The method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. This demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules.The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl The structure and biological properties of RNAs are a function of changing cellular conditions. Here, Baird et al. report a high-throughput Förster resonance energy transfer (FRET) method to rapidly compare RNA structure modulation by cognate and non-cognate ligands across multiplexed solution conditions. Many cellular RNAs adopt distinct global and local conformations in response to environmental variables such as temperature, ionic strength, the identity and concentrations of divalent cations and pH, as well as their specific or nonspecific binding to small molecules, proteins and other RNAs1234567101112132+ concentration and ligand binding. We employ this approach to elucidate how the conformation and stability landscapes of a cyclic diguanylate (c-di-GMP) riboswitch are modulated by cognate and non-cognate ligands. In addition to delineating the solution parameter range over which the RNA exhibits maximal response, our multi-dimensional analysis uncovered an alternative, inhibitory riboswitch conformation stabilized by the aminoglycoside kanamycin B, a non-cognate ligand. Additional biophysical experiments demonstrated that the non-native aminoglycoside-induced conformation modulates binding of the cognate ligand to the riboswitch. Our method for rapidly determining conformational and stability landscapes of RNA thus suggests an approach to control riboswitch function through the stabilization of off-pathway conformations using non-cognate ligands. Our method should be broadly and generally applicable for identifying small-molecule ligands exerting conformational control of functional RNAs.We have developed an efficient method to perform multi-dimensional biochemical and biophysical characterization of RNAs in a single high-throughput experiment. Our approach is based on Förster resonance energy transfer (FRET) and can be implemented on a conventional quantitative PCR (qPCR) instrument. We demonstrate that a single experiment probing 19,400 multiplexed solution conditions yields high-information content 3D and four-dimensional (4D) landscapes describing RNA conformation and tertiary structure stability as a function of Mg2+; and a compact conformation stabilized by Mg2+ and c-di-GMP. The latter was elucidated by X-ray crystallography162+ and c-di-GMP concentrations. Bulk measurements . Even collectively, these various experiments only sampled a handful of solution conditions. Nonetheless, their outcomes already hint at complex conformational and energetic landscapes underlying the function of this gene-regulatory RNA. Because of its important role in bacterial physiology, the c-di-GMP riboswitch is a potentially valuable target for novel antibacterial compounds. For drug discovery, it is important to develop methods more efficient than conventional SAXS and FRET to map more comprehensively the molecular behaviour of the RNA and its response to agonists and antagonists (The class I c-di-GMP (c-di-GMP-I) riboswitch16urements  show thaagonists .2+ and c-di-GMP concentrations, we sought to elucidate, in a comprehensive manner, the interaction between these two variables. To achieve this rapidly, we employed a 384-well microplate fluorometer to measure the FRET efficiency (EFRET) of the labelled c-di-GMP aptamer domain under 100 different conditions (ten concentrations of Mg2+ versus ten of c-di-GMP). This experiment readily (<5 min for measurements and<5 min for data processing) produced a multi-dimensional RNA conformational landscape , Mg2+-stabilized (EFRET ∼0.56) and ligand-bound .As previous work indicates that folding of the c-di-GMP-I riboswitch aptamer domain is a function of both Mg2+ FRET titrations  as a function of c-di-GMP concentrations clearly showed that the affinity of this riboswitch construct for c-di-GMP is ∼8 μM in the region (1–1.5 mM Mg2+) of maximal conformational response  subdivides the landscape into four regimes, with a fifth regime located at the intersection of these midpoints. First, at the lowest Mg2+ and c-di-GMP concentrations, the RNA is unfolded. Second, an increase in c-di-GMP concentrations alone produces negligible ligand binding. Third, higher Mg2+ concentrations produce a c-di-GMP binding-competent RNA state. Fourth, highly stabilizing Mg2+ concentrations dampen the conformational response to ligand binding (KD and KMg plots (∼1.4 mM Mg2+ and ∼6.5 μM c-di-GMP) lies the regime of highest synergy between Mg2+ and c-di-GMP, where the highest slopes in the Mg2+ and c-di-GMP titrations are observed , whereas at 10 mM Mg2+ it appeared to unfold it (2+) should weaken the interaction between the RNA and the aminoglycoside. Moreover, if the aminoglycoside were purely a nonspecific cationic counterion to the anionic RNA, it should promote its folding (higher EFRET). Control experiments using single-fluorophore-labelled constructs ruled out the possibility that kanamycin B aberrantly affects the fluorescent properties of the dyes folding of this riboswitch.Based on the ability of several aminoglycosides to promote the folding of a three-way junction in the central domain of the 16S rRNA, it was proposed that the positively charged aminoglycosides interact with RNA primarily through nonspecific charge neutralizationnfold it . These rthe dyes . Given tEFRET increases at nearly all measured Mg2+ concentrations  under conditions of low Mg2+ (<1 mM) and high kanamycin B concentrations  for which the mean distance between our FRET probes (positioned to report on the native folding transition) does not vary linearly. Overall, features of the kanamycin B landscape suggest the presence of structural state(s) not populated in the c-di-GMP landscape.To further characterize this riboswitch–aminoglycoside interaction, we determined the corresponding 3D conformational landscape , which dtrations , kanamyctrations . The lantrations . At low ecreased . At neared EFRET . At 1.1 ed EFRET , cyan anFRET  was observed in the conformational landscape of the RNA in the presence of kanamycin B (Tm3° (∼60 °C). Control experiments with singly labelled RNA constructs clearly demonstrate that Tm3° measurements reflect conformational stability rather than artefactual dye fluorescence in the presence of kanamycin B, over the temperature range examined  at up to 2 M concentration  in the presence of various ligands including the polyamines spermine and spermidine  correlated with their respective valence and charge density; spermine (4+) compacted the RNA more than spermidine (3+). This is consistent with the activity of nonspecific charge neutralization. If the primary mode of interaction between the riboswitch and kanamycin B were nonspecific charge neutralization, at minimum similar compaction should be induced by the polycationic kanamycin B (5+) as observed in the presence of spermine and spermidine. In contrast, addition of kanamycin B did not produce large changes in Rg. The RNA persisted in an expanded conformation (Rg ∼29.5 Å) that was not disrupted by the addition of up to 1 mM Mg2+, a condition under which (in the absence of the aminoglycoside) the RNA compacts readily . Pre-incubation of the riboswitch with kanamycin B resulted in a small decrease (∼1.4-fold) in c-di-GMP-binding affinity . Further binding . Inspect binding . In the  binding , grey, dWe have developed a FRET-based method to examine the effect of multiple environmental variables on the conformation of an RNA in a single rapid, high-throughput experiment. Our method generates empirical high-information content RNA conformational and stability landscapes using a fluorescence microplate reader and conventional qPCR instrumentation. The biophysical approach we describe here is distinctly different from and, therefore, complementary to the majority of high-information content nucleic acid structure–function characterization methods described to date, which have implemented massively parallel sequencing technologies to explore the genotype–phenotype  landscapes of functional nucleic acids333536EFRET to c-di-GMP at low Mg2+ concentration (2+ concentration (2+), the riboswitch–kanamycin B interaction would not have been identified using conventional ligand titration or thermal melting assays , lyophilized, resuspended in water and stored at −20 °C.The c-di-GMP-I riboswitch aptamer domain constructs  used in 2 and fractionated on a 24-ml bed-volume Superdex 75 (GE) size-exclusion chromatography column equilibrated in the same buffer. The bimolecular RNA (30 kDa) was well separated from the excess Cy3-containing RNA (10.2 kDa). Absorbance was recorded at 260, 540 and 640 nm to monitor RNA, Cy3 and Cy5, respectively. The areas under the Cy3 and Cy5 curves pertaining to the bimolecular peak were compared and the Cy5:Cy3 molar ratio was determined .The bimolecular RNA used for fluorescence measurements was prepared as follows. A 200 μl mixture of 5 μM Cy5-containing RNA and 7.5 μM Cy3-containing RNA were incubated overnight in folding buffer  containing 2 mM MgCl2 concentrations were prepared. A unique folding buffer was dispensed into each of ten columns in a 384-well microplate; the same Mg2+ folding buffer was present in ten rows of each column. Dual-labelled RNA was added to each of the 100 wells to a final concentration of 25 nM. For experiments containing c-di-GMP, 250 nl of a different concentrated ligand stock solution were dispensed to each of the ten rows using a mosquito Crystal (TTP Labtech). This resulted in final concentrations across the rows ranging from 0.1 to 25 mM Mg2+ and down the columns ranging from 0 to 235 μM c-di-GMP in a final volume of 21.25 μl. The microplate was covered with an aluminum seal, centrifuged for 1 min at 700 r.p.m., and incubated on an orbital shaker at 200 r.p.m. overnight at room temperature. For experiments containing kanamycin B, the RNA was incubated overnight in the absence of ligand. Then, 250 nl of each ligand stock were dispensed using a mosquito Crystal (TTP Labtech) as described above to yield a 10 × 10 matrix ranging from 0.1 to 25 mM Mg2+ and from 0 to 198 μM kanamycin B in a final volume of 25.25 μl. The microplate was covered with an aluminum seal, centrifuged for 1 min and incubated on an orbital shaker at 200 r.p.m. for 2.5 h. To evaluate the fully unfolded RNA, EDTA was added to a final concentration of 4 mM to additional RNA samples containing 0.1 mM Mg2+.Folding buffers  containing ten different MgClSamples for thermal unfolding experiments were prepared similarly to the method described above for multiplexed FRET measurements. 20 μl of the samples containing c-di-GMP (described above) were transferred to a 384-well qPCR plate and centrifuged at 1,000 r.p.m. for 1 min before measurement on the qPCR machine. Samples containing kanamycin B were prepared as described above with slight modifications. The RNA was incubated in the presence of ligand overnight on an orbital shaker (200 r.p.m.) at a final concentration of 25 nM RNA in 25.25 μl. The plate was centrifuged for 1 min before measurement.EFRET measurements was observed for data obtained on the qPCR machine, likely due to the sensitivity of the optical readout and background signal from the qPCR plate. The higher noise in the EFRET signal precluded fitting the Mg2+ titration and c-di-GMP titration data at room temperature. However, the determination of Tm3° was robust when compared with samples prepared at various concentrations and volumes of RNA. An increase in RNA concentration and volume  did allow for robust measurements of EFRET on the qPCR machine with reduced noise and of sufficient quality to fit two Mg2+ titrations (in the absence and presence of c-di-GMP). The fits were in agreement with measurements made on the microplate fluorometer.Higher noise in EFRET=IA/(ID+IA). Origin 9.1 software (OriginLab) was used to generate smoothed 3D surface landscapes. The EFRET data from the 10 Mg2+ titrations were smoothed using adjacent-averaging with a window of five points (Origin 9.1). The smoothed data were plotted as a 3D surface and coloured according to the range of EFRET values. Seven data points from the c-di-GMP landscape were removed because of significantly lowered Cy3 and Cy5 reads, suggestive of insufficient volume in the well. From the kanamycin B landscape, the EFRET at 25 mM Mg2+ was unexpectedly lower than the EFRET at 10 mM Mg2+. The data in all other Mg2+ concentrations were robust and in agreement with Mg2+ titrations performed on other days. A disproportionate loss of signal of both Cy3 and Cy5 intensities at 25 mM Mg2+ was responsible for the lower EFRET and was attributed to sample adherence to the microcentrifuge tube used during sample preparation of this sample.Room temperature fluorescence measurements were recorded using a Tecan Infinite 200 Pro microplate reader with a single excitation filter corresponding to Cy3 excitation (535±12.5 nm) and two emission filters corresponding to Cy3 and Cy5 emissions (595±17.5 and 670±12.5 nm). The apparent FRET efficiency50−1. Cy3 and Cy5 (via FRET) emissions were measured 194 times over this range and a FRET melting curve was generated using one excitation filter 520±10 nm (Cy3) and two emission filters at 586.5±10 nm (Cy3) and 682±14 nm (Cy5). Changes in fluorescence signal outside the cooperative tertiary unfolding transition were observed because of photobleaching and the temperature-dependent quantum yields of the dyes. The EFRET thermal unfolding curves (194 data points) were smoothed using an FFT filter using a 10-point window and cutoff frequency of 0.193 (Origin 9.1). Derivatives of the smoothed EFRET thermal unfolding curves were calculated and peaks corresponding to Tm3° were identified using Origin 9.1 (OriginLab). The Tm3° landscape surface was generated by smoothing the Mg2+ titration Tm3° data using adjacent averaging (Origin 9.1).A ViiA7 (Applied Biosystems) qPCR machine was used to simultaneously record melting curves for the RNA tertiary structure under each solution condition from 20 to 70 °C using a ramp rate of 2.5 °C min2 concentrations were prepared. Each sample contained 23 μl of buffer and 2 μl of RNA. Samples were prepared in triplicate. Following overnight incubation in the Mg2+-containing buffers, 1 μl of ligand stock or water was added. Final concentrations were 25 nM RNA in 10 Mg2+ concentrations between 0.17 and 25 mM Mg2+. Each Mg2+ titration was performed in 0 and 200 μM kanamycin B. Samples were incubated for 1.5 h followed by recording fluorescence emissions using a Tecan M1000pro. For Cy3, the excitation wavelength used was 535±20 nm and the emission wavelength was 595±20 nm. For Cy5, the excitation wavelength used was 635±20 nm and the emission wavelength was 670±20 nm. Subsequently, 21 μl of each sample were transferred to a 384-well qPCR plate and data were recorded following a total incubation time of 5 h in the presence of ligand. Thermal measurements employed a 2.5 °C min−1 ramp rate and the following filter sets: Cy3 singly labelled RNA, 520±10 nm (excitation) and 586±10 nm (emission); Cy5 singly labelled RNA, 640±10 nm (excitation) and 682±14 nm (emission).Singly labelled RNA constructs were prepared and purified identically to dual-labelled constructs. Explicitly, the Cy3-labelled oligo red,  was anne2+ titrations were fit using a semi-empirical Hill-type analysis described previouslyEFRET=0.17, in accordance with that observed in the presence of 4 mM EDTA. The c-di-GMP titrations were fit to a two-state binding model to obtain KD,app. In addition, the c-di-GMP titrations were fit using a Hill analysis to obtain measures of the slope of each titration.All MgEFRET values for Mg2+ titrations were observed for samples prepared on different days. This difference was likely due to variations in the molar ratio of the purified bimolecular construct or photobleaching of one of the dyes during sample preparation. Despite different EFRET values, similar KMg parameters were obtained from fits to the Mg2+ titrations. Furthermore, nearly identical Tm3° were measured from the thermal unfolding curves, suggesting that the unfolded and folded states were identical, despite the minor variations observed in EFRET. To establish the inherent error in EFRET measurements, data prepared on different days were compared and the Mg2+ titrations in the absence of ligand were fit using a semi-empirical Hill equation. Next, the EFRET values were normalized using the equation Sn=(S−Slow)/(Shigh−Slow), where Sn is the normalized signal, S is the observed signal, Slow is the signal of the unfolded RNA and Shigh is the signal of the folded RNA. Slow for all experiments was fixed to match the unfolded EFRET value obtained in 4 mM EDTA. Using data normalized from ten experiments performed on different days over the period of several months the average standard deviation across all EFRET values was calculated (±0.012). The error was largest (±0.02) near the Mg2+ midpoint (∼1–2 mM), where small changes in the [Mg2+] due to pipetting error result in slightly larger EFRET differences. In the low and high end of the curve, the standard deviation is ±0.01. Using the normalized parameters, the data in the kanamycin B landscape were scaled to match the EFRET range of the c-di-GMP landscape.Slightly different −1 RNA, 25 mM NaCl, 25 mM KCl and 20 mM HEPES-KOH, pH 7.5 with either 2 or 10 mM Mg2+. Thermal melting was performed on an Agilent 8543 spectrophotometer over a temperature gradient from 20 to 90 °C in 0.1 °C increments with a 12-s equilibration at each temperature before absorbance measurements. Absorbance (260 nm) and derivative data curves were smoothed using a Savitzky-Golay 41-point filter (Origin 9.1).A bimolecular riboswitch construct, identical to that used in fluorescence experiments except lacking the fluorophores, was purified by size-exclusion chromatography as described for preparation of the labelled RNA. A 750-μl purified sample of the bimolecular riboswitch RNA was prepared in a folding buffer with final concentrations of 10 μg ml2 and 20 mM HEPES-KOH, pH 7.5. The higher Mg2+ concentration was used in this folding buffer (3 mM MgCl2) in order to compensate for the high concentration of RNA2+. The RNA was purified on the column and the RNA concentration was determined before preparation of samples for SAXS experimentation. SAXS samples in 1 mM Mg2+ were prepared by mixing 35 μl of purified RNA (1.98 g l−1) with 63.5 μl of the size-exclusion chromatography buffer. To prepare SAXS samples in 0.01 mM Mg2+, 350 μl of the purified RNA were concentrated to a final concentration of 6.7 g l−1 using an Amicon Ultra centrifugal filter (Millipore). 