to functionally activate translation in bacteria by removing structural constraints in the crRNA RBS region, we sought to examine how changes in the in-cell SHAPE-Seq reactivities of the RBS region relate to changes in gene expression. However, the AG-rich region between nucleotides 36–46 in crR10 and crR12 has the potential to contain multiple Shine-Dalgarno (SD) sequences. Since the exposure of the RBS turns on gene expression, we hypothesized that the dominant six-nucleotide SD sequence would exhibit the largest reactivity increase. To find this sequence, we summed reactivities over a six nucleotide sliding window for the crR12/crR10 ON and OFF states and looked for the biggest difference between them . We found that nucleotides 36–41 showed the largest overall increase, with the most notable increases occurring at nucleotides 36–39 in both crR12 and crR10 Figure ,D.We next sought to use in-cell SHAPE-Seq to examine a modified version of the natural sRNA translation repression system from the insertion sequence 10 (IS10) transposon . In the We began by characterizing the in-cell structures of RNA-IN and RNA-OUT individually. Our first observation was that the nucleotides in the RNA-IN S4 5′ UTR were highly reactive and likely unstructured in the cell Figure . In addiE. coli cells expressing the RNA-IN reporter construct with the RNA-OUT antisense construct. Initially, we performed three replicate experiments with the S4/A4 pair, but observed varying RNA-IN reactivity patterns, despite each replicate exhibiting roughly the same level of translation repression (∼85%) . A closer analysis of the raw SHAPE-Seq (+) and (−) channel fragment distributions revealed large spikes at position 26 in both channels, suggesting that RNA-IN S4 was being cleaved between positions 25 and 26 when in complex with RNA-OUT A4. To further confirm this effect was due to cognate RNA–RNA interactions, we examined orthogonal pairs of RNA-IN/RNA-OUT (i.e. pairs S4/A3 and S3/A4) and found no spikes at position 26 or major changes in reactivity compared to the individually measured RNAs .We then characterized how RNA-OUT binding to RNA-IN leads to translation repression by performing the full in-cell SHAPE-Seq structure–function measurement in Previous work showed that the wild-type RNA-IN/RNA-OUT duplex is targeted by RNAse III between nucleotides 15–16 of RNA-IN and 22–23 of RNA-OUT for degradation in the cell . HoweverTo characterize the cellular RNA–RNA interactions that lead to translation repression, we performed replicate in-cell SHAPE-Seq experiments with the RNA-IN S4 C24A A25C double mutant and RNA-OUT A4 Figure . SeveralFinally, we examined reactivity changes from the perspective of the antisense RNA-OUT RNAs . As expected, there are no major differences in the reactivity map of RNA-OUT A4 when the orthogonal RNA-IN S3 is present. However, unlike in the riboregulator system, we did observe subtle reactivity changes in RNA-OUT A4 in the presence of RNA-IN S4 C24A A25C, despite the copy number difference.E. coli cells: 5S rRNA, RNase P and the btuB mRNA riboswitch domain . To remedy this, we first amplified the ssDNA libraries without this primer for 15 cycles to amplify the target of interest, then added primer I for another 15 cycles to build the rest of the adapter required for sequencing . We confirmed the additional cycles did not alter the resulting reactivities .To further test the capabilities of in-cell SHAPE-Seq, we targeted three endogenously expressed functional RNAs that are present at varying levels in We first examined the highly abundant 5S rRNA . As seenE. coli RPR derived from comparative sequence analysis  to cleave the 5′-leader from precursor tRNAs (pre-tRNAs) . The RNaanalysis   is present   . Most inormation . While tE. coli cells. Our range of examples demonstrate that these reactivity spectra can be used to corroborate existing models of RNA folding and interactions, as well as suggest refinements to our understanding of RNA systems that are less well studied. We thus anticipate in-cell SHAPE-Seq to be useful for the study of a broad array of endogenous RNAs.Overall, these results indicate that in-cell SHAPE-Seq can be used to obtain nucleotide-resolution reactivity maps for endogenous transcripts directly in in vitro SHAPE-Seq experiments  and the RNA-IN/OUT systems . In many cases, the trends in reactivities across the molecules were consistent, with quantitative differences at isolated positions. The biggest deviations were seen when we examined the RNA-IN/OUT complex, which showed significantly lower in-cell reactivities in the region surrounding the RBS of RNA-IN . Overall, the similarity between the in-cell and in vitro refolded SHAPE-Seq reactivities suggests that for these regulatory RNAs the complex cellular environment does not play a significant role in altering structures from their equilibrium states.Our ability to characterize cellular RNA structures with in-cell SHAPE-Seq gave us an opportunity to compare our results with reactivities generated with eriments  to studyeriments . Interesin vitro SHAPE-Seq experimental comparisons for 5S rRNA, which is routinely used as a benchmark for in vitro RNA folding   . In conture S24)  51). In. Inin vie Figure , inset. in vitro SHAPE-Seq, including the use of highly specific reverse transcription priming sites to target select RNAs, PCR selection against ligation dimers and off-target cDNAs , and a flexible platform for rapid functional characterization of RNA regulators in E. coli. All of these improvements enabled deep read coverage for many in-cell SHAPE-Seq experiments in a single MiSeq run with less effort than current in-cell next-generation sequencing-based techniques , where a large number of changes were observed that matched well with the known interactions of 5S rRNA within the ribosome . While these systems are designed to interact with ribosomes in the cell, these interactions may be too fleeting, or not present at high enough abundance, to be detected within the population of RNAs probed in these experiments, as was the case with the antisense RNAs for these systems . Consistent with our results, while this manuscript was under review, a complementary in-cell SHAPE probing technique called icSHAPE was used to show that the agreement between in vitro and in-cell RNA folds was closer than previously expected, especially near translation initiation regions (Finally, this work enabled us to study how the complex cellular environment can affect RNA folding. This was most clear in a comparison between SHAPE-Seq reactivities from E. coli such as S. cerevisiae, M. musculus or A. thaliana (in vitro SHAPE-Seq experiments offers a way to reveal interactions and structural changes that may be present in the cellular environment as we demonstrated with 5S rRNA. Further, we have provided a detailed step-by-step protocol in the Supplementary Methods to facilitate the application of in-cell SHAPE-Seq to other systems, including RT primer design guidelines. We expect that in-cell SHAPE-Seq will be an easily approachable tool for biologists and engineers to uncover relationships between the sequence, structure and function of RNAs in the cell.We anticipate in-cell SHAPE-Seq to be applicable to studying cellular RNA structure–function relationships within a broad array of mechanistic and cellular contexts, including other organisms beyond thaliana ,20,23,24thaliana ,22 allowthaliana . In addi


