Fungal RNA samples are usually isolated from fungal mycelia grown in liquid culture, which relies on prolific growth of the fungus in liquid media. The fungal biomass is then collected by vacuum filtration, which can result in low recovery for samples with reduced biomass due to poor growth in liquid media.Here we report an alternative culturing method, based on growth on solid media which is independent of the ability of a fungus to grow in liquid culture. We show that growth on solid media overlayed with a nylon membrane is superior to other culturing methods, producing large amounts of biomass and allowing for easy harvesting of fungal mycelia. Furthermore, we show that mycelium harvested with this method yielded high-quality RNA, superior to RNA isolated from liquid grown mycelium. We also show that inclusion of a second chloroform extraction step in the procedure significantly increases RNA yield.This method is particularly useful for fungal species that show poor or no growth in liquid media, but are easily cultured on solid media. Culturing can be performed on small petri dishes, which significantly reduces handling and therefore allowing growth and isolation of RNA from multiple strains in a high throughput manner. The obtained RNA samples are of high quality in sufficient quantities for several northern blot experiments or quantitative RT-PCR experiments. For isolation of nucleic acids from mycelia, the fungus is generally grown in liquid media because Fusarium oxysporum f.sp. conglutinans and the mycorrhizal fungus Rhizoctonia solani. For comparison, fungal biomass was also obtained by growth in liquid static and liquid shaking cultures and on solid agar without a nylon membrane overlay. RNA was isolated from the harvested fungal materials using TRIzol Reagent and the isolated RNA samples were compared for their quality and quantity.We tested the method with two different fungal species, the necrotrophic fungus F. oxysporum spores or with a small agar plug from a fresh plate of R. solani: i) 10 ml of liquid 1/2 strength Potato Dextrose Broth (PDB)  in a 50 mm petri dish kept static, ii) 25 ml of liquid 1/2 strength PDB in a 250 ml flask kept shaking at 200 rpm after inoculation, iii) 10 ml of 1/2 strength Potato Dextrose Agar (PDA)  in a 50 mm petri dish without nylon membrane overlay, iv) 10 ml of 1/2 strength PDA in 50 mm petri dish with nylon membrane overlay. All cultures were incubated at 28°C for 4 days  with equal results, however other types of membranes are likely to perform equally well. All growth methods were carried out in biological duplicates, with the membrane overlay culture further duplicated for analysis of differences between one and two chloroform extraction steps.Fungal biomass was obtained by inoculating the following media with 5 μl of a -80°C glycerol stock of F. oxysporum in static liquid culture was poor  was added and the sample ground further until the slurry had thawed. The sample was split into two equal volumes and transferred to 2 ml Eppendorf tubes. After 5 min incubation at room temperature (RT) 300 μl of chloroform was added, the tube shaken vigorously for 15 seconds and allowed to stand at RT for 3 minutes. The sample was then centrifuged at 13,000 × g for 15 minutes at 4°C. One nylon membrane-derived sample was subjected to a second chloroform extraction as above. The supernatant from the final extraction step was transferred to a clean 1.5 ml Eppendorf tube and the RNA precipitated with 500 μl isopropanol at −20°C for 2 hours or longer . Precipitated RNA was collected by centrifugation at 13,000 × g for 15 minutes at 4°C, the pellet washed with 1 ml of ice cold 75% ethanol and air dried briefly at RT. The RNA pellets were resuspended in 20 μl of nuclease free water and the two duplicate tubes combined.The harvested fungal material was ground to a fine powder in liquid NRNA concentration and purity were measured using a NanoDrop Spectrophotometer (Biolab ND-1000)  , as these will provide a resilient surface for removal of the biomass. Using an overlay technique and subsequent removal of the biomass with a scalpel, removes the need for centrifugation and washing steps that are required when using liquid cultures, an advantage even for fungi that grow well in liquid culture. Indeed we obtained higher RNA yields from mycelium grown on a membrane compared to mycelium grown in liquid culture for  Figure A, furtheF. oxysporum lines in parallel and routinely obtained ~100 μg of total RNA from mycelia grown for 4 days on 10 ml of solid media overlayed with a Hybond membrane, performing two chloroform steps. The isolated RNA was used in Northern blot hybridisation experiments , 5′ RACE and RT-PCR analyses. Figure We have routinely used this method to isolate total RNA  from multiple The authors declare that they have no competing interests.US designed the study, carried out experiments and wrote the manuscript. NAS carried out experiments and helped drafting the manuscript. MBW designed the study and wrote the manuscript. All authors read and approved the final version.


Electrical Impedance Tomography (EIT) is used as a fast clinical imaging technique for monitoring the health of the human organs such as lungs, heart, brain and breast. Each practical EIT reconstruction algorithm should be efficient enough in terms of convergence rate, and accuracy. The main objective of this study is to investigate the feasibility of precise empirical conductivity imaging using a sinc-convolution algorithm in D-bar framework.At the first step, synthetic and experimental data were used to compute an intermediate object named scattering transform. Next, this object was used in a two-dimensional integral equation which was precisely and rapidly solved via sinc-convolution algorithm to find the square root of the conductivity for each pixel of image. For the purpose of comparison, multigrid and NOSER algorithms were implemented under a similar setting. Quality of reconstructions of synthetic models was tested against GREIT approved quality measures. To validate the simulation results, reconstructions of a phantom chest and a human lung were used.Evaluation of synthetic reconstructions shows that the quality of sinc-convolution reconstructions is considerably better than that of each of its competitors in terms of amplitude response, position error, ringing, resolution and shape-deformation. In addition, the results confirm near-exponential and linear convergence rates for sinc-convolution and multigrid, respectively. Moreover, the least degree of relative errors and the most degree of truth were found in sinc-convolution reconstructions from experimental phantom data. Reconstructions of clinical lung data show that the related physiological effect is well recovered by sinc-convolution algorithm.Parametric evaluation demonstrates the efficiency of sinc-convolution to reconstruct accurate conductivity images from experimental data. Excellent results in phantom and clinical reconstructions using sinc-convolution support parametric assessment results and suggest the sinc-convolution to be used for precise clinical EIT applications. Electrical impedance tomography is a new non-invasive imaging technique in which the conductivity distribution inside a body is reconstructed via knowledge of injected current patterns and resulted induced voltages through finite number of electrodes placed on its surface . This moReconstructing the conductivity images in EIT involves solving forward and inverse problems . The solAlgorithms for solving the forward problem of EIT use Finite Element Methods (FEM), Boundary Element Methods (BEM) and Finite Difference Methods (FDM). Existin1. Linearized iterative methods such as Calderon’s method , back-pr2. Non-linear iterative methods such as equation error formulation , output 3. Layer stripping methods  which ar4. Direct algorithms such as D-bar  and BlocD-bar method is a new direct methodology, which was firstly introduced in the constructive proof of Nachman . This meO(N6) arithmetic computations on N-point grids which is huge even for advanced ultra-fast computers  × . This provides the required knowledge to define the sinc-points via definition of region-related mapping functions in the next step of algorithm. In the second step of algorithm, the two-dimensional convolution integral in the right-hand-side of equation (13) is decomposed into four two-dimensional convolution integrals ri, i = 1, ., 4.Recall from the previous section that the support of scattering transform may be embedded in a disk of radius R. In the first step of the sinc-convolution algorithm the required bounds of two-dimensional convolution integral are determined as i, greater than one-fourth of the maximum amplitude:Preliminarily, a one-fourth amplitude set γq are computed and the distances from the medium center to them are calculated as rt and rq respectively. Then the following performance measuring parameters are calculated.A one-fourth threshold could guarantee to detect most of the visually significant effects in reconstructed conductivity images. The center of gravity of γ and γAt with conductivity σtin a medium with conductivity σr[• Amplitude response (AR) measures the ratio of image pixel amplitude in the target to that in the reconstructed image. For a circular target of area tivity σrIn this study, this parameter is normalized so that it AR = 1 for a circular target with • Position error (PE) represents the extent to which reconstructed image truly represents the position of the circular target in the medium. This parameter is computed as :C centered at center of gravity of γq, the ringing could be obtained by [• Ringing (RNG) measures the degree of opposite sign area surrounding the main reconstructed target area. For a circle ained by :• Resolution (RES) is a measure of the smallest visible object within the reconstructed image. This parameter is be defined as :Aq and A0 denote the number of pixels in γq and entire reconstructed image respectively.where q which did not fit within a circle of an equal area. This parameter is computed as [• Shape deformation (SD) measure quantifies the fraction of γputed as :C denotes a circle centered at COG of γq with an area equivalent to Aq.where All three methods were implemented within MATLAB and computations were performed in a Laptop with 2.4 GHZ CPU and 2 GB RAM. The methods were separately applied to the datasets extracted from aforementioned simulated and real models. To fairly compare the quality of reconstructed conductivity images, iteration parameters were set in a common range for all methods. In addition, same-size grids in k-plane were used in implementation of sinc-convolution and MG.The following two steps were used to evaluate the quality of sinc-convolution images.First, the synthetic reconstructions were evaluated via efficiency parameters of the preceding section. Next, reconstructions of physical and clinical models were used to validate the parametric assessments.The supremum of reconstruction errors and the required computation times for reconstructions of the synthetic chest phantom using MG and sinc-convolution with different levels of discretization in k-plane were measured according to equation (24) and then enlisted in third and fourth columns of Tables Comparing corresponding accuracies of the reconstruction methods, one can notice that in each case the accuracy of the sinc-convolution method is much better than that of the MG, especially in reconstructions with large grids in k-plane.Next, for each discretization level in Tables -3 in 1871 seconds, the MG can only achieve low accuracies not better than 10-2 in 3290 seconds, which is considered as a very poor performance. Now, it is predictable that to reconstruct higher resolution conductivity images in k-plane, the performance of the sinc-convolution would be finer than that of the MG.Moreover, observing the computation times of sinc-convolution and MG in the fourth column of Tables The plots in Figures • The amplitude response of all three methods increase from the center of medium toward the boundary. Remarkable oscillations appear in the amplitude response of MG and NOSER respectively when the target is in the midway point and closest point to the boundary. Despite these two methods, the amplitude response of sinc-convolution is approximately uniform. This consistency guarantees that the same value of conductivities in different parts of the body contribute equally to the conductivity images produced by sinc-convolution.