10 μl of this RNA stock were mixed with 88.5 μl of folding buffer containing 0 mM Mg2+. Buffer samples were prepared identically to RNA sample preparation but with the addition of appropriate folding buffer instead of RNA stock. Samples were shipped to the Advanced Photon Source with frozen cold packs. At the ID-12 BESSRC beamline, the samples were centrifuged briefly before addition of 1.5 μl of appropriate ligand (20 mM stock). The final concentration of each ligand  was 300 μM. The final RNA concentrations were 0.69 and 0.67 g l−1 for the samples in 1 and 0.01 mM Mg2+, respectively. The samples were incubated at room temperature for >11 h before data collection. Twenty consecutive exposures of 1 s were recorded while the sample was continuously flowed. Data were collected for buffer samples without RNA immediately before samples containing RNA. Data were processed using Igor Pro (Wavemetrics) as described elsewhereA 200-μl sample of unimolecular riboswitch RNA was prepared in folding buffer with final concentrations of 615 μM RNA, 25 mM NaCl, 25 mM KCl, 3 mM MgCl2 and 0 or 200 μM kanamycin B . An excess of the four folding buffers containing Mg2+ and kanamycin B at the aforementioned concentrations was prepared for concentration and washing of the RNA samples. The samples were concentrated tenfold using Amicon Ultra centrifugal filter units (Millipore) and washed with appropriate folding buffer. This process was repeated ≥5 times to thoroughly equilibrate the RNA in the appropriate buffers. The equilibrated samples were incubated overnight at room temperature. The following day the RNA concentrations were determined using absorbance measurements at 260 nm, including a multiplicative factor of 1.35 to correct for hypochromicity. The RNA samples were diluted in the appropriate folding buffers to achieve a final concentration of 50 μM RNA. The c-di-GMP titrant was prepared to a final concentration of 500 μM by adding 0.6 μl of 50 mM c-di-GMP to 59.4 μl of appropriate folding buffer. Hence, the buffers of the sample and the titrant were not more than 99.0% identical and a minor endothermic signal was observed as the titration curves became saturated with ligand. Raw ITC data were exported and processed using NITPICThe unimolecular riboswitch construct at a final concentration of 45 μM in 500 μl was mixed with folding buffer containing 2 or 10 mM MgClHow to cite this article: Baird, N. J. et al. Rapid RNA–ligand interaction analysis through high-information content conformational and stability landscapes. Nat. Commun. 6:8898 doi: 10.1038/ncomms9898 (2015).Supplementary Figures 1-13, Supplementary Tables 1-3, Supplementary Notes 1-7 and Supplementary References


Transporters comprise the largest family of membrane proteins in human organism, including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of xenobiotic and endogenous molecules. Transporters are widely expressed in various human tissues and are routinely evaluated during the process of drug development and approval. Over the past decade, increasing evidence shows that drug transporters are important in both normal physiology and disease. Currently, transporters are utilized as therapeutic targets to treat numerous diseases such as diabetes, major depression, hypertension and constipation. Despite the steady growth of the field of transporter biology, more than half of the members in transporter superfamily have little information available about their endogenous substrate(s) or physiological functions. This review outlines current research methods in transporter studies, and summarizes the drug-transporter interactions including drug-drug and drug-endogenous substrate interactions. In the end, we also discuss the therapeutic perspective of transporters based on their physiological and pathophysiological roles. Movement of many drugs and endogenous molecules across the cell membrane is governed by protein transporters. In general, membrane transporters are divided into two major superfamilies—the ATP binding cassette (ABC) family and the solute carrier (SLC) family. To date, more than 400 transporters have been annotated in the human genome. The ABC transporters are primary active transporters that utilize the energy from ATP hydrolysis to transport substrates across the membrane. SLCs can be either facilitative transporters, which transport their substrates down the gradient across the membrane, or secondary active transporters, which transport their substrates against the gradient across the membrane by coupling a downhill transport of another substrate. Major drug transporters are listed in Table The biological function of transporters has been investigated using multiple approaches. Among them, metabolomic studies have been quite valuable for substrate identification of transporters systemically, which identify the alterations of many metabolites or their substrates directly. Alternatively, the uptake of substrate candidates can be evaluated by radio-tracer or radio-free LC/MS quantification. This method was used to identify thiamine and ergothioneine as the physiological substrates of OCT1 (SLC22A1) and OCTN1 (SLC22A4) respectively . Clinical genomics also reveal involvement of transporters in bioconversion and toxicity of drugs. One of the most compelling examples of the critical role of transporters in drug toxicity stems from a GWAS encompassing 85 patients with myopathy and 90 normal control individuals  that has