RNA titers of 6 of the 7 non-GII 4 strains remained unaffected after alcohol exposure. Compared to GII strains, GI strains were more susceptible to ethanol than to isopropanol. At 90%, both alcohols reduced RNA titers of 8 of the 9 GII.4 strains by ≥ 1 log10 RNA copies/ml. After exposure to 70% ethanol, RNA titers of GII.4 Den Haag and Sydney strains decreased by ≥ 1.9 log10, whereas RNA reductions for GII.4 New Orleans strains were < 0.5 log10. To explain these differences, we sequenced the complete capsid gene of the 9 GII.4 strains and identified 17 amino acid substitutions in the P2 region among the 3 GII.4 variant viruses. When comparing with an additional set of 200 GII.4 VP1 sequences, only S310 and P396 were present in all GII.4 New Orleans viruses but not in the ethanol-sensitive GII.4 Sydney and GII.4 Den Haag viruses Our data demonstrate that alcohol susceptibility patterns between different norovirus genotypes vary widely and that virolysis data for a single strain or genotype are not representative for all noroviruses.Alcohol-based hand sanitizers are widely used to disinfect hands to prevent the spread of pathogens including noroviruses. Alcohols inactivate norovirus by destruction of the viral capsid, resulting in the leakage of viral RNA (virolysis). Since conflicting results have been reported on the susceptibility of human noroviruses against alcohols, we exposed a panel of 30 human norovirus strains (14 GI and 16 GII strains) to different concentrations  of ethanol and isopropanol and tested the viral RNA titer by RT-qPCR. Viral RNA titers of 10 (71.4%), 14 (100%), 3 (21.4%) and 7 (50%) of the 14 GI strains were reduced by > 1 log Noroviruses are the leading cause of epidemic and endemic gastroenteritis in people of all ages worldwide , 2. The Alcohol induced virolysis of non-enveloped viruses is characterized by destruction or serious damage of viral capsids, which results in leakage of viral RNA a process referred to as virolysis , 17. RNain vitro have demonstrated that the P2 domain binds to histoblood group antigens for which it has highly conserved binding pockets. Deletion of just one amino acid from one of the P2 epitopes has been shown to eliminate the binding capability of norovirus virus-like-particles (VLPs)[Most human norovirus infections are caused by viruses that belong to genogroup GI and GII viruses. Of these, GII.4 viruses have caused the majority of the infections over the last decade , 20. Thees (VLPs). We recees (VLPs). Based oNorovirus positive stool specimens (14 GI and 16 GII) were obtained from acute gastroenteritis outbreaks that were submitted to CDC from 2010 to 2013. Additionally, three stool samples from human volunteers (GI.1 viruses) were kindly provided by Dr. Christine Moe at Emory University. A 10% suspension of each stool sample was made in phosphate-buffered saline  and clarified by centrifugation at 11, 337×g  for 10 min. The clarified supernatant was then filtered through a 0.45-μm pore size Millex-MP filter  to remove solid particles. Each filtered sample was stored at -80°C until use. To minimize the possible effect of individual stool matrix, each norovirus strain was pooled separately with three strains from a different genogroup. For example, three pools of the same GI strain were prepared each mixed with a different GII virus.Different concentrations ) of ethanol and isopropanol  were prepared in distilled water. A suspension test was used to measure the efficacy of different alcohols against human noroviruses as described previously. BrieflyTM Ribonuclease and 15 μl of 10×RNAse buffer  for 1 h at 37°C. After stopping the reaction was stopped by adding 2 μl of RNase inhibitor  [To remove viral RNA from damaged viral packages, 100 μl of neutralized alcohol (or PBS)-treated pooled GI/GII samples were incubated with 33 μl of DI water, 1μl of RNase Onebad, CA) , viral Rbad, CA) , 25. Untbad, CA)  and to cTo identify amino acids that possibly could explain why GII.4 New Orleans viruses, and not the genetically closely-related GII.4 Den Haag and GII.4 Sydney viruses, were resistant to ethanol, we sequenced the complete ORF2 gene of 8 of the 9 GII.4 viruses that were included in the panel. For one virus strain (2010746526), no sufficient sample was left to obtain the complete ORF2 sequence. In addition, complete ORF2 sequence of an additional 200 GII.4 viruses from norovirus outbreaks in the US that occurred between 2009–2016 were determined . ORF2 seThe deduced amino acid sequences of the VP1 sequences including reference GII.4 viruses GII.4 Den Haag (JN400607), GII.4 New Orleans (GV445325), and GII.4 Sydney (JX459908) were then aligned using Clustal W. The alignment was then mapped onto the X-ray structure of the P domain of a representative GII.4 virus (PDB ID: 3SLD) using Chimera software  to infer10 (Nd/N0), where N0 is the number of RNA copies detected in untreated samples and Nd is the number of viral particles or RNA copies in the alcohol-treated samples. TaqMan real-time RT-PCR data were expressed as the mean of at least three replicates from each independent experiment. Univariate linear regression models were fitted to compare overall virolysis patterns between norovirus strains, and the Mann-Whitney test was used to determine significant differences in RNA reduction levels using PASW Statistic 18 software  [Reduction of viral RNA reduction was determined by calculating the logork, NY) . P valueStool specimens from human volunteers without any personal identifying information that had been determined IRB-exempt were used in this study.10 RNA copies/ml, respectively. The titers of 4 (3 GI.6 and 1 GI.7) of the GI strains were > 1.8 log10 RNA copies/ml reduced after exposure to 90% isopropanol, whereas no RNA