• For position error, the plots show that when the target moves from the center to the boundary, the PE in MG, NOSER and sinc-convolution increases from −0.3, -0.1 and −0.1 to 0.2, 0.2, and 0.5 respectively. It is clear that the variance of PE in sinc-convolution curve is the closest one to zero. Therefore, the positions of objects are expected to be well recovered in the images reconstructed by sinc-convolution algorithm.• The ringing plots indicate that for all three reconstruction algorithms, this artifact is increased as the target moves from the center of medium toward the boundary. The curves show that, for each position of the target, the maximum RNG is found in the image reconstructed by NOSER.• Resolution plots show that the resolution of the NOSER and sinc-convolution are more uniform and considerably less than that of MG. It is clear that the RES of sinc-convolution is fractionally lower than NOSER. Therefore, one may expect to observe most of the conductivity details in sinc-convolution reconstructions.• Shape deformation plots show that the SD of the target in sinc-convolution reconstructions is considerably less than that in images produced by each of other two algorithms. The optimum points for shape deformation in all three methods are near the boundary electrodes.Aforementioned results evince the suitability of the sinc-convolution algorithm for experimental impedance imaging. In the following, reconstruction of experimental phantom tank via sinc-convolution is presented and compared with that of MG and NOSER.Figure The relative errors in reconstructing heart and lung, using under-test methods are enlisted in the second and third columns of Table Let define degree of truth (DT) of reconstructions as:γrec and γ respectively denote the reconstructed and true conductivity. For each reconstruction experiment in the first column of Table where It is clear that the representative results of this experiment in Figure • The overall size, position, and the orientation of the organs in the image produced by sinc-convolution are more accurate than that in Figures • The sinc-convolution image recovers the separation between the two lungs well while MG and NOSER images do not; MG algorithm overestimates that distance and NOSER underestimates it.• The distortion and blurring of the heart and lungs which are respectively evident in the MG and NOSER images are not appeared respectively in the sinc-convolution image..• The degree of ringing artifact in sinc-convolution image of Figure As can be seen, the representative results of this experiment agree very well with accuracy assessment plots in Figure Two-dimensional conductivity images of the spontaneously breathing neonate chest are reconstructed using all three methods. The results are depicted in Figure It is worth noting that tidal volumes in the neonate’s left lung were reported less than those in his right lung . That isNote that, since exact information about the conductivity distribution inside the neonate’s chest is not available, no parametric evaluation and comparison could be planned. However, the representative results of this experiment and their correspondence to parametric evaluations confirm the feasibility of precise clinical EIT reconstruction using sinc-convolution.The feasibility of accurate practical conductivity image reconstruction via use of sinc-convolution algorithm in D-bar framework was investigated in this study. In the meantime, the performance of this algorithm was compared with two practical methods including, multigrid and NOSER. In this regard, a two-fold scenario was employed. In the first step, the quality of sinc-convolution reconstructions from noisy boundary data collected on specific synthetic models were evaluated against GREIT agreed accuracy parameters. Results show that the amplitude response and resolution of images are relatively better in sinc-convolution reconstructions. In addition, the effect of the distortions like position error, ringing and shape deformation is considerably reduced in the images produced by sinc-convolution method. Moreover, comparing the convergence rate of the sinc-convolution with that of MG shows that the new sinc-convolution method is computationally more efficient than its D-bar based competitor.In the second step, conductivity images of an experimental phantom chest were reconstructed using all three methods. Excellent agreement between their qualities and parametric assessment results support the sinc-convolution suitability for experimental EIT. As a complementary experiment, two-dimensional conductivity images of the chest cross-section of a spontaneously breathing neonate were reconstructed using all three methods. A watchful comparison shows that the related physiological problem is best revealed in sinc-convolution images. In addition, position, size and orientation of organs are well recovered in sinc-convolution images.These reasons, suggest the sinc-convolution as an efficient algorithm for precise clinical EIT applications.The best constant conductivity approximation to the measured boundary data can be computed according to the following formula, which is found in ,21.ρ denote the resistivity , then for a medium of homogenous resistivity, the voltage on the l th electrode from the kth current pattern is proportional to the voltage arising from a constant distribution of one. That isLet Ulk} denote the set of measured voltage data and Vlk(ρ) the calculated voltage on the electrodes. To find the best constant resistivity fit to the data, one must solve the minimization problemLet {ρbest to this minimization problem is given byThe solution The best constant conductivity is then EIT: Electrical impedance tomography; PI: Product integral; MG: Multi-grid; CR: Convergence rate; DT: Degree of truth; AR: Amplitude response; PE: Position error; RES: Resolution; RNG: Ringing; SD: Shape deformation.The authors declare that they have no competing interests.MA designed and performed the experiments and numerical modeling; ARNN analyzed the experiments and numerical modeling. Both of authors read and approved the final manuscript.


The order of authors is incorrect. Please view the correct author order, order of affiliations and Citation below:1, Pengcheng Liu 1, Li Wei 2, Jinshen Wang 2, Jin Qi 2, Shengmei Feng 2, Lianfu Deng 2Ming Cai1 Department of Orthopaedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China2 Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases with Integrated Chinese-Western Medicine, Ruijin Hospital, Jiao Tong University School of Medicine, Shanghai, ChinaCai M, Liu P, Wei L, Wang J, Qi J, et al. (2014) Atp6v1c1 May Regulate Filament Actin Arrangement in Breast Cancer Cells. PLoS ONE 9(1): e84833. doi:10.1371/journal.pone.0084833


Escherichia coli that resides in the 5′ untranslated region of the alx gene and controls its translation in a pH-dependent manner. Under normal growth conditions, this RNA region forms a translationally inactive structure, but when transcribed under alkaline conditions, it forms an active structure producing the Alx protein. We identified two distinct transcriptional pause sites and proposed that pausing at these sites interfered with the formation of the inactive structure while facilitating folding of the active one. Alkali increases the longevity of pausing at these sites, thereby promoting folding of the translationally active form of alx RNA. We show here that mutations that modify the extent and/or position of pausing, although silent with regard to structure stability per se, greatly influence the dynamics of folding and thereby translation. Our data illustrate the mechanistic design of alx regulation, relying on precise temporal and spatial characteristics. We propose that this unique design provides an opportunity for environmental signals such as pH to introduce structural changes in the RNA and thereby modulate expression.Previously, we described a novel pH-responsive RNA element in  RNA conformation plays a major role in processes such as transcription termination, RNA processing and translation initiation. Hence, the folding of RNA is an important determinant in regulation of gene expression in many cellular systems, in both prokaryotes and eukaryotes. Furthermore, the structure of RNAs may change in response to environmental signals. Thus, understanding the mechanisms by which RNAs assemble to form active units is of considerable interest.Escherichia coli non-coding RNAs, including signal recognition particle (SRP), RNase P and transfer-messenger RNA (tmRNA) (An increasing body of evidence suggests that much of the regulation of gene expression that is based on RNA folding is accomplished during the elongation phase of transcription and involves pausing of RNA polymerase (RNAP) . Pausing (tmRNA) . Importa (tmRNA) .alx gene, encoding a putative transporter, and controls its translation in a pH-dependent manner. In cells grown under neutral conditions, the PRE-alx transcript forms a translationally inactive structure, whereas growth under high pH results in the formation of an active structure and production of Alx  precedes the n of Alx . The pH-te PC in , RNAP sete PC in . Once foEscherichia coli MC4100 cells were grown at 37°C (200 rpm) in Luria-Bertani broth (LB) medium (pH 6.8) or in potassium modified LB medium  ( pH 8.4) . Ampicilalx-PRE-alx'-'lacZ , PT7-PRE-alx' in pGEM4 (pSA66), Palx-PRE-alx' in pGEM4 (pSA64) and Palx-PRE-alx' in pZE12 (pSA65) has been described previously  as template and two tail-to-tail divergent primers (5′ phosphorylated) of which one carried the desired mutation. The PCR product was subjected to blunt-end ligation and then used as template to amplify the desired fragment for sub-cloning. To construct Palx-PRE-alx' deleted for bases 12–91 of PRE (pSA64 Δ12–91), divergent primers spaced by the site of the deletion were used instead of tail to tail. Mutant A137G was constructed in two steps using overlapping divergent primers, both carrying the desired mutation. The first two PCR reactions contained each, one of the primers, the corresponding fragment end primer used to generate the wild-type fragment and plasmid carrying Palx-PRE-alx'-'lacZ (pSA60) as template. The products from these two reactions were used as template in a second PCR reaction containing the fragment end primers used to generate the wild-type fragment. The resulting fragment was then sub-cloned.Mutants A600 of 0.4. β-galactosidase activity was assayed as described (Overnight cultures were diluted 1:100 in fresh LB or LBK medium and grown to ODescribed .RNA was synthesized from pSA66 (wild-type and mutants) and probed as described previously .RNA synthesized from pSA66 (wild-type and mutants) was incubated for 30 min at 22°C and then analyzed on 5% non-denaturing polyacrylamide gels as described previously .E. coli RNAP  at pH 7.2 in 50 µl reactions containing 20 mM Tris–HCl, 150 mM KCl, 10 mM MgCl2, 5 mM dithiothreitol (DTT), NTPs (250 µM each), 20 units RNase inhibitor (Takara Bio) and 2.5 µg of plasmid DNA carrying Palx-PRE-alx' . The mixture was preincubated for 5 min at 37°C before the addition of enzyme. After 5 min of synthesis at 37°C, 1.7 µl of dimethyl sulfate (DMS) (diluted 1:10 in ethanol) was added. The reactions were stopped 5 min after the addition of DMS by phenol/chloroform extraction followed by ethanol precipitation in the presence of 0.3 M sodium acetate and 1 µl of Quick-Precip (Edge BioSystems). To detect the modified sites, the treated RNA was annealed with 0.6 pmol end-labeled primer  and then subjected to primer extension (at 43°C for 60 min) in 15 µl reactions containing 0.5% (v/v) Tween 20, 50 mM Tris–HCl (pH 8.3), 40 mM KCl, 5 mM MgCl2, 10 mM DTT, deoxynucleotide triphosphate (dNTPs) (1 mM each) and 25 units Expand reverse transcriptase (Roche). The extension products were separated on 6% polyacrylamide, 7.8 M urea gels, alongside