been shown to be involved in the hepatic uptake of statins. GWASs have also associated SLCO1B1 and SLCO1B3 variants with bilirubin levels and unconjugated hyperbilirubinemia  cells. The colon carcinoma cell line Caco-2 are especially useful for the study of intestinal transporters, since these cells exhibit morphological and functional similarity with the enterocytes lining the small intestine  of a membrane transporter frequently begins with an o Bröer, . If combThe primary goal of transporter assays is to determine the kinetics of substrate transport. For the primary active transporter, like the ABC transporter, substrate translocation across the cell membrane is directly correlated to ATP hydrolysis. Thus ATPase assays that measure the inorganic phosphate release using colorimetric measurement can be utilized to assess active transporter kinetics , encoded by SLC46A1, incorporates folate by coupling a proton co-transport down the proton gradient at pH 5.5, however, this transport activity is absent at neutral pH , TALEN and CRISPR/Cas-based genomic engineering provides powerful and convenient tools to generate transgenic or knockout animals for a gene of interest . Potential drug-drug interactions (DDIs) may cause severe, adverse reactions in patients due to alteration in ADME by transporters. Most DDIs via transporters involve, but are not restricted to, the main transporters in drug ADME: P-gp, BCRP, OATP1B1/1B3, OAT1/3, OCT1 and OCT2  to exogenous or endogenous stimuli. Altered drug ADME during inflammation has long been primarily ascribed to changes of enzymes in drug metabolism. An example of this is decreased expression of P450, the main drug metabolizing enzyme in liver, following inflammation. Moreover, recent studies have also shown that the expression and activity of numerous transporters are also changed in inflammatory condition. In experimental cell and animal models, inflammatory conditions induced down regulation of MDRs, MRPs and OATPs. Consequently, the ADME of drugs which are substrates of these transporters would be broadly affected  due to cholestasis. The transporter BSEP plays an important role in the disposition and exportation of bile salts from liver. BSEP inhibition by drugs can cause accumulation of bile salts in liver, thus leading to cholestasis. There is a strong correlation between inhibition of the Oct1 KO mice provide a model to investigate the function of Oct1 systemically. Thiamine is converted to thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in cytosol, which serves as the essential co-enzyme for energy generation and glucose metabolism. Due to reduced thiamine uptake in liver, thiamine-related changes were observed in the serum of Oct1 KO mice. These included reduced levels of TMP and TPP and higher levels of pyruvate and alpha-ketoglutarate, which are the substrates of pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (OGDH)  and OCT1 might play a critical role in the uptake of serotonin in liver , the homologue of mammal SLC13A5, confers extended lifespan in a caloric restriction-like manner (Rogina et al., Slc13a5 in mice results in reduction in the liver uptake of citrate from circulation accompanied by an increase in mitochondrial biogenesis, lipid oxidation, energy expenditure and decrease in hepatic de novo lipogenesis. The loss of Slc13a5 also protects mice from high-fat diet induced obesity and insulin resistance (Birkenfeld et al., Drosophila INDY, leaving mitochondria citrate transporter activity unchanged (Sun et al., Vibrio cholera at 3.20 Å. This provides the structural basis for understanding the substrate binding properties as well as conformational change upon substrate binding (Mulligan et al., In 2+-insulin hexamers in the secretory granules of the pancreas, which releases the physiologically active form of insulin in the serum driven by a pH change (Xu et al., 2+ transport activity than the Trp325 ZnT8 (Nicolson et al., Zinc transporters are reported to be involved in the pathophysiology of cancer, cardiovascular diseases and Alzheimer’s disease making them possible therapeutic targets for treatment of these diseases (Prasad et al., The exact relationship between ZnT8 activity and diabetes risk, however, remains controversial. Zn ions have several effects on the regulation of insulin production and clearance. Co-secretion of Zn ions with insulin from beta cells inhibits insulin production in an autocrine and paracrine fashion through the K-ATP channel (Bancila et al., Intriguingly, a recent GWAS reported that 12, rare truncating mutations in ZnT8 were surprisingly protective against developing diabetes and, collectively, explained a 65% reduction in diabetes risk (Flannick et al., Transporter biology is a rapidly changing field in pharmaceutical research. Identifying the function of transporters in physiological and pathological conditions is garnering significant attention for drug development. Many drugs, nutrients and metabolites are moved by transporters not only through the plasma membrane but also among subcellular organelles like vesicles, ER, lysosome and mitochondria. Transporters remain one of the most underestimated fields for drug targets. Within the hundreds of annotated transporters in human genome, more than half are orphan transporters with unknown substrates or function. The nature of these transporters, including their expression and localization in organs and tissues, subcellular localization, endogenous substrates, largely remains elusive. As discussed in this review, transporters can be of significant therapeutic potential in the treatment of various diseases. A comprehensive dissection of the physiological roles of the transporter could be a key step to excavate this “gold mine”. Our growing knowledge of transporter biology will greatly accelerate the discovery of novel drug targets and development of first-in-class therapies.