reduction was observed for 50% ethanol, and for 50% and 70% isopropanol  except for one GII.12 strain, of which titer was reduced by 1.4 log10 RNA copies/ml. Exposure to 90% alcohols reduced RNA titers of all 9 GII.4 strains by ≥ 0.9 log10 RNA copies/ml. Overall, exposure to 70% ethanol as well as 90% ethanol and 90% isopropanol resulted in significant RNA reductions for GII strains (P < 0.001) with an average of 1.2 ± 1.1, 1.4 ± 0.9, and 1.0 ± 0.8 log10 RNA copies, respectively.After exposure to alcohol, RNA titers of 6 of the 7 non-GII.4 strains were unaffected  and GII.4 Sydney (n = 4) viruses were reduced by >1.9 log10 RNA copies/ml whereas the titers for GII.4 New Orleans (n = 3) viruses were reduced by less than 0.5 logopies/ml . After eopies/ml .The median RNA reductions of the three types of GII.4 variant strains  were not different after exposure to 90% isopropanol. However, after exposure to 50%,70%, 90% ethanol and 50% isopropanol, the median RNA reductions of GII.4 Den Haag and Sydney strains were significantly higher than for the GII.4 New Orleans viruses (P ≤ 0.001) . InteresWe sequenced the complete capsid genes of 8 of 9 GII.4 viruses that were analyzed for their resistance to alcohols. The P1 and S domain had 4 and 0 amino acid changes, respectively, while the P2 region had 17 amino acid substitutions  . Of thosAppropriate hand hygiene is the single most effective way to prevent norovirus transmission. In the laboratory, the effect of ABHS on norovirus has been studied primarily by using the Norwalk virus (GI.1) reference strain , 31. We According to the Klein-DeForest scheme, a typical dose response relationship exists between the concentration of alcohol and the level of virus inactivation . The typThe P2 hypervariable subdomain of noroviruses is surface exposed and contains histoblood group antigen as well as important antigen binding sites that interact with the human host , 35. We The physical and chemical properties of viral capsid proteins may also affect interactions with environmental matrices which may lead to different environmental behaviors  and different disinfection patterns , 38. RepIn summary, we found that after exposure to 70% ethanol several norovirus GII.4 strains showed no reduction (< 0.5 log) in viral RNA titer whereas other norovirus GII.4 variants showed a 1.9–3 log reduction. Interestingly, GII.4 New Orleans viruses, which in the P2 domain differ only in 17 amino acids, compared to GII.4 Den Haag and GII.4 Sydney viruses, showed almost no reduction in viral RNA titers after exposure to alcohol. These differences in susceptibility correlated with the consistent presence of two amino acids S310 and P396 located on the protruding (P2) domain of the GII.4 New Orleans capsids. To confirm the importance of these amino acids among GII.4 viruses in protection against capsid degradation by alcohols, additional experiments, ideally using infectious clones to introduce specific amino acids in a backbone of a strain that is less sensitive to alcohol are required. Since the ratio between RNA reduction and infectivity reduction by alcohols remains unknown , 17, the


Changing one RNA strand in polygons automatically induced the stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares and pentagons. When immunological adjuvants were incorporated, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced in vitro and in animals up to 100-fold, while RNA polygon controls induced unnoticeable effect. The RNA nanoparticles were delivered to macrophages specifically. The degree of immunostimulation greatly depended on the size, shape and number of the payload per nanoparticles. Stronger immune response was observed when the number of adjuvants per polygon was increased, demonstrating the advantage of shape transition from triangle to pentagon.Modulation of immune response is important in cancer immunotherapy, vaccine adjuvant development and inflammatory or immune disease therapy. Here we report the development of new immunomodulators  In the-in-hand ,3–5, foo-in-hand , branch -in-hand , loop–re-in-hand , ‘sticky-in-hand ,18,19 an-in-hand . These m-in-hand . Recentl-in-hand ,23. In R-in-hand ,25. As s-in-hand , square--in-hand ,28, RNA -in-hand ,9 or pRN-in-hand ,9,29,30,-in-hand , RNA bun-in-hand ,31 and f-in-hand ,16 utili-in-hand . Overall-in-hand ,11, rece-in-hand .In this report, we introduce a conceptual approach to the rational design of stable RNA architectures by stretching the 60° AOB angle (∠) Figure  of the tvia endosomal Toll-like receptor 9 (TLR9)  are immunostimulatory DNA molecules that mimic the immunostimulatory activity of bacterial DNA ,39. CpG 9 (TLR9) ,41. Upon9 (TLR9) . Recentl9 (TLR9) , DNA pol9 (TLR9) , DNA ori9 (TLR9) , Y-shape9 (TLR9)  and DNA 9 (TLR9)  to delivin vitro T7 transcription using polymerase chain reaction (PCR) generated DNA templates. RNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE) and were either Cy5 whole body RNA labeled (Mirus Bio LLC) or 5′-end [γ-32P] ATP (PerkinElmer) labeled, as previously described  was primarily used for designing polygon models using Swiss PDB viewer, as previously described . RNA strescribed .2). Samples were annealed for 1 h in a thermocycler with controlled, slow cooling (1°C/min) from 80 to 4°C. All RNA polygons harboring CpG ODNs were assembled from their corresponding 2′F-U/C modified strands in one pot procedure.RNA polygons were assembled in one pot by mixing equimolar concentrations (1 μM) of four RNA strands for the triangle, five RNA strands for the square and six RNA strands for the pentagon in 1× TMS buffer  native polyacrylamide gels in the presence of 0.5 × TMS buffer. Gels were run at constant 90 V, +4 °C. Gels were imaged with Typhoon FLA 7000  to visualize RNA strands. TGGE analysis was performed on 7% native PAGE in a buffer containing 50 mM TRIS pH 8.0, 100 mM NaCl and 0.2 mM MgClescribed ,26,28. ABoiling resistance assay was performed in 10 μl containing 1 μM preassembled polygons in TMS buffer or in the presence of 8 M urea. Samples were incubated at 100 °C for several minutes, then snap cooled on dry ice to prevent refolding following evaluation on 7% native PAGE at 4 °C. Individual experiments were repeated several times to reduce error.Quantification analysis was performed using ImageJ . Equal-s2. Cells were then seeded on 24-well plates or 96-well plates at a density of 5 × 105 cells/ml and cultured overnight before use.Mouse macrophage-like RAW 264.7 cells were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin at 37°C in humidified air containing 5% CO5 cells per well and cultured