sequencing reactions.RNA was synthesized using alx-PRE-alx'  was incubated at 37°C for 10 min in the presence of 10 µCi of α-32 P-ATP, 20 mM Tris–HCl, 150 mM KCl, 10 mM MgCl2, 2.77 mM DTT, 1 µM ATP, 2.5 µM GTP, 20 µg/ml BSA, 150 µM GpC dinucleotide (Sigma), glycerol to 3.33% (v/v) (including glycerol of the enzyme storage buffer) and 0.5 unit E. coli RNAP in a 45-µl volume. Thereafter, aliquots of 9 µl were transferred to pre-warmed tubes, and transcription was resumed by adding 1 µl of pre-warmed solution containing NTPs (2.5 mM each), 20 mM Tris–HCl, 150 mM KCl, 10 mM MgCl2 and 150 µg/ml rifampicin (to block re-initiation). The reactions were stopped by freezing in liquid nitrogen. Single round transcription reactions with PCR generated templates were performed similarly, with the following modifications—reactions were conducted at 30°C  or 25°C  or 20 min . Band intensities were measured using the Quantity One program (Bio-Rad).Single round transcription reactions were performed at pH 7.2 as described in  with sli or 25°C b, the inr 20 min b and 225alx' RNA (wild-type and mutants) was probed in vivo in LB medium as described previously for pSA65  upstream of the first pause site, which were expected to affect pausing efficiency and/or position. The mutations designed were assumed to have a minimal effect, or none at all, on the stability of either the active or the inactive structure . As single point mutations can perturb separation that is based on RNA length, we estimated the position of pausing by using suitable RNA size markers, each carrying the corresponding point mutation. This mapping shows that mutations A137G and C135U shift the location of pause C two to three nucleotides forward, relative to wild-type, whereas in mutant A132G, the natural position of pause C is maintained . The data showing that shifting the site forward is accompanied by a rise in alx translation endorse the mechanistic perception we previously suggested for alx regulation. Given that typically 12 ± 1 nucleotides of the nascent RNA are sequestered within the transcription elongation complex . These results suggest that an increase in the rate of pausing by RNAP accompanied by a substantially slower pause escape increase the proportion of transcripts in which the formation of stem C is inhibited, thereby allowing the formation of the translationally active structure. Pause extent measurements of mutants A137G and C135U show that these mutants exhibit pause characteristics similar to wild-type . The calculated ratios in A132G, C135U and A137G RNAs are 2.8, 3.4 and 4.1, respectively, whereas wild-type RNA exhibits a ratio of 1.8. On the basis of these measurements, the active to inactive ratios of these high-level expression mutants are higher than that of wild-type RNA. Our results show that shifting the pause site farther from hairpin S or increasing the extent of pausing leads to an increase in the formation of the alternative, translationally active structure, correlating with the rise in expression observed in vivo.The differences in RNA folding caused by the changes in transcriptional pausing were investigated in assays of cotranscriptional structure probing. In these assays, the structures of the different mutant RNAs were determined using DMS during synthesis with oli RNAP . To learin vitro with RNA folding occurring in vivo, we compared the in vivo RNA structure of the mutants with wild-type. Notably, position A137 in mutant C135U is modified in vivo in contrast to the in vitro assays. The in vivo probing shows that the modification ratios of A137/A131 and A153/C155 increase . Conversely, cotranscriptional binding of FMN to ribD RNA results in formation of a transcription terminator. Two pause sites located between the aptamer and the expression platform delay the regulatory decision, extending the time frame for FMN binding , Chromatin and RNA [1796/12]. Funding for open access charge: The Israel Science Foundation founded by The Israel Academy of Sciences and Humanities [911/09] and Deutsche Forschungsgemeinschaft research grant [VO 875/5-1].This work was supported by the Israel Science Foundation founded by Conflict of interest statement. None declared.


Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.Respiratory syncytial virus (RSV) is an RNA virus in the Family  Respiratory syncytial virus (RSV) is a major pathogen of infants with the potential to cause severe respiratory disease. RSV has an RNA genome and one approach to developing a drug against this virus is to gain a greater understanding of the mechanisms used by the viral polymerase to generate new RNA. In this study we developed a novel assay for examining how the RSV polymerase interacts with a specific promoter sequence at the end of an RNA template, and performed analysis of RSV RNA produced in infected cells to confirm the findings. Our experiments showed that the behavior of the polymerase on the promoter was surprisingly complex. We found that not only could the polymerase initiate synthesis of progeny genome RNA from an initiation site at the end of the template, but it could also generate another small RNA from a second initiation site. In addition, we showed that the polymerase could add additional RNA sequence to the template promoter, which affected its ability to initiate RNA synthesis. These findings extend our understanding of the functions of the promoter, and suggest a mechanism by which RNA synthesis from the promoter is regulated. Respiratory syncytial virus (RSV) is the major cause of respiratory tract disease in infants and young children worldwide, causing 3.4 million cases of severe acute lower respiratory infection, and between 66,000 and 199,000 deaths per annum Mononegavirales, Family Paramyxoviridae. In general terms, RSV shares the strategy for gene expression and genome replication that is used by all non-segmented negative strand (NNS) RNA viruses RSV has a single stranded, negative sense RNA genome and is classified in the Order in vitro assays that reconstitute RNA synthesis using purified components e.g. in vitro for the rhabdoviruses in vitro have been unsuccessful. However, available data indicate that the N protein must be locally and transiently displaced to allow the RdRp to engage the RSV RNA template in its active site Although the promoter regions of RSV and other NNS RNA viruses have been thoroughly mapped and the viral proteins involved in RNA synthesis have been identified, a detailed understanding of the molecular mechanisms underlying transcription and genome replication initiation lags significantly behind that of other RNA viruses. In part, this is due to the lack of tractable assays for studying polymerase behavior. Although the minigenome system is a valuable tool, because it is an intracellular assay it is largely limited to studying the final, stable products of these processes, and cannot be used to examine unstable RNA intermediates. It is also not possible to manipulate intracellular conditions to isolate specific steps in RNA synthesis initiation. In addition, from a drug discovery perspective, it is an expensive and time-consuming assay that is not readily applicable to a high-throughput screening approach. Study of positive strand RNA viruses has been helped enormously by the development of in vitro using isolated components. To date, the only recombinant NNS virus RdRps that have been expressed and purified in functional form are those of VSV, Chandipura and Sendai virus in vitro RNA synthesis assay described here is not yet known. Because the L/P preparations were not completely pure, and because there was variation in the relative levels of full-length and truncated L proteins, the experiments described in To enable detailed analysis of the mechanisms involved in RSV RNA synthesis initiation, an assay was developed in which RSV RNA synthesis was reconstituted 32P]ATP label. Although the M2-1 protein has been shown to bind P and RNA and affect transcription of mRNAs longer than ∼200 nts, it was not included in these experiments because it has been shown to have no effect on either transcription or replication initiation N812ATo determine if the isolated RdRp was capable of performing RNA synthesis on a naked RNA template, L/P complexes were incubated with an RNA oligonucleotide representing the 3′ terminal 25 nts of the TrC promoter  in the p32P]GTP rather than [α-32P]ATP. In this case, the in vitro RNA synthesis reaction also resulted in products of 8–10 and 21 nts in length  in place of the 3′ hydroxyl group was tested in a reaction containing all four NTPs. The presence of 3′ PMN should abrogate 3′ terminal nt addition, while not preventing the ability of the RdRp to use the RNA as a template. The 3′ PMN TrC RNA generated significant levels of the RNAs≤23 nts, but the 26–28 nt RNA products were not detected , the RNA synthesis reaction was performed without UTP. As shown in 32P]ATP or [α-32P]GTP as a label (32P]ATP (32P]ATP label). The presence of these bands suggested that either the NTP stocks were impure, or that the RdRp had poor fidelity in this assay, allowing it to insert an alternative NTP instead of UTP. Products less than 11 nts in length could also be detected, but their abundance was not affected by the presence or absence of UTP, indicating that these were premature termination products, rather than RNA initiated from downstream sites . In contrast, RNA products labeled with [γ-32P]GTP were readily detected. In reactions lacking UTP, a product of 11 nts could be detected . In back-priming, the 3′ end of the RNA interacts with an internal sequence to form a hairpin structure, and the RdRp adds nts to the 3′ terminus using the folded RNA as a template in vitro with some specificity, it was of interest to determine if this occurred during RSV infection. In the context of an RSV infection, the TrC sequence is at the 3′ end of the antigenome. To our knowledge, no one has previously identified additional sequences at the 3′ terminus of the RSV antigenome. However, antigenome 3′ terminal sequences are rarely determined directly, but instead are inferred from the genome sequence Having shown that nts were added to the 3′ end of the TrC RNA in vitro RNA synthesis assay. Both RNA templates contained a 3′ terminal PMN group to ensure that neither was subject to further 3′ modification. Analysis of the RNA generated from these templates showed that the presence of a 3′ terminal CUG extension was highly deleterious to RNA synthesis, indicating that the 3′ extension inhibited access of the RdRp to the promoter (32P]ATP label into RNA synthesized from the +CUG template (data not shown). Thus, these data indicate that the 3′ terminal extension can inhibit antigenome promoter activity.The presence of additional sequence at the 3′ end of almost half of the antigenome sequences that we examined indicated that the 3′ extension plays a role in RSV replication. The only known function of antigenome RNA is as a template for RNA synthesis. Therefore, we examined if the additional nts at the 3′ end of the TrC sequence affected promoter activity. The 1–25 TrC RNA template was compared to a “+CUG” RNA template, which contained 1–25 nts of TrC sequence and a 3′ CUG extension, using the de novo RNA synthesis from the TrC promoter, and 3′ extension of the RNA by a back-priming mechanism. Analysis of RNA isolated from RSV infected cells provided confirmatory evidence that there is a +3 initiation site within the TrC promoter, and the antigenome RNA can be modified by 3′ extension. These findings suggest that the interactions between the RSV RdRp and the promoter are more complex than previously thought. They also demonstrate clear differences between RSV and the prototype NNS RNA virus, VSV, but a surprising similarity with the more distantly related Borna disease virus, which has also been shown to elongate the 3′ ends of replicative RNAs Mononegavirales, the details of these processes should be considered on a case-by-case basis for each virus.This is the first report of purification of functional RSV polymerase from a recombinant source and the first time that an assay of this kind has been applied to study initiation of paramyxovirus RNA synthesis. Experiments