Isolation of pure RNA from woody perennials, especially fruit crops such as grapevine rich in complex secondary metabolites, has remained very challenging. Lack of effective RNA isolation technology has resulted in difficulties in viral diagnosis and discovery as well as studies on many biological processes of these highly important woody plants. It is imperative to develop and refine methodologies with which large amounts of pure nucleic acids can be readily isolated from woody perennials.We compared five commonly used RNA isolation kits in isolating total RNA from twelve species of woody perennials. We made modifications to select RNA isolation systems to simplify and improve their efficiency in RNA isolation. The yield and quality of isolated RNAs were assessed via gel electrophoresis and spectrophotometric measurement. We also performed RT-PCR and RT-qPCR to detect several major viruses from grapevines.Two of the kits were shown to be the best in both the yield and quality of the isolated RNA from all twelve woody species. Using disposable extraction bags for tissue homogenization not only improved the yield without affecting quality, but also made the RNA isolation technology simpler, less costly, and suitable for adoption by many potential users with facility limitations. This system was successfully applied to a wide range of woody plants, including fruit crops, ornamentals and timber trees. Inclusion of polyvinylpyrrolidone in the extraction buffer drastically improved the performance of the system in isolating total RNA from old grapevine leaves collected later in the season. This modification made our system highly effective in isolating quality RNA from grapevine leaves throughout the entire growing season. We further demonstrated that the resulting nucleic acid preparations are suitable for detection of several major grapevine viruses with RNA or DNA genomes using PCR, RT-PCR and qPCR as well as for assays on plant microRNAs.This improved RNA isolation system would have wide applications in viral diagnostics and discovery, studies on gene expression and regulation, transcriptomics, and small RNA biology in grapevines. We believe this system will also be useful in diverse applications pertaining to research on many other woody perennials and recalcitrant plant species.The online version of this article (doi:10.1186/s12985-015-0376-3) contains supplementary material, which is available to authorized users. Woody plants, such as timber trees, fruit trees, and ornamental trees, are of great importance to people, not only economically and ecologically but also bio-aesthetically . Woody fFor the control of viral diseases in woody fruit crops, the most important strategy is prevention through the use of virus-free propagation materials during orchard and vineyard establishment. Development and implementation of rapid, highly sensitive and reliable diagnostic methods is the key to plant health in woody fruit crops and constitutes an integral component of the virus-free certification programs in many countries. Currently, the official methods for diagnosis of woody fruit crop viruses are largely biological indicator indexing and serological methods . These mAs one of the most powerful technologies ever developed in recent years, deep sequencing makes possible the global identification of virtually all viruses and viroids present in an infected plant, which would otherwise be impossible by conventional, sequence-specific RT-PCR-based assays . Deep seThe success of deep sequencing, RT-PCR and RT-qPCR is contingent upon quality RNAs. Unfortunately, isolation of highly pure RNA from woody plants has been very challenging and problematic compared to herbaceous plant species. This is due to the presence of high levels of secondary metabolites, such as polyphenols and polysaccharides, in woody plants. Polyphenolic substances react with RNAs and form insoluble complexes, thus severely interfering with RNA isolation, resulting in low yield and poor quality RNAs. Consequently, the isolated RNAs are not suitable for downstream applications such as RT-PCR, RT-qPCR and deep sequencing. These issues were circumvented through the use of double-stranded RNAs to serve as template for viral genome sequencing and PCR-based diagnosis –25. HoweA number of RNA isolation kits have been developed by biotech companies and used for isolation of RNAs from diverse plant species, mostly annual, herbaceous plants –35. ThesThe objectives of this study were to compare and refine RNA isolation methodologies that are commercially available for use in isolation of sufficient quantities of quality RNAs from a wide range of woody plant species. We have identified the best commercial kits for the isolation of RNAs . We have further simplified the RNA isolation technology so that they could be adopted by a large spectrum of users. Finally, we have also made significant improvement to the RNA isolation methodology, so that it is suitable for the detection of both RNA and DNA viruses using grapevine tissues collected throughout the growing season.Five commonly used commercial RNA isolation kits were compared for their effectiveness in isolation of RNA from woody plants. These five kits are TRIzol Reagent (Life Technologies), RNeasy Plant mini kit (Qiagen), Spectrum™ Plant Total RNA kit (Sigma), AccuPrep viral RNA extraction kit (Bioneer) and Plant/fungi total RNA kit (Norgen BioTek). These kits were chosen due to their advantages in certain aspects in RNA isolation. RNeasy plant mini kit has been used for a long time for isolating RNA or cleaning up crude RNA preps isolated using various methods from different plant species, including woody plants , 34, 37.The five kits were first compared for their performance in isolating total RNA from young peach leaves. The RNA integrity was assessed by visualization of ribosomal RNA bands on 1.5 % MOPS/formaldehyde gel. As shown in Fig. V. vinifera cv. Chardonnay). The results showed that the Spectrum plant total RNA kit from Sigma gave the best RNA yield of 39.9 μg per isolation from young leaves, followed by kits from Norgen and Bioneer . Subsequent PCR amplification was carried out with primers RSP21 and