overnight. Then, RNA nanoparticles harboring different numbers of CpG ODNs were diluted in Opti-MEM medium  and added to the cells. The cells were continually cultured for 8 h at 37 °C in humidified air containing 5% CO2, and the cell culture supernatant were collected and stored at −80 °C until use. The concentration of TNF-α and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) using Mouse ELISA MAX™ Deluxe sets , following protocols provided by the manufacturer.RAW 264.7 cells were plated into 24-well plates with the density of 2.5 × 10in vivo immunostimulation, RNA triangular nanoparticles harboring CpG ODN, RNA triangular nanoparticles, or CpG ODN were dissolved in phosphate buffered saline (PBS) and administrated to the mice via tail vein injection at 2 mg/kg (CpG ODN per body weight). The same volume of PBS was injected into a mouse as a control. Blood samples were collected 3 h post-injection by cardiac puncture. Serum was prepared by centrifugation at 12 800 g for 10 min. Serum TNF-α and IL-6 levels were determined by enzyme-linked immunosorbent assay (ELISA) using Mouse ELISA MAX™ Deluxe sets , following protocols provided by the manufacturer.Male CD-1 mice (4–5 weeks old) were purchased from Charles River Laboratories. All animal procedures were approved by the Institutional Animal Care and Use Committee at University of Kentucky and were performed in accordance with guidelines issued by the National Institutes of Health for the care of laboratory animals. For 2 overnight. The culture medium was removed and the cells were washed with Opti-MEM medium twice to remove dead cells. RNA nanoparticles harboring Cy3-labeled CpG DNA or Cy3-labled CpG DNA only were diluted in Opti-MEM medium and added to the cells. After 4 h incubation at 37 °C in humidified air containing 5% CO2, the cells were washed twice with PBS and fixed with 4% formaldehyde. ProLong® Gold Antifade Reagent with DAPI  was used to stain the cell nucleus and mount the samples. Alexa Fluor® 488 phalloidin  was used to stain actin. The images were obtained on a Olympus FV1000 confocal microscope .RAW 264.7 cells were seeded on glass coverslips in 24-well plates and cultured at 37 °C in humidified air containing 5% COin silico design of the RNA triangle, square and pentagon 2D polygons. During the computer modeling we used the particular angle of the 3WJ formed by H1 and H2 as an inner angle of the polygons as we hypothesized that the angle could be stretched to a more open conformation. Throughout this report the intra-helical angle between H1 and H2 is denoted as ∠AOB, as shown in figure The structural features of the recently discovered ultrastable pRNA 3WJ module from the bacteriophage Phi29 DNA packaging motor were utilized for KD gels, and KD values were determined to be 18.8, 20.3 and 22.5 nM for triangles, squares and pentagons, respectively .Each polygon was composed of a different number of RNA strands classified as short strands  and long strands  Figure . By incrThese results demonstrate that each RNA nanoparticle assembles into the desired nanostructure, and indicated by a stretching of the 60°∠AOB to wider conformations. The assembly of RNA strands into specific-shaped nanoparticles based on the 60°∠AOB of the pRNA 3WJ motif was controlled by modulating the number of short external stands and the length of the long internal strand.To further evaluate the size and shape of the resulting RNA assemblies, structural characterization of each polygon was conducted by AFM. AFM images of the pRNA 3WJ based polygons revealed that the shapes of resulting polygons were similar to their predicted, theoretical 3D models Figure . The est2 area, resulting in 48 triangular particles, 33 square particles and 17 pentagon nanoparticles . The estimated number of triangular nanoparticles adsorbed on the mica was ∼1.5 times more than that of the square and three times more than that of the pentagon. However, native PAGE revealed that the yields among the three polygons were almost equal. The difference in adsorption amounts between polygons on the mica surface was presumably due to variation in their sizes and 3D conformations, resulting in different dynamic and physical properties.Quantification analysis was performed to compare apparent yields between polygons observed on AFM mica surface. Equal concentrations (1 nM) of the polygons were deposited on a mica surface and correctly folded polygons were manually counted in a 0.5 μmhttp://www.malvern.com], while AFM imaging can produce images larger than the real diameter due to tip size used  in presence of 0.2 mM MgCl2: triangle TM = 56°C, square TM = 53°C and pentagon TM = 50°C  resulted in a higher thermostability and resistance in chemical degradation and the change in stability was in large part due to the stretching of the ∠AOB.The stabilities of polygons were studied using a perpendicular TGGE (Biometra GmbH). This convenient technique has garnered widespread use for measuring melting temperatures of RNA nanoparticles with multiple strands ,26,28,55C Figure . The trie Figure . The quaCpG oligonucleotide is an immunological adjuvant popularly used as vaccine adjuvant or immunotherapy reagent for disease control and treatment . To evalThe extracellular immunostimulatory efficacy of RNA polygons was evaluated by measuring the release of cytokines TNF-α and IL-6 after addition to mouse macrophage-like RAW 264.7 cells Figure  and B, ain vivo, nanoparticles were administered to CD-1 mice by injection into the tail vein at 2 mg/kg (CpG oligonucleotide per body weight), following level determination of cytokine TNF-α and IL-6 levels after 3 h post-administration in collected blood serum. Figure in vivo. These data are in agreement with the in vitro stimulation of murine RAW 264.7 cells.To examine whether RNA nanoparticles retain their immunostimulatory activity Previously, it has been demonstrated that the CpG oligonucleotide can be readily recognized by TLR9 on the endosomal membrane of macrophages, resulting in cellular uptake of the CpG adjuvants ,59. To iThis observation was further confirmed by confocal microscopy images Figure  revealinCollectively, both flow cytometry and confocal imaging demonstrated that all RNA nanoparticles with different shape and harboring CpGs have much stronger binding and cellular entry to the macrophage-like RAW 264.7 cells compared to free CpGs. In addition, cellular uptake was highly dependent on the number of CpG per polygon. With the increasing number of CpG per polygon, more efficient cell binding and entry was observed  harboring one CpG compared to square (∼11 nm) and