with this assay revealed that the RSV RdRp is capable of two different RNA synthesis activities: initiation of It is accepted that normally the RSV template RNA is encapsidated with N protein. However, the fact that our experiments showed that the RSV polymerase was able to recognize the RNA in a sequence specific manner in the absence of N, and modify the TrC RNA apparently by using an RNA secondary structure, reveals insight into the molecular details of the polymerase-template complex. These findings suggest that although the antigenome RNA is normally encapsidated with N protein, there are occasions during the RSV replication cycle when the RdRp can interact with the RNA directly. The ability of the RSV RdRp to recognize the promoter in the absence of N protein is not necessarily surprising, as prior studies have shown that there is no requirement for the RSV promoter to be in phase with N protein The TrC promoter would be expected to direct RNA synthesis initiation from the +1 position, to yield the genome RNA, and RNA initiated from +1 could be readily detected in RSV infected cells. However, RNA initiated at +3 was also detected. Primer extension analysis showed that this RNA was truncated within a short distance from the promoter and consistent with this, RNA transcripts of 21–25 nts in length could be readily detected in infected cells. The function of the small RNA initiated from +3 is not yet known. However, previous studies suggest that Tr-specific RNA might play a role in subverting the cellular stress granule response de novo initiation mechanism, and apparently does not depend on prior initiation at +1 . However, it is possible that repair can occur through a back-priming mechanism in a cellular environment. Finally, it is possible that the 3′ terminal additions are part of a regulatory mechanism to modulate promoter activity. Consistent with this idea, the data shown in in vitro assay. It is important to note that in this assay the RNA was naked, and so the effect of the 3′ extension might not reflect the situation in RSV infected cells. For example, in the in vitro assay, the 3′ extension is likely to have created an RNA secondary structure that prevented access of the RdRp to the promoter. In contrast, in infected cells the antigenome RNA would be expected to become completely encapsidated in N protein, eliminating RNA secondary structure. Comparison of wt and mutant templates containing the extensions using the minigenome system (in which the template RNA does become encapsidated) indicated that, the 3′ terminal UG and CUG additions slightly increased RNA synthesis from both the +1 and +3 positions, while a G extension had no effect (data not shown). However, we also found that the 3′ termini of the input wt and mutant minigenome templates had the potential to be modified in the transfected cells, and so it is not clear how much weight can be attributed to these results. Nonetheless, it is interesting to speculate that the ability of the RdRp to add nts to the antigenome terminus and the effect of the nt extension are linked by the encapsidation status of the antigenome RNA. For example, one possibility is that 3′ terminal extension occurs when encapsidation of the newly synthesized antigenome RNA lags behind RNA synthesis. In this scenario, the putative hairpin structure formed by 3′ terminal extension might function to prevent initiation of de novo RNA synthesis until the antigenome RNA becomes fully encapsidated, at which point it might become an even more efficient promoter.It remains somewhat unclear what role the 3′ terminal extension plays in RSV replication. Unfortunately, because of the multifunctional nature of the TrC promoter in directing RNA synthesis and encapsidation, it is probably not possible to generate a mutant virus in which the 3′ terminal extension activity is ablated without also affecting other aspects of genome replication. Therefore, we can only speculate on the significance of antigenome 3′ terminal extension during RSV infection. In the case of Borna Disease virus, the function of the additional nts at the genome and antigenome ends is to compensate for cleavage of the 5′ ends of replication products, which allows the virus to avoid detection by RIG I In summary, the findings presented here indicate that the behavior of the RSV RdRp at the TrC promoter sequence is more complex than previously realized, directing initiation of RNA synthesis from two sites, and having the capacity to add nts to the 3′ end of the antigenome template by a back-priming mechanism. We speculate that the advantage of having greater complexity in polymerase-promoter interactions is that it might offer an opportunity for temporal or environmental regulation of RSV RNA expression.N812A was generated by QuickChange site-directed mutagenesis (Aligent Technologies). RSV L or LN812A, were cloned in the pFastBac Dual vector (Invitrogen) together with the RSV A2 P ORF, which was tagged with a hexahistidine sequence, separated from the ORF by a tobacco etch virus (TEV) protease cleavage site. Recombinant baculoviruses were recovered using the Bac-To-Bac system (Invitrogen) and used to infect Sf21 cells in suspension culture. RSV L/P protein complexes were isolated from cell lysates by affinity chromatography, TEV protease cleavage, and size exclusion. Isolated L/P complexes were analyzed by SDS-PAGE and PageBlue staining (Fermentas) and the L protein concentration was estimated against bovine serum albumin reference standards. The bands migrating between 160 to 250 kDa and ∼35 and 50 kDa were confirmed to be RSV L and P polypeptides by excising each band and performing trypsin digestion followed by liquid chromatography, tandem mass spectrometry (LC/MS/MS). The MS/MS spectra were analyzed using SEQUEST software using the RSV A2 sequence as a reference. The identity of a band migrating between 70 and 80 kDa was determined by performing SDS-PAGE and Western blot analysis using an hsp70/HSC70 specific antibody (Santa Cruz).A codon-optimized version of the RSV (strain A2) L protein ORF was chemically synthesized (GeneArt) and the mutant LN812A ORFs were introduced under the control of the T7 promoter in pTM1. Minigenome RNA analysis was performed using plasmid MP-28, which encodes a replication competent dicistronic CAT minigenome template Codon optimized wt and L2, 5 mM DTT, 40 U RNase inhibitor, and NTPs, including either 1 µl of [α-32P]ATP, CTP, GTP or UTP (as indicated in the legend) or [γ-32P]GTP (∼10 µCi), in a final volume of 50 µl. The RNA and NTP concentrations used for each experiment are indicated in the figure legends. Reactions were incubated at 30°C for 3 h, heated to 90°C for 3 min to inactivate the RdRp and cooled briefly on ice. Reactions containing [α-32P]NTP were combined with 10U calf intestinal alkaline phosphatase, incubated at 37°C for 1 h and RNA was isolated by phenol-chloroform extraction and ethanol precipitation. Reactions containing [γ-32P]GTP were combined with 7.5 µl 10% SDS, 0.5 µl 500 mM EDTA and 10 µg proteinase K and incubated at 45°C for 45 min before phenol-chloroform extraction and ethanol precipitation. The RNA was analyzed by electrophoresis on a 20% polyacrylamide gel containing 7 M urea in tris-borate-EDTA buffer, followed by autoradiography. On each autoradiogram, the nt lengths of the RNA products were determined by comparison with a molecular weight ladder generated by alkali hydrolysis of a 32P end-labeled RNA oligonucleotide representing the anticipated 25 nt Tr RNA product. This marker is shown in RNA oligonucleotides representing nts 1–25 of either wt or mutant TrC promoter sequence or its complement (Tr sequence) were purchased (Dharmacon). All oligonucleotides contained an –OH group at the 3′ terminus, unless stated otherwise, and an –OH group at the 5′ terminus. The RNA oligonucleotides were combined with RSV L/P (containing ∼100 ng of L protein) in transcription buffer containing 50 mM Tris HCl at pH 7.4, 50 mM NaCl, 5 mM MgCl32P end labeled DNA oligonucleotides of sequence and length equivalent to cDNAs corresponding to RNAs initiated at positions +1 and +3 on the antigenome. The method for detecting small genome sense RNA by Northern blot analysis was adapted from a protocol described by Varallyay and co-workers 32P end-labeled locked nucleic acid modified DNA oligonucleotide specific to nts 5–32 (relative to the 5′ terminus) of genome sense RNA (5′- GAGATATTAGTTTTTGACACTTTTTTTC - 3′) in the same buffer at 62°C. Blots were washed with 6× SSC twice for 15 minutes at room temperature, and twice for 10 minutes at 62°C, and the RNA was detected by autoradiography.HEp-2 cells were infected with RSV at an MOI of 5 or mock infected and incubated at 37°C for 17 h. Total intracellular RNA was isolated using Trizol (Invitrogen). Primer extension reactions were carried out as described previously 6 HEp2 cells were infected at an MOI of 5. At 17 h post infection, the supernatant was replaced with media containing 2 µg/ml actinomycin D and cells were incubated at 37°C for a further 1 h. Following an ice cold PBS wash, cells were treated for 1 min with PBS supplemented with 250 µg/ml lyso-lecithin, on ice. Cells were scraped into 400 µl of ice cold Buffer A , disrupted by repeated passage through an 18G needle and incubated on ice for 10 min. Following centrifugation at 2400× g for 10 min at 4°C, the resulting pellet was disrupted in 200 µl of ice cold Buffer B  by repeated passage through an 18G needle and then incubated on ice for 10 min. The sample was centrifuged and the resulting pellet was disrupted in 200 µl of Buffer B supplemented with 0.5% deoxycholate, 1% tween 40 as described above. Following a 10 min incubation on ice and a repeat centrifugation, the supernatant enriched for viral RNPs was collected and RNA was extracted using Trizol (Invitrogen). The purified RNA was tailed with either A or C residues using E. coli poly A polymerase (NEB), followed by heat inactivation of the enzyme, according manufacturer's instructions. First strand cDNA synthesis was performed using primers 5′ GAGGACTCGAGCTCAAGCATGCATTTTTTTTTTTTTTT, or 5′ GAGGACTCGAGCTCAAGCATGCATGGGGGGGGGGGGGGG, which hybridized to the poly A or poly C tail, respectively, and Sensiscript reverse transcriptase (Qiagen), according to manufacturer's instructions. To determine the sequence of the antigenome 3′ terminus, purified cDNA was PCR amplified using primer SLNQi 5′-GAGGACTCGAGCTCAAGC and a TrC specific primer Tr1: 5′-GCAGCACTTTTAGTGAACTAATCC. The resulting product was subjected to a second round of hemi-nested PCR using primer SLNQi and primer Tr2: 5′-GCAGTCGACCATTTTAATCTTGGAG. PCR products were gel purified and either sequenced directly or cloned into a pGEM vector for sequencing of individual cDNA clones. Analysis of the genome 3′ terminus was performed as described above, using the same cDNA preparation and primer SLNQi, but with NS1 specific primers: 5′-GCACAAACACAATGCCATTC and 5′-GCAGTCGACGTATGTATCACTGCCTTAGCC.3′ RACE and sequence analysis of antigenome and genome RNA  was perf