RSP22 targeting the capsid protein gene of Grapevine rupestris stem pitting-associated virus (GRSPaV) , young and mature grapevine leaves were ground in liquid nitrogen to fine powder using a mortar and pestle and a portion of the resulting fine powder (50 mg) was used in RNA isolation without further grinding in the extraction buffer. For method B, 100 mg of leaf tissue was ground in an extraction bag containing 1 ml of lysis solution with a hand-held homogenizer until the tissue was completely macerated. We used 100 mg of leaf tissue in this method because only half of the resulting lysate could be collected after grinding. The collected lysate, of 500 μl in volume, was then processed using kits from either Sigma or Norgen. As shown in Fig. We then investigated the suitability of the improved procedure for RNA isolation from a wide range of woody plants. The woody plants tested were trees - paper birch, Norway maple, white pine and white spruce, fruit trees – cherry, nectarine, and apple, and ornamentals – lilac and horse chestnut. In addition, strawberry, an important herbaceous perennial species that is difficult with isolation of quality RNA , was alsWe then tested the effectiveness of the established methodology in isolation of total RNA from red-fruited cultivars of wine grapes (Cabernet Franc) collected later in the season showing leafroll disease symptoms. To our surprise the RNA isolation methodology was not effective at all for isolating RNAs from symptomatic leaves. As shown in Table Grapevine red blotch-associated virus (GRBaV) is a recently characterized DNA virus with a seemingly wide distribution in North American [American , 52, 53.American , 53. TheThe effectiveness of the modified method in isolating total RNA from old grapevine leaves collected in late fall was further validated by examining a large number of samples and both red and white wine varieties. These samples included several major wine varieties that are important for the cold climate viticulture and enology in Ontario: Cabernet Franc, Chardonnay, Riesling, Gamay and Gewurztraminer. To compare the effects of different types of tissue on yield and quality of isolated RNAs, we used both leaves and cambium scrapings as source materials for RNA isolation. The average yield of total RNA isolated from old leaves of 19 samples was 7.0 μg, higher than that from cambium scrapings (5.5 μg), with similar quality: 2.02 for A260/A280 and 1.98 for A260/A230 for RNAs from leaf tissues, and 2.07 for A260/A280 and 1.95 for A260/A230 for RNAs from cambium scrapings. RT-PCR was conducted to detect GRSPaV, GLRaV-2, GLRaV-3 and GRBaV using total RNA isolated from leaf and phloem samples  for the detection of several major viruses that infect grapevine. Lastly, we have shown that the same isolation system was effective not only in isolating RNA but also small DNA molecules as judged by successful amplification of GRBaV, a recently discovered DNA virus from grapevines showing red blotch symptoms.Eriobotryya japonica) [Interestingly, the performance of these five commercial kits is largely plant-specific. For example, when peach leaves were used, four of the systems (with the exception of the Bioneer kit) worked well in generating large quantities of total RNA of satisfactory purity Fig. . In sharaponica) , strawbeaponica) , among oaponica) –58, 60.The challenges in isolation of RNA from grapevines is further demonstrated by the fact that both Spectrum Plant Total RNA kit and Plant/Fungi Total RNA kit failed in isolating quality total RNA from older and symptomatic grapevine leaves, although they worked very well for young and mature leaves collected early in the season. However, we have resolved this issue by modifying the Spectrum Plant Total RNA kit through addition of 2.5 % PVP-40 to the lysis buffer Table ; the impUsing leaf as source material for virus detection has significant advantages as compared with other tissues, such as cambium. First, ample leaf materials are available throughout the growing seasons. Second, collection and processing of leaf tissue are much easier and more convenient than petiole and phloem scrapings. Third, sampling of leaves causes no damage to the vine as compared to sampling of cane cuttings from which cambium scrapings are obtained. Fourth, leaves from multiple canes can be collected from a single vine or from several vines, making test of composite samples possible. This advantage becomes more prominent, as several samples from different parts of a vine are usually collected in virus testing in consideration of the uneven distribution of viruses in the infected grapevines –64. FurtGrapevine red blotch-associated virus, has been identified in grapes with red blotch disease [Badnavirus (family Caulimoviridae) was discovered in grapevine showing vein-clearing and decline syndromes [Only RNA viruses had been reported to infect grapevines up till 2011. However, a DNA virus and two retro-like viruses have been recently identified in grapevine as a result of the application of deep sequencing technologies , 8, 9. F disease , 14, 52.The genomes of several economically important woody plants  have been sequenced. The subsequent endeavors would be to determine, through gene profiling and comparative genomics/transcriptomics, the function of genes and genetic elements that regulate gene expressions in order to answer the question of what genes make a tree that is fundamentally different from herbaceous plants . The impIn closing, we have shown that both Sigma and Norgen kits were highly effective in isolating quality total RNA from grapevines and a wide range of other woody perennials using young leaf tissues. We have further demonstrated that this RNA isolation procedure can be substantially simplified through the use of disposable BIOREBA bags during sample homoge nization, without the need for mortar and pestle and liquid nitrogen as recommended by vendors. Further modification of the system by including PVP-40 in the lysis buffer enabled the successful use of our method on both young