pentagon (∼13 nm). However, upon increasing the numbers of CpG per RNA nanoparticle the cytokine induction was affected more by pentagon as the number or local concentration of CpG is highest in the pentagon. This study illustrates the importance of the size and shape of RNA nanoparticles for improvement of activity of CpG based vaccines adjuvants targeting infectious diseases and cancer cells, as well as for increasing immune responses by the innate and adaptive immune systems. The RNA nanoparticles harboring CpGs are safe, effective, versatile and easy to manufacture, offering new solutions to address the unmet needs in current vaccines and adjuvants design and development. Recent findings on the thermodynamically ultra-stable (In addition to the discovery of the unique approach for the rational design of stable RNA nanoparticles, it has been demonstrated that each RNA polygon has the potential to serve as multivalent nanocarriers for vaccine adjuvants, particularly of CpG oligodeoxynucleotides. The designed RNAs self-assemble into distinct, non-toxic homogeneous nanoparticles with high chemical, thermal and intracellular stability. We found that the size and shape of the RNA nanostructures are important factors for the induction of immunostimulatory processes a-stable ,11 and ha-stable  RNA nanoPDB ID: 4KZ2.Supplementary Data are available at NAR Online.


Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation -3. The R Recenprotein)  and usedprotein) . Notwithin vivo and in vitro due to several advantages over the competing techniques [Protein complementation (PC) and particularly fluorescent PC is widely used for protein-protein interaction studies both chniques ,8,9. Thichniques -3,10-14.Recently, we described a method for RNA visualization in live cells that uses a combination of fluorescent protein complementation and high affinity interaction of an RNA-binding protein with an aptamer -3. In thHere we present another approach to RNA visualization in live cells based on fluorescent protein complementation. Two different RNA aptamers are added as tags to the RNA molecule of interest; they bind with high affinity two RNA-binding peptides (∼20 amino acid residues) fused with the fragments of a split EGFP .There are several reasons for attempting these modifications. First, the ribonucleoprotein complex which is assembled on the target RNA is smaller as compared with that containing reassembled eIF4A protein. Smaller detection tools should provide lower probability of interference with function. Second, eIF4A protein is a component of the translation machinery in eukaryotic cells and it also has close homologs among bacterial proteins. Therefore, it cannot be excluded that its over-expression may interfere with essential biological functions in live cells. Short viral peptides on the other hand have no homologs in bacterial or in eukaryotic cells. Therefore their expression in a cell will less likely interfere with the cellular functions. Finally, an alternative design of the RNA recognizing complex adds more flexibility to the PC approach and expands the set of RNA-binding molecules applicable for RNA labeling methods.2.2.1.Custom-synthesized peptides, HTLV-1 Rex, HIV-1 Rev and bacteriophage λN, were purchased from GeneScript,  . RNA apt2.2.NcoI and AvrII, which placed the insert after the first T7 promoter region and before the T7 terminator region  and β-EGFP , respectively. A multi-step PCR was performed to create a DNA fragment that encodes two fusion proteins, HTLV-1 Rex peptide-α-EGFP and bacteriophage λN peptide-β-EGFP, plus T7 promoter in between  under the control of the T7/LacO promoter, and cloned in E. coli strain BL21 (DE3). As a result, RNA was transcribed from this plasmid as a short untranslated transcript without a ribosome binding site and containing two adjacent aptamers separated by 10Ts. As a negative control, DNA sequence encoding two identical RNA aptamer sequences binding HTLV-1 Rex peptides was synthesized and similarly cloned into pETDuet-1 vector. DNA templates and primers are listed in To clone aptamers, a DNA sequence was designed which encodes two RNA aptamers, one of which binds the λN peptide and the other the HTLV-1 Rex peptide. The aptamer sequences were separated by a dT2.3.E. coli BL21 (DE) 3 − dcm ompT hsdS (rB− mB−) gal λ(B F (DE3)) (Stratagene). As a negative control, the cells were transformed with a plasmid expressing two identical aptamers. Single colonies of transformed cells were grown first at 37 °C in LB medium supplemented with antibiotics for 3 h. Then, cultures were diluted 300-fold into fresh LB medium containing the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) and grown overnight at room temperature. Optical density of the cultures (OD600) was between 0.4 and 0.6 at the time of examination.Two plasmids encoding protein and RNA components of the complementation complexes were co-expressed in 2.4.600 = 0.4 to 0.6) were washed once with PBS buffer prior to assaying. In total 100,000 cells have been analyzed.Fluorescence measurements were obtained with a Becton-Dickinson FACSCalibur flow cytometer with a 488-nm argon excitation laser and a 515–545 nm emission filter (FL1). 500 μL of the cells induced overnight  with 100x magnification objective controlled by IPLab v.3.7 software . An ND4 filter was used to reduce cell photo-damage. Pseudo-green color was added according to the fluorescence level in the florescent images. Image processing was performed using ImageJ 1.36 B software .3.3.1.To detect RNA in live bacterial cells we tagged it with two different RNA aptamer sequences which bind two different viral peptides with high affinity. Each peptide was expressed in the cell as a fusion with one of the two fragments of a split enhanced green fluorescent protein (EGFP) . InteracTo develop an efficient PC method based on two aptamers/two peptides interactions, several issues should be considered. The affinity of each interacting peptide/aptamer pair should be high enough (in the nanomolar range) and ideally it should be comparable for both pairs. There should be no cross-reactivity between the two peptide/aptamer pairs. Also, there should be no interaction between the RNA-binding peptides in absence of RNA, which could otherwise bring the fragments of a detector protein together and thus increase the non-specific background. The RNA-binding peptides should be of comparable length to avoid distorting the assembly of the protein complementation complex. Finally, the placement of the two RNA aptamers on the target RNA should allow accessibility of the