This review aimed to answer whether the vasopressors are useful at the early phase of hemorrhagic shock. Data were taken from published experimental studies and clinical trials. Published case reports were discarded. A search of electronic database PubMed was conducted using keywords of hemorrhagic shock, vasopressors, vasoconstrictors, norepinephrine, epinephrine, vasopressin. The redundant papers were not included. We identified 15 experimental studies that compared hemorrhagic shock resuscitated with or without vasopressors, three retrospective clinical studies, and one controlled trial. The experimental and clinical studies are discussed in the clinical context, and their strengths as well as limitations are highlighted. There is a strong rationale for a vasopressor support in severe hemorrhagic shock. However, this should be tempered by the risk of excessive vasoconstriction during such hypovolemic state. The experimental models must be analyzed within their own limits and cannot be directly translated into clinical practice. In addition, because of many biases, the results of clinical trials are debatable. Therefore, based on current information, further clinical trials comparing early vasopressor support plus fluid resuscitation versus fluid resuscitation alone are warranted. The use of vasopressors, such as norepinephrine for the hemodynamic management of hemorrhagic shock, may be considered in the early phase of resuscitation and is a common practice among several prehospital and hospital emergency teams in Europe except in the United Kingdom . NeverthPublished experimental studies and clinical trials. Published case reports were discarded.A search of the electronic database PubMed was conducted using the keywords of hemorrhagic shock, vasopressors, vasoconstrictors, norepinephrine, epinephrine, and vasopressin. The redundant papers were not included.We identified 15 experimental studies that compared hemorrhagic shock resuscitated with or without vasopressors, three retrospective clinical studies, and one controlled trial. Given the great heterogeneity in the already reported studies, it was not possible to perform a formal metaanalysis. Therefore, experimental and clinical studies are discussed in the clinical context and their strengths as well as limitations are highlighted.a fortiori in the case of cardiac arrest, the usefulness of rapid vasopressor injection to restore arterial blood pressure and redirect cardiac output toward vital organs is undisputable. Well before this phase of extreme exsanguination, animal models show that anesthetic agents exert a significant effect on the vasoconstrictive response to hemorrhage and that stable and normal blood pressure values seen in conscious animals are no longer observed in anesthetized animals following withdrawal of similar blood volumes [The early phase of hemorrhage is characterized by sympathetic system activation resulting in compensatory venous and arterial vasoconstriction, aimed at normalizing arterial blood pressure . Initial volumes . In this volumes . In addi volumes . Blood w volumes  and oxid volumes . Thus, i volumes . TherefoIn addition to their usefulness in the case of vasodilatation or insufficient vasoconstriction, vasopressors may be useful to restore hemodynamic parameters along with adequate vital organ infusion, thereby reducing the need for continuous fluid infusion, which may result in side-effects, such as tissue edema. The occurrence of a systemic inflammatory response, in particular an acute respiratory distress syndrome (ARDS), appears to be the primary cause of death on Day 3 in trauma patients . In a seLastly, in the case of abdominal lesions, early vasopressor infusion allowing portal output to decrease via splanchnic vasoconstriction (this effect being particularly pronounced following vasopressin use) is likely to result in decreased hemorrhagic loss from splanchnic blood vessels, while maintaining adequate infusion of other organs.The question raised regarding vasopressor support for hemorrhagic shock is perhaps somewhat simplified, because it places all trauma and even all polytrauma patients under the same label of hemorrhagic shock. Shock in polytrauma patients is likely to be very different from single, hemorrhagic lesion. In these cases, the problem is not only hemorrhage, but also the multiple tissue injuries associated with prolonged shock, due to both hypovolemia as well as alveolar and tissue hypoxia, which subsequently trigger a hyperinflammatory response ,17. In tAlthough theoretically tempting, these concepts arguing for vasopressor support for hemorrhagic shock are only speculative, and animal and human studies must be further scrutinized to determine whether vasopressor agents are efficacious and at the same time devoid of detrimental ischemic side-effects.. compared crystalloid administration alone versus crystalloid + phenylephrine or vasopressin [In animal models, when given at the early stage, vasopressors were shown to be capable of restoring a quasi-normal hemodynamic status by mobilizing nonconstraint venous blood volume and thus normalizing arterial blood pressure, cardiac output, and preload parameters, such as pulse pressure variations . Howeveropressin . Vasopreopressin . As the opressin ,27, thisopressin . These lopressin , the conIn experimental models of massive hemorrhage, several recent studies reported that vasopressin infusion was associated with survival benefits compared to fluid administration alone or to other vasopressor agents (epinephrine or norepinephrine) -33. ThesDespite the use of clinical relevant models of hemorrhagic shock , the exp = 0.04) with a mortality rate at day 5 of 25% versus 13%, respectively (p = 0.19) [To date, no clinical prospective study conducted in the hemorrhagic shock or traumatology setting compared different therapeutic strategies with and without vasopressor use. One, single, descriptive study reported on dopamine use in a general care protocol for polytrauma patients with life-threatening hemorrhage from pelvic fractures associating early arteriography ± embolization along with vasopressor treatment initiated within the first hour of hospital admission . When co = 0.19) .Despite numerous theoretical arguments in favor of the relatively early vasopressor use in association with fluid infusion in hemorrhagic shock management, there is still insufficient clinical evidence to validate this strategy. Experimental data available to date clearly indicate that vasopressor support cannot be replaced by fluid loading. In contrast, when given in association with fluids, the use of vasopressor agents appears to offer advantages, at least in some experimental models. Nevertheless, the type of vasopressor and the precise timing still need to be defined. Lastly, no clinical studies have validated any vasopressor support for the management of hemorrhagic shock. The use of norepinephrine advocated by some teams appears reasonable, but it must be kept in mind that this recommendation is entirely based on expert opinions ,39. The NCT00379522), may provide an answer.Following this discussion on vasopressor support, one should acknowledge that fluid loading is the first step to be considered in the management of hemorrhagic shock. Based on current information, further clinical studies that are designed to compare early vasopressor support plus fluid resuscitation versus fluid resuscitation alone are warranted. In this context, the prospective European study conducted to assess the impact of vasopressin infusion as a salvage therapy in prehospital hemorrhagic shock that persists despite standard treatment, including a first line vasopressor (Vasopressin In Traumatic Shock (VITRIS) trial, The authors declare that they have no competing interests.All authors contributed to the drafting of the manuscript and approved the final version.


Escherichia coli. The foundation of this sensor is carbon nanotubes decorated with gold nanoparticles that are modified with a specific, surface adherent ribonucleiuc acid (RNA) sequence element. The multi-step sensor assembly was accomplished by growing carbon nanotubes on a graphite substrate, the direct synthesis of gold nanoparticles on the nanotube surface, and the attachment of thiolated RNA to the bound nanoparticles. The application of the compounded nano-materials for sensor development has the distinct advantage of retaining the electrical behavior property of carbon nanotubes and, through the gold nanoparticles, incorporating an increased surface area for additional analyte attachment sites, thus increasing sensitivity. We successfully demonstrated that the coating of gold nanoparticles with a selective RNA sequence increased the capture of E. coli by 189% when compared to uncoated particles. The approach to sensor formation detailed in this study illustrates the great potential of unique composite structures in the development of a multi-array, electrochemical sensor for the fast and sensitive detection of pathogens.This study examines the creation of a nano-featured biosensor platform designed for the rapid and selective detection of the bacterium  In an eE. coli and 40,000 Salmonella infection cases were reported; demonstrating a clear justification for developing a means of pathogen detection. As current pathogen detection and identification procedures require approximately 48 hours, there is a critical need for earlier detection of pathogens, before they can rapidly spread and induce harmful effects. Using Fourier Transform Infrared Spectroscopy, a new technique has been developed to detect the presence of E. coli within 6 hours [E. coli. Breakthrough advances are needed to further reduce these detection windows to an appropriate microbial scale, and expand sensor capabilities to include additional classes and orders of pathogens.As the field of sensor technology has developed, the need for a whole-cell, real-time mechanism for the detection of specific bacteria has emerged. In 2010 alone, according to the Centers for Disease Control (CDC) and Prevention, over 70,000 Salmonella [E. coli O157:H7 strains [in vivo system for generation and their restriction to only working under physiological conditions. Recently, aptamers have emerged as a more advantageous target recognition agent when compared to antibodies [One such technological advancement is the utilization of cell specific molecular recognition systems for detection, a field that has been rapidly evolving. Previous studies have successfully demonstrated increased identification of lmonella  and E. c strains ,11 throutibodies . Aptamertibodies  and havetibodies .E. coli strain, as demonstrated by So and colleagues [E. coli to the RNA coated gold nanoparticles with high affinity and laid the groundwork for future biosensor development from this established composite model. Furthermore, the inclusion of additional capture elements targeted to sense different pathogens could easily be integrated to produce a multi-arrayed sensor.The goal of this study was to successfully conjugate a pathogen specific aptamer with a novel base material in an effort to develop a unique bacterium targeted biosensor. A platform base of CNTs was selected as they have previously been utilized in electrochemical sensor formation for biomolecules , due to lleagues . This st2.2.1.E. coli bacterial strain was obtained from ATCC. Cultures were inoculated by the addition of 10 μL of frozen stock solution to 6 mL of sterile Luria Bertani (LB) broth, comprised of 20 g of powder dissolved in 1 L of water. The cells were then incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. The next day, the cells were washed by centrifugation and re-suspended in phosphate buffered saline (PBS) at a final concentration of 106 cells/mL.Unless otherwise noted all chemicals were purchased from Fisher Scientific. DH5α 2.2.Highly oriented pyrolitic graphite sheets (Molecular Imaging) were cut and sanded into rectangles to serve as the platform base. A nano-hair type layer of strongly grafted CNT was fabricated on these surfaces through a two-step process  and used2.3.4  solution and an additional minute of stirring. Next, 5 mL of 0.1% sodium citrate (Na3C6H5O7) solution and 0.01% sodium borohydride (NaBH4) were introduced, which instantly turned the solution from pale yellow to maroon. This solution was continuously stirred for 2 minutes to allow freshly synthesized NPs on CNTs to stabilize. The solution was then removed and the samples, still attached to the side of the beaker, were rinsed with distilled water. After washing and air drying, the samples underwent SEM and energy dispersive spectroscopy (EDS) spectra analysis to verify gold nanoparticle synthesis.The resultant CNT/graphite composites were cut into smaller pieces and adhered to the side of a beaker using double sided carbon tape. This allowed for the CNTs to be fully immersed within the solution without risk of contact and damage by the magnetic stir bar. For gold nanoparticle synthesis, 43.75 mL of water was stirred for 3 minutes followed by the addition of 3 mL of a 0.4% HAuCl2.4.The surface-specific RNA sequence used was based off of a sequence discovered by So and colleagues  using a TAT AGT GAG TCG TAT TA – 3′SS-DNA: 5′ - TAA TAC GAC TCA CTA TAG GGA GA GGG AGA GCG GAA GCG TGC TGG GTC GCA GTT TGC GCG CGT TCC AAG TTC TCT CAT CAC GGA ATA CAT AAC CCA GAG GTC GAT GGG GGG GGG GGG – 3′LS-DNA: 5′ - ® T7 Transcription Kit containing 2′-F-UTP and 2′-F-CTP (Epicentre Biotechnologies) at 37 °C for 6 hours and stored at −20 °C to prevent degradation.In brief, SS-DNA and LS-DNA were combined and annealed at 94 °C for 2 min then transcribed using a DurascribeAfter successful DNA transcription, a thiol group was hybridized on the end of the RNA, which facilitated its attachment to the gold nanoparticles. This is due to the high affinity thiol has to gold and was accomplished through a compliment nucleotide hybridization of cytosine and guanine. A 12 cytosine chain containing a thiol group on its end was annealed at 94 °C for 2 minutes to the 12 guanine spacer on the RNA transcribed product sequence. For verification of successful transcription as well as concentration determination of the RNA, absorbance at 260 nm was recorded using a NanoDrop spectrophotometer.2.5.10 M−1·cm−1, l = 1 cm)Prior to introduction on the final platform, RNA was first attached to the colloidal gold nanoparticles in solution. These particles were synthesized using the same process as stated earlier on the carbon nanotube samples. After synthesis, the particle concentration was determined using UV-Vis analysis and the Beer-Lambert Law (A = εcl), where A = absorbance in optical density units, ε = exstinction coefficient (at the surface plasmon wavelength), l = path length through sample, and c = concentration of nanoparticles.  