corresponding RNA-binding peptides to the cognate RNA aptamers. This implies that a flexible linker of sufficient length should be placed between the RNA aptamer tags.It is known that the high-affinity and high-specificity interactions of many RNA-binding proteins with the corresponding RNAs are determined by the peptide sequences which contain arginine-rich motifs (ARMs) . Recent 3.2.in vitro experiments the increasing concentrations of the ARM peptides were combined with RNA aptamers at fixed concentrations. The complexes were then qualitatively analyzed by a non-radioactive gel-shift assay  was fused to the N-terminus of the HTLV-1 Rex peptide (16 aa-long) via a flexible linker consisting of serine and glycine residues ,22. We u–158 aa wE. coli cells expressing the entire complementation complex and appropriate controls were grown overnight at room temperature in the presence of the inducer, isopropyl-ß-D-thiogalactopyranoside (IPTG) for co-expression of proteins and RNA. Fluorescence of these cells was compared with fluorescence of the cells expressing two protein fusions in RNA absence and cells expressing two protein fusions plus an incorrect combination of the two aptamers (two HTLV-1 Rex peptide-binding aptamers linked with the T10-linker).Our experiments with the complementing fusion proteins containing short viral peptides, as well as the fragments of the split eIF4A protein, revealed that background fluorescence caused by spurious self-assembly of the protein fragments can be modulated by cell growth conditions and IPTG concentration . These tE. coli cells expressing two fusion proteins induced with 1 mM IPTG displayed high fluorescence in the absence of RNA target, and there was also no difference in fluorescence distribution in the cells expressing correct or incorrect aptamer sequences  the source of background fluorescence is the same and it can be modulated by changing conditions of cell growth, specifically by concentrations of the inducer, IPTG, and by changing the temperature of cell culturing. The slightly higher background in the PC system using viral peptides can be likely explained by the aggregation of the arginine-rich peptides. However, it does not change the major trend characteristic to both PC systems (compare the results in


R2 non-LTR retrotransposons insert at a specific site in the 28S rRNA genes of many animal phyla. R2 elements encode a single polypeptide with reverse transcriptase, endonuclease and nucleic acid binding domains. Integration involves separate cleavage of the two DNA strands at the target site and utilization of the released 3′ ends to prime DNA synthesis. Critical to this integration is the ability of the protein to specifically bind 3′ and 5′ regions of the R2 RNA. In this report, alanine mutations in two conserved motifs N-terminal to the reverse transcriptase domain were generated and shown to result in proteins that retained the ability to cleave the first strand of the DNA target, to reverse transcribe RNA from an annealed primer and to displace annealed RNA when using DNA as a template. However, the mutant proteins had greatly reduced ability to bind 3′ and 5′ RNA in mobility shift assays, use the DNA target to prime reverse transcription and conduct second-strand DNA cleavage. These motifs thus appear to participate in all activities of the R2 protein known to require specific RNA binding. The similarity of these R2 RNA binding motifs to those of telomerase and group II introns is discussed. Non-LTR retrotransposable elements, also known as long interspersed nuclear elements (LINEs), are highly abundant genomic parasites of eukaryotes. One of the best-characterized members of this class is R2, an element which exclusively inserts into a highly conserved sequence of the 28S rRNA genes ,2. The eThe reverse transcriptase encoded by R2 (R2-RT) has unusual properties when compared to the RTs of retroviruses. First, priming of DNA synthesis from the 3′ hydroxyl exposed by the DNA cleavage does not require complementarities between the DNA target and the RNA template ,6. SeconIn addition to the specificity of the R2 endonuclease for the 28S gene target site, R2-RT has high specificity when binding RNA. The only RNA sequences utilized in the TPRT reaction are those that contain the 3′ untranslated region (3′ UTR) of R2 . This 3′Therefore, the R2 protein, group II intron protein and telomerase share the property of specifically binding an RNA molecule that will be used as a template for reverse transcription primed by the 3′ end of a DNA strand. Critical RNA binding domains for both group II introns and telomerase have been shown to be immediately N-terminal to the fingers region of their reverse transcriptase domain . In thisBombyx mori R2 expression construct pR260  of sodium dodecyl sulphate (SDS) polyacrylamide gels using bovine serum albumen as the protein concentration standard.Alanine replacement mutations of the ct pR260  in the -eviously ,10 with 32P-ATP . PCR amplifications, gel purification and elution of target DNA was performed as described previously . This DNA substrate was generated by PCR amplification from clone pB109  with a feviously  with the2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.01% Triton X-100, 10%–12% glycerol with 4 ng labeled target DNA and variable amounts of R2 protein as described in the figure legends (All assays were performed in a 13 μl volume containing 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 5 mM MgClThe ability of wild type and mutant R2 proteins to extend DNA primers annealed to either RNA or DNA templates was assayed as described previously . PolymerThe primer, 5′ CTGGCGAAAGGGGGATGTGC 3′, was 5′ end labeled and annealed to a 320 nucleotide single stranded DNA template or this DNA template annealed to a 100 nt RNA block as described previously . While a slight reduction in product levels was sometimes detected (data not shown) all mutant proteins retained significant ability to conduct the TPRT reaction. More dramatic reductions were obtained by combining the three alanine mutations in the -1 region into one construct and the two mutations in the 0 region into a second construct. The activities of these two mutant proteins are described below and the proteins are referred to as the -1 and 0 region mutations.To insure the mutations did not influence the overall structure or stability of the R2 protein, the -1 and 0 region mutant proteins were first tested for their ability to specifically cleave the first strand of the DNA target. Previous studies have shown that the R2 protein in the absence of RNA can cleave only one strand of the DNA target site . This fiTo determine if the mutations affected the basic nucleotide polymerase activity of the protein, the 0 and -1 mutants were tested for their ability to extend a DNA primer annealed to a template. As diagramed in Figure As shown in Figure in vitro assays  Figure . Unlike As shown in Figure EMSA were next performed to directly determine whether failure of the TPRT assay was a failure of the mutant R2 proteins to bind the 3′ UTR RNA or a failure to position the RNA properly for TPRT. These gel shift experiments were conduct only with R2 RNAs, as it is difficult to detect wild-type R2 protein binding to non-R2 RNA ,6,9,20. Shift assays done with the -1 and 0 mutations revealed target DNA binding in the absence of RNA at levels similar to that observed for the wild-type protein. In the case of the 0 mutant, higher levels of more slowly migrating bands were observed indicating a greater tendency for additional protein subunits to bind to the DNA target at the highest protein concentration (lane 15). In the presence of the R2 RNA, the shifted complexes observed for the -1 mutant were identical to those observed for the protein only assays, suggesting a greatly reduced ability of this protein to bind RNA. In the case of the 0 mutant protein, no RNA-induced super-shifted band was observed at low protein concentrations also consistent with a reduced ability of this protein to bind RNA. However, a range of slower migrating bands was observed at higher concentrations of protein in the presence of RNA compared to the absence of RNA (compare lanes 15 and 18), suggesting a residual level of RNA binding was still possible in the 0 region mutation.In addition to the ability of the R2 protein to bind RNA corresponding to the 3′ UTR of the R2 element, the R2 protein can also bind a 300 bp region of RNA from near the 5′ end of the R2 element . This 5′As shown in lanes 3–6 of Figure Cleavage of the two strands of DNA at the R2 insertion site is conducted by different R2 subunits Figure  4). Pro. Pro4). Comparison of R2 protein sequences from diverse metazoans revealed two conserved regions (0 and -1) upstream of the series of common motifs that can be identified in all reverse transcriptases . AnalysiWe previously suggested that the 0 motif might enable the R2 protein to bind more extensively to template sequences upstream of the active site of the RT domain, thus, allowing the R2-RT to undergo template jumps and displace an RNA strand annealed to a DNA template . Evidencin vitro assays were conducted with crude cell lysates. Because no other activity could be assayed, there was no means to insure that the mutant L1 proteins were stabile. Second, the L1-RT activity being assayed was not dependent upon the addition of a primer, thus, there is uncertainty as to how DNA synthesis was initiated in these assays. Based on the experiments presented here and the similarity to telomerase and the RT of mobile group II introns described below, we suggest that RNA binding is an important role of the 0 motif in most other non-LTR retrotransposons including L1.The 0 motif can be found in all lineages of non-LTR retrotransposons ,29. For The RNA binding motifs N-terminal to the universal RT motifs have been identified in both mobile group II introns and in telomerases. Group II introns have a conserved motif (labeled RT0 in ref. The more extensive characterization of RNA binding by telomerase has included X-ray diffraction studies of the isolated RNA binding domain as well as the complete telomerase ,32. Two Phylogenetic comparisons based on the sequences of the conserved domains in all reverse transcriptase-like enzymes, suggest that non-LTR retrotransposons, telomerase and group II introns are phylogenetically related and separate from the reverse transcriptases of LTR retrotransposons and retroviruses ,21,25,33


The paper aims to examine the influence of human resource management (HRM) practices on bank efficiency using Malmquist index of total factor productivity. The model comprises HRM index that represents the quality of HRM practices. The results are decomposed into three efficiency scores, namely, technical efficiency, pure efficiency, and scale efficiency. In this study, panel data for 44 banks in Bangladesh are used for the period 2008-2013. This paper reveals that foreign banks are ahead in converting the influence of HRM practices into efficiency scores (0.946>0.833). On the other hand, domestic banks performed better than foreign banks in terms of pure efficiency and scale efficiency. But, in terms of technical efficiency, the domestic banks are regressed by 6.7% annually whereas foreign banks are progressed with a yearly value of 5.8%. The results are robust, because the Mann-Whitney test and Kruskall-Wallis test (non-parametric tests) also confirm the same results. This study emphasizes HRM practices in the banking industry to ensure efficiency in the long-term scenario. Domestic banks are suggested to ensure continuous development in HRM practices in order to compete with foreign banks. In recent years, human resource management (HRM) practices are considered to be a key strategic partner in all organizations to attain critical business objectives . ExpliciBangladesh is a Next Eleven developing country in the world economy. Its total population is 159,147,170 placing it eighth globally . Now, inOrganizational performance (OP) has been mostly measured in analyzing quantitative aspects (bank efficiency and performance) rather than quality aspect in the banking studies. To discover the connection between bank efficiency and management quality aspects, we implement a nonparametric frontier method of data envelopment analysis (DEA) developed by Färe,Grosskopf and Roos . Here, wIn our previous study , we examIn this paper, section 2 briefly represents the overall scenario of the banking industry in Bangladesh. Section 3 compromises a succinct assessment of the existing literature regarding prime issues of HRM practices and efficiency measurement techniques to attain bank efficiency. The next section consists of variables and the data set. Section 5 contains the methodology that describes the suggested HRM component in Malmquist index decomposition. The outcomes of the investigation are described in Section 6. The findings of study, the shortcoming of the study and directions for future research are also deliberated in section 7 and 8 respectively. The last section contains the conclusion including suggestions for the banking sector.After the emergence of Bangladesh in 1971, Bangladesh