was added to the mixture and stirred for 20 minutes. Additional NaCl (40 μL of 2M) was then added to the solution and stirred for another 20 minutes. This was repeated a final time and left to incubate overnight protected from light. The next day the particles are centrifuged at 12,000 × g for 15 minutes and re-suspended in 100 μL of a 100 mM NaCl and 25 mM tris acetate solution. Lastly, the particles were washed two additional times. This process was conducted on gold nanoparticles without the addition of RNA to serve as a control. To confirm attachment, Dynamic Light Scattering (DLS) and Zeta Potential analyses were conducted on a Malvern Zetasizer.2.6.E. coli to quantify targeted RNA attachment. All experiments were conducted in triplicate to verify findings. First, at room temperature, 400 μL of 106 concentration of DH5α in PBS was combined with 100 μL of the coated particles. This experiment was run in parallel with 100 μL of bare gold nanoparticles to allow for direct comparison. The samples were rocked for 20 minutes then spun down at low speed, to pellet the bacteria but not the unbound particles. The cellular pellet was suspended in sterile water for UV-Vis measurements. One milliliter of the sample is placed in a quartz cuvette and absorption is measured from ∼200 nm to 800 nm. Baseline absorbance spectra from excess RNA and E coli were removed from the overall absorption to ensure only readings from the nanoparticles were measured.After RNA coating, the gold particles were then exposed to 3.3.1.The present study was aimed at developing a novel sensor to detect bacteria based on carbon nanotubes functionalized with gold nanoparticles with a ribonucleic acid (RNA) sequence attached as a capture element. Highly oriented pyrolitic graphite (HOPG) was used as the base carbon structure due to its ability to withstand high temperatures as well as its relative inertness. To confirm CNT growth on the graphite and visualize the packing density SEM imaging was conducted. As seen in To ensure the presence of gold nanoparticles, their successful attachment to the CNT surface, and even distribution, SEM images were obtained . These i3.2.E. coli, it had to first be verified that RNA was effectively bound to the surface of the gold nanoparticles. To accomplish this task, the hydrodynamic diameter and the surface charge of the gold nanoparticles was measured both prior to and post RNA introduction, as successful attachment of RNA would alter both characteristics. These results are shown in Prior to the introduction of 3.3.E. coli preferentially bound to the RNA coated gold nanoparticles over bare ones. This was achieved by isolating the cellular component after co-incubation and analyzing the gold content. A marked increase in gold associated with the RNA coated samples would signify greater coupling and a higher degree of specificity between E. coli and the particles. E. coli. When further analyzed, this data confirms a 189% enhanced bacterial binding in the presence of E. coli specific RNA versus the uncoated particles; calculated by dividing the difference in peak absorbance by the uncoated absorbance value.The next step was the proof of concept experimentation to ascertain if 3.4.E. coli strain. Following introduction of E. coli to the biosensors, they were washed and SEM imaged to evaluate extent of microbial association. As seen in E. coli molecules, however no significant difference was observed between the bare and RNA coated sensors. After further investigation, we believe that this lack of RNA specificity is due to interactions between the RNA and the carbon nanotube surface. It has been shown that CNTs interact with the conformation of nucleic acids [E. coli binding, as well as an amperometric analysis of the overall structure.After independently confirming gold nanoparticle growth on the bound CNTs and successful RNA coating of gold nanoparticles, the final step was to construct and assess the effectiveness of the complete biosensor. Therefore, following synthesis of gold nanoparticles on the graphite bound CNTs the particles were subsequently functionalized with the thiolated RNA sequence specific for the DH5α ic acids  due to tic acids . We spec4.E. coli sequence to gold nanoparticles. These RNA coated gold nanoparticles were shown to enhance E. coli capture by 189% when compared to bare gold nanoparticles. Following full construction of the composite sensor, preliminary tests showed no statistical significance with the addition of RNA, which we suspect is due to interactions occurring between the RNA and CNTs. Even still, we believe this work has great consequence as it lays the ground work for future molecular recognition based sensor constructed from nanomaterials.There exists a strong need within the sensors community to develop the capability for the real-time sensing mechanism for foreign pathogens. The work presented in this study defines a composite bases sensor that incorporates specific RNA sequences to selectively bind a microbial agent. CNTs were successfully grown on a graphite substrate and subsequently served as a point of synthesis for gold nanoparticles. In solution, we successfully attached a thiolated RNA that recognizes Furthermore, this research demonstrated that carbon substrates, in bulk and nano-scale, were able to be utilized in the development of a novel sensor to detect pathogens in the environment. We are confident from this work that utilizing novel nanostructures for the application of a bio-sensor in bacterial detection and identification is attainable. This same sensing approach outlined here can easily be applied to other bacterial surface-specific RNA sequences, attaching them in the same manner, and ultimately creating an electrochemical multi-array sensor to determine the strain of bacteria as well as their aqueous solution concentration. Additionally, this process can be extended to other microorganisms to identify and quantify targets through the development of a hand-held multi-array sensor.


Drosophila melanogaster exposure to menthol produced controversial results due maybe to methodological differences. Here, we compared the oviposition-site preference of wild-type D. melanogaster lines freely or forcibly exposed to menthol-rich food. After 12 generations, oviposition-site preference diverged between the two lines. Counterintuitively, menthol ‘forced’ lines showed a persistent aversion to menthol whereas ‘free choice’ lines exhibited a decreased aversion to menthol-rich food. This effect was specific to menthol since the ‘free choice’ lines showed unaltered responses to caffeine and sucrose. This suggests that the genetic factors underlying Drosophila oviposition site preference are more rapidly influenced when flies have a choice between alternative sources compared to flies permanently exposed to the same aversive substance.Food choice and preference relies on multiple sensory systems that are under the control of genes and sensory experience. Exposure to specific nutrients and nutrient-related molecules can change food preference in vertebrates and invertebrates. For example, larval exposure of several holometabolous insects to menthol can change their adult response to this molecule. However, studies involving  Drosophila melanogaster  or presented to a choice between the two types of food. In the last case, we took advantage of the short generation duration of this species to establish ‘choice lines’, showing either a preference, or an aversion, to oviposit on menthol. Many Drosophila studies have used experimental selection of complex behaviour to identify the gene(s) underlying behavioural response. For example, microarrays performed on choice lines have pinpointed some of the genes underlying geotactism (D. melanogaster for many years (D. melanogaster lines consistently on either menthol-rich food (‘forced lines’), or a diet based on their initial oviposition preference phenotype . Our data suggest that oviposition-site decision response is differently affected between forced and choice lines.Animals raised in the laboratory are exposed to a constant diet contrarily to wild-type animals that have a broader choice of food sources in nature. Laboratory strains of ood cues . Howeverotactism , aggressotactism  or sex potactism . Geneticny years . In our Drosophila melanogaster Meigen were used: the widely used laboratory strain Canton-S (CS) and the Dijon2000 strain (Di2) established in 2000. Since initial data obtained with the two strains showed no significant differences in behavioural response to menthol, they were pooled. Subsequent experiments were performed with the Di2 strain. Flies were raised on a yeast–cornmeal–agar medium and kept at 24±0.5°C with 65±5% humidity on a 12-hr light:12-hr dark cycle. All experiments were performed under similar conditions. Flies were collected and sexed 0–4 hour after emergence under light CO2 anaesthesia and held until 4 to 5 day-old in groups of 30 same-sex individuals in glass vials with fresh plain food. Unless mentioned, all other flies including those of parental lines were kept in a menthol-free environment.Two wild-type strains of v/v) ethanol and kept at 4°C. Menthol solution was added to fresh lab food and is designated hereafter as menthol-food (M-food). A similar volume of ethanol (90% v/v) was added to the control diet menthol-free food (Plain-food  =  P-food). Egg-laying food devices (see hereinafter) were used immediately or kept at 4°C to be used until two days later. To assess the optimal proportion of menthol to be used in our tests, we performed preliminary multiple-choice tests using a range of menthol containing food, and measured the number of eggs laid on each type of food. Concentrations ranging from 0 to 0.01% induced no food preference , whereas an aversive effect was detected from 0.1% menthol . At the menthol concentration of 0.5%, females rarely oviposited on M-food (MPI = −1). All the experiments presented here were thus carried out with a menthol concentration of 0.1%. To assess the specificity of the choice procedure (carried out with menthol), we also measured egg-laying behaviour of F24 and F25 choice-line females on food enriched either with caffeine (16 mM)  or sucrose (10 mM) . To obtain and test enough individuals, we carried this experiment a few generations after our choice procedure had resumed with F24+F25 flies .We used pure racemic menthol . A 250 mg/ml solution was prepared by dissolving menthol in 90% (2-anaesthetized) were kept overnight in a dual-choice egg-laying device .Groups of 4–5-day-old flies composed of 25 adult females and 25 adult males (previously COg device . This dei) fertilized eggs laid and (ii) surviving females were counted after 12 hours. This allowed us to determine the average number of eggs laid per female . The comparison of the numbers of eggs laid on M-food (Nm) vs P-food (Np) was used to compute the menthol preference index (MPI = [Nm−Np]/[Nm+Np]). In theory, MPI can vary between −1 (strong aversion against menthol) and +1 (strong attraction to menthol).The numbers of  was measured daily during their first 5 days of adult life using the parameters described above. Since the number of eggs laid on days 4 and 5 were relatively low, the data obtained for the two days were pooled to permit statistical analysis.To test a possible effect on female fecundity, we recorded, between F0 and F12, the total number of eggs laid by control, P-forced and M-forced females on P-food or on M-food during 12 h .vs choice exposure to menthol. The ‘forced’ procedure consisted to raise individuals during their complete development, generation after generation, either on M-food .Two distinct experimental procedures were designed to assess the effect of forced ‘choice’ procedure consisted in keeping the progeny left either on M- or on P-food, generation after generation on that food. More precisely, parental flies  had the choice to lay eggs in the M- vs P-food dual-choice device. Only the eggs laid on M-food were used to initiate the F1 ‘choice M-line’  and to the F26–F36 generations. Therefore, the choice procedure was carried out for F1–F12, F20–F25 and F37–F48 generations. During the interruption of the choice procedure, P- and M-choice lines were, respectively, kept on P- and M-food at 18°C. Oviposition behaviour was finally checked in F58 choice-line females.The To control the effect of each experimental procedure, parental strains (unexposed ‘control lines’), kept in similar laboratory conditions, were tested at the beginning and during the course of our experiments. Data from all lines  were obtained by testing at least 4 sublines per line  and each generation was initiated by at least 2 replicates per subline. To avoid potential bottlenecks in choice lines, a generation was established with at least 100 eggs per vial.p-value based on a Monte Carlo computation) completed by Conover–Iman multiple pairwise comparisons . In addition, to visualize the change of menthol preference from F0 to F12 in each experimental procedure, we used the EPFs on M- and on P-food in the two lines to build a contingency table (19×2 for forced exposure and 25×2 for choice exposure) used to perform a Chi-square analysis with computation of the adjusted residuals. Adjusted residual test provides a fair indication of the importance of the cells to the ultimate Chi-square value and reveals how far the observed values are from the expected ones. They have a standard normal distribution (i.e. mean = 0 and SD = 1), and an adjusted residual higher than 1.96 or lower than −1.96 indicates that the observed value is significantly larger, or smaller, than the expected one . In choice experiments, the number of eggs found on oviposition sites was compared using Wilcoxon signed ranked test. Intra-generational difference in the MPIs (EPFs) of P- and M-females were assessed using Mann–Whitney test, whereas MPIs (and/or EPFs) throughout F0–F12 generations were compared using Kruskal–Wallis test . MPI and EPF were considered as dependent variables, whereas ‘Food type’  was considered as a qualitative independent variable and ‘Generation’ as an independent quantitative variable. When the ANCOVA result was significant, we used the Ryan–Einot–Gabriel–Welsch (REGWQ) test to compare food type effect. For choice lines, ANCOVA only included generations with choice procedure: the first analysis was carried out on [F0–F12], the second analysis on ([F0–F12]+[F20–F25]), and the third one on ([F0–F12]+[F37–F46]).MPI (and/or EPF) during the five first days of adult life of the F12 females, preference index of F24+F25 P- and M-choice females toward caffeine and sucrose and female fecundity  were assessed using Kruskal–Wallis test as previously described.vs unexposed lines), MPIs were always negative (−0.38 to −0.42) in ‘M- vs P-food’ dual-choice tests  and ‘indifferent’ (−0.03 to +0.06) in ‘P- vs P-food’ dual-choice tests . Note that the number of eggs laid per female (EPF) increased by about 50% from F0 .In all our tests, females had the choice to oviposit either on menthol-rich food  and the number of eggs per female  in the two lines.To determine the long-term effect of the ‘forced’ exposure to menthol, a wild-type strain was permanently kept (during 52 generations) either on M-food (‘M-forced line’) or on P-food  = 250; p = 0.008), the MPI of the M-forced line was always as negative as the MPI of the P-forced line  and menthol preference did not change between F1 and F12 generations for P- and M-lines (2 = 0.02 (slope = −0.19) and 0.07 (slope = 0.12); p = 0.691 and 0.463, respectively). EPF increased in forced lines during F10–F12 generation; however, without any difference between the M- and P-lines . The fecundity slightly increased after eight generations (F9) in the two forced lines .Except for F1 . The parental strain used to initiate the choice lines showed a similar aversive MPI as the parental strain used to initiate the ‘forced’ lines. The ‘choice procedure’ differently affected the MPI of the M- and P-choice lines . After two generations (F3), the response of M-choice line was generally less negative than in the P-choice line . In other words, the strong aversive effect induced by menthol during the first generations gradually disappeared. The progressive change in menthol preference of M-choice line . Note that the correlation for the M-choice line was negatively impacted by the F10 and F11 generations data  More rarely, a strong attraction to menthol was noted (at F24 and F25: MPI = +0.5 corresponds to 75% eggs laid on M-food), but these ‘reversed preference’ data points might be outliers, not representative of the general trend. Overall, the three lines differed for their EPF . The fecundity rapidly increased (between F0 and F1) in the two choice lines and remained elevated at least until F12 .Next, we measured the behavioural effect of menthol exposure on P- and M-choice lines . Each substance, mixed with food, was presented alongside P-food , the response of P- and M-choice lines to caffeine and sucrose was not altered .To determine the specificity of the choice procedure (performed with menthol-rich food), we measured the response of P- and M-choice lines to caffeine and sucrose. This was carried out with F24+F25 females pooled, four generations after our choice procedure had resumed  affecting the attraction and oviposition responses of D. sechellia flies to the odorant molecules emitted by its specific host fruit, Morinda citrifolia  the behaviours related to egg-laying  (ii) the exposure period (iii) the diet  the experimental design  , (ii) the period , (iii) tnogaster  and specjavensis ), or  was unexpected. We do not know whether these sporadic changes are biologically meaningful. Possibly, this non-linear response between generations may reflect differential alteration of the genes underlying the multiple sensory systems integrating the perception of menthol (see below) and leading to the global behavioural response of the female. In any case, since this strongly contrasts with the highly stable (repulsive) response shown by forced lines, it suggests that the system underlying female oviposition choice was differently affected by our two experimental procedures.The choice procedure induced relatively fast changes: after 12 generations, the repulsive effect of menthol was significantly decreased in the M-choice line. If the decreased aversion of F12 flies of the M-choice line was somewhat expected, the strong reversal preference to menthol , this phenomenon may be a result of a biased sampling of the early progeny laid by the most precocious egg-laying females. D. pseudoobscura lines selected for reduced dispersion of oviposition sites also showed a rapid fecundity increase (2–3 generations) in ‘high dispersion’ lines (The variation of fecundity observed during the course of the choice experiment may be a side effect of our choice procedure. The positive relationship between the variation of both the MPI and EPF in F12 females of different ages  suggestsn’ lines . ConversIn summary, our study reveals a striking difference in oviposition preference on menthol-rich food according to the experimental procedure: the choice procedure induced a faster – but non-permanent – change whereas the forced procedure had no major effect. Our next goal will consist of characterizing some of the genetic factors and the neural pathways underlying the change of the behavioural response to menthol in choice lines.


Staphylococcus aureus is a gram-positive cocci and an important human commensal bacteria and pathogen. S. aureus infections are increasingly difficult to treat because of the emergence of highly resistant MRSA (methicillin-resistant S. aureus) strains. Here we present a method to study differential gene expression in S. aureus using high-throughput RNA-sequencing (RNA-seq). We used RNA-seq to examine gene expression in S. aureus RN4220 cells containing an exogenously expressed transcription factor and between two S. aureus strains (RN4220 and NCTC8325-4). We investigated the sequence and gene expression differences between RN4220 and NCTC8325-4 and used the RNA-seq data to identify S. aureus promoters suitable for in vitro analysis. We used RNA-seq to describe, on a genome wide scale, genes positively and negatively regulated by the phage encoded transcription factor gp67. RNA-seq offers the ability to study differential gene expression with single-nucleotide resolution, and is a considerable improvement over the predominant genome-wide transcriptome technologies used in S. aureus. Staphylococcus aureus (S. aureus) is a pathogenic bacterium that can cause a variety of infections, most notably of the skin [S. aureus infections can be difficult and costly to treat due to antibiotic resistance, especially in the Methicillin-Resistant Staphylococcus aureus (MRSA) strains [the skin . S. aure strains -4.S. aureus [S. aureus, RNA-seq was recently used to identify small non-coding RNAs [S. aureus have largely used microarray techniques to examine global gene expression changes [High-throughput studies have been particularly useful to examine global gene expression in . aureus -7. The a. aureus . RNA-seq. aureus ,13, but ing RNAs  and to sing RNAs , but tra changes ,17. It h changes ,13.S. aureus colonizes the nasal cavity of 30% of the human population, but under certain circumstances can invade tissues and cause disease [S. aureus to act as both a commensal bacterial and a pathogen, studies have attempted to identify the key pathways regulating pathogenicity in this organism. A regulatory RNA, termed RNAIII, is thought to be the main effector of the switch to pathogenic growth as it controls the expression of secreted toxins. Levels of RNAIII are regulated by the Agr proteins [ disease . Given tproteins ,19.S. aureus strains, lytic bacteriophages have been suggested as potential therapeutic agents [S. aureus phages and identified proteins with antimicrobial activity [S. aureus RNAP, but not E. coli RNAP, through an interaction with the global housekeeping transcription factor σA; (2) does not block the functions of σA, including DNA recognition and core RNAP binding; but (3) interferes with the interaction between the core RNAP α subunit C-terminal domain (α-CTD) and UP-element sequences that are only required for transcription at a small subset of promoters [Due to the lack of traditional therapies to treat highly resistant c agents ,21 or asactivity -24. One activity . Subsequromoters . Thereforomoters .S. aureus in the competent lab strain RN4220, and between S. aureus strains. To identify S. aureus genes repressed by gp67, we expressed gp67 in S. aureus cells. In addition to the relative gene expression data that would be provided by microarray, we used the RNA-seq data to identify Single Nucleotide Polymorphisms (SNPs) and to quantitatively evaluate the relative levels of gene expression between loci within the same sample. We examined the differences in the transcriptome of S. aureus strains NCTC8325-4 and RN4220 and used the RNA-seq data to identify a putative orphan CRISPR element in these strains.In this work, we establish an RNA-seq approach to study differential gene expression in RN4220 was obtained from Peter Moyle in Tom Muir’s lab at The Rockefeller University. pRMC2 and NCTC8325-4 were a generous gift from Sivaramesh Wigneshweraraj at Imperial College, London.S. aureus expression vector pRMC2 [S. aureus strain RN4220 by standard electroporation [Gp67 was cloned into the or pRMC2  using prporation  and tran600 = 0.4) using the RNeasy kit from Qiagen. Briefly, 2x108 cells were removed from growing cultures, immediately added to 2 volumes of BioStabilize solution (Qiagen) and incubated for 5 minutes at room temperature. Cells were then collected by centrifugation, resuspended in TE buffer containing 1mg/ml lysostaphin and 200µg proteinase K and incubated for 15 minutes at room temperature. 