Bank (BB)—the central bank of the country was established through Bangladesh Bank Order 1972. The banking industry started its journey with 2 state-run specialized banks (SDBs), 6 nationalized commercial banks (NCBs) and 3 foreign commercial banks (FCBs). The growth of the banking system in Bangladesh has moved through 3 stages. All the banks of Bangladesh were nationalized during the first phase of BB (1972–1982). Moreover, the credit focused on public investment and farming. Investment rates were decidedly controlled by the central bank and yearly inflation was 8.9% . The thiIn the present day, the banking sector of Bangladesh can be categorized into 5 groups. Group 1: There are 4 State Owned Commercial Banks (SOCBs). These banks, together, control more than 54% of total deposits. These are majorly or fully owned by the Bangladesh government. Group 2: There are 5 Specialized Banks (SDBs) that are established for special purposes, such as industrial or agricultural development. SDBs are also majorly or fully owned by the Bangladesh government. Grameen Bank, a Nobel-prize winner, is a specialized micro-finance institution. It is the pioneer of micro-credit concept worldwide and have made a considerable contribution towards the empowerment of women and reduction of poverty in Bangladesh. Group 3: Private Commercial Banks (PCBs) are the highest growth sector due to better service and products. There are 39 PCBs in Bangladesh. The 31 conventional PCBs perform banking in interest-based operations. There are also 8 PCBs that are performing banking based on Islamic Shariah . Group 4: There are 9 Foreign Commercial Banks (FCBs). The branches of these foreign banks are unified with the overseas branches. Group 5: There are also 4 non-scheduled banks. These are Probashi Kollyan Bank, Karmashangosthan Bank, Jubilee Bank, and Ansar VDP Unnayan Bank.http://www.bb.org.bd/). All commercial banks in Bangladesh provide savings account, current account, tele-banking, internet banking, home-banking, and SWIFT account service as like commercial banks in other countries.As of 2014 (June), the total number of bank branches was 8,794. Among these branches, 57.11%  are situated in the rural areas and 42.89%  are situated in the urban regions. The SOCBs hold 3,536 branches, SDBs hold 1,496 branches, PCBs hold 3,692 branches, and FCBs hold 70 branches . Data are collected from BankScope data base from 2008 to 2013. In this study, an overall quality index of HRM is achieved through nominated 4 indicators; i.e., annual HR recruitment (HRRec), HR compensation budget (HRCom), HR training and development budget (HRTrD), and employee relations budget (HREre). The variables of this study are designed from the background work by Chang and Huang ; Chou anIn the recent years, extensive use of DEA has been employed for performance evaluation. The reason behind is that a DEA model can convert multiple inputs/outputs in performance index ignoring the uncovered relationship of data among the DMUs . Moreovext), and the output vector by (yt), the production technology can be defined as:In this study, the methodology is motivated from our earlier study . If the λ stands for the radial factor that adjusts the output vector’s position. We can duplicate zt.According to Chou, Shao and Lin  and CoelConsidering two consecutive time frames  and combining the distance function of (Eff), technical changes (Tech), and quality index (QI).At this point, we include the quality index and Roos  within tThe Malmquist index of total factor productivity is expected to be liable for the quality issue in the output function as all 3 elements are in a multiplication form. So,Note that, a value of 1 explains that the efficiency remains equal compared to the period (t) in (t+1). Again, a value of more than 1 represents progress and less than 1 indicates a regress in efficiency as a relative measure.Eff, DVRS is the output distance function for variable returns to scale. The initial part of the QI, Tech, PE and SE. Now, After further decomposition of +1,xt+1)]Here, Dat;We duplicate a) alone, the following LP can be used for the a0) and QI (a1) need to be calculated.The technique for calculating HRM practices  was employed.In the above model, the quality index In the first section of the empirical results, using the In the second section of the empirical results, In the last section of the empirical results, we test robustness of the derived data from DEA calculation in two different independent sample groups (domestic and foreign banks). Coakes and Steed  demonstrThe parametric t-test results show that foreign banks are scored 0.946 whereas domestic banks are marked with 0.833 in The major findings of this research are categorized in three clusters. First, Tables Second, Last but not least, in The detail records of HRM practices and related investment are remain publicly unavailable to the external audiences’ use . LikewisThis paper examines the use of traditional DEA, applying a quality index of HRM within the traditional Malmquist index model to a dataset of 44 banks of Bangladesh, from 2008–2013. We modify the conventional DEA model combining HRM as the quality index to measure bank efficiency. The superiority of this model is that it allows the calculated frontier to detect the best practice banks examining the direct impact of HRM practices.This paper explores the importance of HRM practices in bank efficiency. In terms of pure efficiency, 8 out of 9 foreign banks in Bangladesh have found averagely progressed over the years. In total, foreign banks scored higher progress in scale efficiency compared to that of domestic banks. In view of the HRM quality index, only two foreign banks scored a positive change and not a domestic bank has found progressed over the study period. Comparing the domestic banks’ efficiency, domestic banks of Bangladesh have found progressed in terms of technical change. Most significantly, not a domestic bank has found progressed in HRM practices. The above results have also confirmed by the test of Mann-Whitney test for ensuring its robustness. The test indicates that the domestic banks are less efficient than the foreign banks in term of quality indicator of HRM practices.This paper contributes in 4 key areas of existing literature. Firstly, it fills the literature gap by inspecting the direct effect of HRM practices by the selected 44 banks in their respective efficiencies. Secondly, the scare literature on HRM and bank efficiency especially in Bangladesh context despite its enormous importance in the international trade and business . Thirdly