100µl zirconia beads (0.1mm) were added to lyse the cells in a bead beater at top speed for 3 x 2minutes, with a 1-minute rest on ice. The lysate was centrifuged briefly to remove the beads and the remaining procedure was carried out to the manufacturer’s specifications. Purified RNA was quantified using a spectrometer (NanoDrop).RNA was purified from cells at mid-log phase growth .Expression values are reported as fragments-per-kilobase-of-gene-per-million-mapped reads (FPKM). Data were visualized using the Integrated Genomics Viewer .Transcripts were quantified by assessing the total number of reads for the entire transcript using the program cuffdiff, part of the Cufflinks suite of tools for sequencing-based transcript assembly and quantification. Briefly, reads were assigned to transcripts as described above and the samples to be compared were evaluated for variance and tested for differential expression. P-values  were detRNA-seq data have been submitted to GEO (accession number GSE48896).In vitro transcription assays were performed as described .S. aureus promoters in vivo, we used RNA-seq to examine differential gene expression in S. aureus. We cloned gp67 into an inducible expression cassette (pRMC2) [S. aureus RN4220 cells to create RN4220-pRMC2 and RN4220-pRMC2-gp67. Addition of inducer to media inhibited growth of cells containing pRMC2-gp67, as previously described, but had no effect on cells containing only empty vector [To determine the effects of the G1 phage-encoded transcription factor gp67 on all  (pRMC2)  and trany vector ,27. RNA RNA-seq techniques are standardized in eukaryotic samples . BecauseS. aureus cells and that our two samples differ predictably.To ensure that our cDNA library contained mRNA in addition to the small structured RNAs that remained after rRNA reduction, we tested for the presence of gp67 specific mRNA from cells containing pRMC2-gp67 and cells containing empty vector . Only ceS. aureus NCTC8325 annotated genome sequence. While the sequence for RN4220 is available, it differs from NCTC8325 only by 121 SNPs and several indels that cluster around phage insertion sites. The NCTC8325 genome is more fully annotated and more amenable to use by the software required to map RNA reads and compare expression levels.The cDNA library was then sequenced using Illumina technology. Before analyzing the data, we searched for RNA reads that mapped to the gene for gp67. We only identified RNA reads mapping to gp67 from the sample containing pRMC2-gp67 . We thenS. aureus RNAP and inhibit cell growth [≈91%) of transcripts were unchanged (p>0.05) in cells expressing gp67 compared with control cells containing only empty vector. This targeted effect of gp67 is in agreement with structural and biochemical data [To understand the effects of gp67 on global transcription levels, we searched for genes that were differentially expressed in pRMC2-gp67 cells. gp67 is known to bind to l growth ,24,27. FS. aureus RNAP and those indirectly affected by disruption of other regulatory factors or alterations in mRNA stability and degradation. We therefore sought to directly test gp67 at S. aureus promoters shown to be susceptible to inhibition in vivo. However, very few S. aureus promoters have been examined in vitro [in vitro were not modulated by gp67 expression in vivo [RNA-seq analysis, like microarrays, reports only the steady state level of RNA in cells. Additional sample preparation is required to identify primary transcripts  or to main vitro ,19,36 an in vivo . Identifin vivo [in vivo. However, some mRNAs in our data show clear evidence for a long 5’ UTR  just upstream of the mapped 5’ end of the mRNA. This information is not provided by standard microarray analysis that reports only on RNA expression within the coding region.mRNA processing enzymes can remove 5’ and 3’ UTRs from mRNAs in cells. Mapping promoter start sites requires enriching for primary transcripts that have not undergone processing in vivo . For ourg 5’ UTR . MoreoveS. aureus in vitro transcription system [in vitro and gave RNA products of the expected size on a urea-PAGE gel  were ribosomal proteins or proteins otherwise involved in translation , was found to differ from the NCTC8325 genome at 12 locations, and RN4220 had 121 SNPs, including those previously identified in NTCT8325-4. Importantly, our RNA-seq analysis validates several SNPs known to cause functional differences between these two strains as unique to RN4220, such as the frame shift in the virulence transcriptional regulator AgrA  and the nuclease . This munuclease . The SNPnuclease , RimM  are bacterial RNA elements that allow to an adaptive response to phage infection . CRISPRsS. aureus is not thought to have a functional CRISPR system. No genes in the S. aureus genome have any homology to previously identified Cas proteins. Genomic searches for putative CRISPR elements in the S. aureus NCTC8325 genome reveal only five weak hits [eak hits .S. aureus genome including both coding and non-coding regions. This element may be an orphan CRISPR, and reintroduction of Cas genes into S. aureus may activate this putative RNA element.We used our RNA-seq data to determine whether RNA was expressed at any of the putative CRISPR loci. While four of the five putative CRISPR elements were located in annotated ORFs, and contained no signal for an RNA element in our RNA-seq data, one putative CRISPR was located in an intergenic region and showed clear evidence for RNA-seq reads . The putS. aureus is of extensive interest due to the pathogenic importance of this organism [S. aureus switches to its pathogenic transcriptional profile may provide novel targets for drug therapy. Studies in S. aureus have used microarray analyses to study differential gene expression in response to exogenously expressed proteins or drugs [S. aureus both between cells expressing and lacking an exogenously expressed protein and between closely related S. aureus strains.Gene expression and regulation in organism -11. A beor drugs ,17,47. HS. aureus phage transcription factor, gp67 [S. aureus genome are most highly expressed in log-growing cells and to identify novel S. aureus promoters for analysis by in vitro transcription.Like microarray analysis, RNA-seq provides relative gene expression levels. We examined the genes downregulated by the expression of a or, gp67 ,27. HoweS. aureus have used the genechip technologies described by Dunman et al. [S. aureus strains to validate their genome-wide transcription quantification method. Their work focused in part on the role of ArgA as a transcriptional modulator by using an ArgA knock out strain. RN4220, examined here, contains a mutation in AgrA known to modulate its activity. Like Dunman et al., we show a significant decrease in RNAIII due to ArgA dysfunction. We also find that protein A (spa) is upregulated in RN4220, in agreement with microarray analysis of an AgrA knock out strain. However, the transcriptional differences between NCTC8325-4 and RN4220 at mid-log growth appear to be more limited than the differences between wild type RN27 and cells containing an AgrA mutation. We also used RNA-seq data from RN4220 carrying an expression plasmid and NCTC8325-4 to compare gene expression and SNPs between the two strains. Recent work has highlighted potential functional differences between these strains [Many studies in n et al. . Dunman  strains ,38,41. GStaphylococcus aureus, and the ability to perform genome-wide analysis of transcription under various conditions, increased our understanding of transcriptional networks in this organism. While microarrays are more cost-effective than high-throughput sequencing, the cost of routine RNA-seq experiments have dropped precipitously in recent years, and are likely to continue to drop [The development of microarray technology in  to drop . RNA-seq to drop , and all to drop . Additio to drop  or in vaE. coli [E. coli [The technology presented here could be easily adapted to mechanistic study of transcription in prokaryotes, as has been done in eukaryotic samples . While hE. coli , this anE. coli . Mapping[E. coli , should S. aureus. RNA-seq provides data that was previously only accessible through multiple, complimentary techniques. Because prokaryotic genomes are generally small, and contain relatively short intergenic distances with limited non-coding regions, we sequenced the majority of the S. aureus genome through RNA-seq analysis of the transcriptome and were able to identify many SNPs, including in non-coding regions. RNA-seq has become increasingly cost effective and we have developed a protocol for sample preparation in Staphylococcus aureus cells. We believe standardization of RNA-seq for prokaryotic samples, and routine transcriptome analysis using high-throughput sequencing, would provide a significant advantage over the current microarray based techniques.This study shows that RNA-seq is a valuable tool to examine gene expression in Table S1Genes significantly downregulated by gp67 expression.RNA-seq was used to quantify gene expression in the presence and absence of gp67 as described in the Materials and Methods. Cuffdiff was used to quantify gene expression at each loci in the NCTC8325 genome and significance was determined by conducting a Benjamini-Hochberg correction for multiple testing. Only genes at which p<0.05 are listed.(PDF)Click here for additional data file.Table S2Genes significantly upregulated by gp67 expression.RNA-seq was used to quantify gene expression in the presence and absence of gp67 as described in the Materials and Methods. Cuffdiff was used to quantify gene expression at each loci in the NCTC8325 genome and significance was determined by conducting a Benjamini-Hochberg correction for multiple testing. Only genes at which p<0.05 are listed.(PDF)Click here for additional data file.Table S3Most highly expressed genes in NCTC8325-4 and RN4220.RNA-seq was used to quantify gene expression in the S. aureus NCTC8325-4 and RN4220. Cuffdiff was used to quantify gene expression at each loci in the NCTC8325 genome and significance between samples was determined by conducting a Benjamini-Hochberg correction for multiple testing. No genes in this table differ significantly between the two strains.(PDF)Click here for additional data file.Table S4SNPs identified as unique to RN4220.SNPs were identified using SAMtools and verified manually be examining the RNA-seq data in Integrated Genomics Viewer.(PDF)Click here for additional data file.Table S5Genes significantly downregulated (a) and upregulated (b) in RN4220 relative to NCTC8325-4.S. aureus NCTC8325-4 and RN4220RNA-seq was used to quantify gene expression in the . Cuffdiff was used to quantify gene expression at each loci in the NCTC8325 genome and significance between samples was determined by conducting a Benjamini-Hochberg correction for multiple testing. Differences of p<0.05 were considered significant and included.(PDF)Click here for additional data file.Table S6Single nucleotide polymorphisms previously identified as unique to RN4220 but also present in NCTC8325-4.SNPs were identified using SAMtools and verified manually be examining the RNA-seq data from both NCTC8325-4 and RN4220 in Integrated Genomics Viewer.(PDF)Click here for additional data file.