and old leaves collected from the field throughout the entirety of the growing season. This improved nucleic acid isolation method would have wide applications in the diagnostics and discovery of viruses and viroids, studies on gene expression and regulation, transcriptomics, and small RNA biology in woody plant species.Vitis vinifera , V. riparia (from Quebec and Manitoba) from growth chamber in the University of Guelph. Grapevine leaves and canes were also collected from 6 varieties of V. vinifera  from early summer (June) to late fall (November) from commercial vineyards in Niagara, Ontario. Leaves from paper birch (Betula paperifera), Norway maple (Acer platanoides), white pine (Pinus strobus L.), white spruce (Picea glauca) and horse chestnut (Aesculus hippocastanum) were collected in May from University of Guelph Arboretum. Leaves of peach (Prunus persica), lilac (Syringa vulgaris), strawberry (Fragaria × ananass), cherry (Prunus avium), nectarine (Prunus persica) and apple  were collected in June from local gardens.Twelve species of diverse woody plants were included in this study. These plants included fruit crops , ornamentals (lilac and horse chestnut) and trees . Grapevine leaves were collected from three varieties of Five commercial kits were selected for this study, which included TRIzol Reagent (Life Technologies), RNeasy Plant mini kit (Qiagen), Spectrum™ Plant Total RNA kit (Sigma), AccuPrep viral RNA extraction kit (Bioneer) and Plant/fungi total RNA kit (Norgen BioTek).Three methods of sample homogenization were investigated.Method A: This was the default method and used in initial experiments for all kits according to the manufacturers’ instructions. Plant leaf tissues were ground in liquid nitrogen to fine powder using a mortar and pestle and 50 mg of plant leaf fine powder was then transferred into a 1.5 or 2 ml microfuge tube and processed following instructions of the individual kit.Method B: Leaf samples were homogenized in an extraction bag with a hand-held homogenizer, both purchased from BIOREABA . One hundred milligrams of leaf tissue was ground in an extraction bag containing 1 ml of lysis solution with a hand homogenizer until the tissue was completely macerated. The bag was rolled up, placed in a 50 ml plastic centrifugation tube and spun down for 10 s in a swing bucket rotor at 4,000 rpm to collect the liquid. The lysate of about 500 μl  was then transferred into a 1.5 ml microfuge tube and processed following instructions of the individual kit.Method C: Leaf samples were homogenized with mortar and pestle without liquid nitrogen. 100 mg of plant leaf tissue was ground in a mortar containing 1 ml of lysis solution with a pestle until the tissue was completely macerated. The lysate of 500 μl  was then transferred into a 1.5 ml microfuge tube and from then on processed following protocol provided by the individual kit for RNA isolation.The protocols of the five kits are briefly described as follows for the convenience of readers. More information about each kit can be found on the manufacturer’s instructions.One milliliter of TRIzol Reagent was added to a 2 ml microfuge tube with 50 mg of plant tissue fine powder. The tube was vortexed vigorously, and then incubated at room temperature for 5 min. Two hundred microliter of chloroform was added and vortexed vigorously, followed by incubation at room temperature for 3 min. The sample was centrifuged at 13, 000 rpm for 15 min at 4 °C. The upper aqueous phase was collected into a new tube and 0.5 ml of 100 % isopropanol was added to the tube. The mixture then was incubated at room temperature for 10 min and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was discarded and the RNA pellet was washed by applying 1 ml of 75 % ethanol, vortexing briefly and centrifuging the tube at 10,000 rpm for 5 min at 4 °C. The wash was discarded and the RNA pellet was air-dried for 5–10 min. The RNA pellet was then resuspended in 50 μl of RNase-free water by pipetting the solution up and down several times, followed by incubation at 55–60 °C for 5–15 min in a water bath.450 μl of buffer RLT or RLC was added to a 1.5-ml microfuge tube containing 50 mg of plant tissue fine powder. The tube was vortexed vigorously and incubated at 56 °C for 3 min. The lysate was transferred to a QIAshredder spin column placed in a 2 ml collection tube, and centrifuged for 2 min at 12,000 rpm. The supernatant was then transferred to a new microfuge tube and 0.5 volume of ethanol (96–100 %) was added and mixed immediately by pipetting. The sample  was then transferred to an RNeasy spin column placed in a 2-ml collection tube and centrifuged for 15 s at 10,000 rpm. The flow-through was discarded. The RNeasy spin column was washed once by applying 700 μl buffer RW1 to the column, centrifuging for 15 s at 10,000 rpm and then discarding the flow-through. The column was then washed twice by applying 500 μl buffer RPE to the column, centrifuging for 15 s at 10,000 rpm, followed by discarding the flow-through. The column was centrifuged at full speed for 1 min to thoroughly remove the wash buffer. For RNA elution the RNeasy spin column was placed in a new 1.5 ml tube and 50 μl RNase-free water was added directly to the spin column membrane. The column was centrifuge for 1 min at 10,000 rpm to elute the RNA.500 μl of lysis solution with β-mercaptoethanol were added to a 2-ml microfuge tube containing 50 mg of plant tissue fine powder. The tube was vortexed vigorously and incubated at 56 °C for 3 min. The lysate was centrifuged at 14,000 rpm for 3 min and then the supernatant was transferred into a Filtration Column seated in a 2 ml collection tube, and centrifuged for 1 min at 14,000 rpm. The clarified flow-through lysate was then transferred to a new microfuge tube and 250 μl of Binding Solution was added and mixed immediately by vortexing. The mixture was applied to a Binding Column seated in a 2-ml collection tube and centrifuged for 1 min at 14,000 rpm. After discarding the flow-through, the column was washed once by applying 500 μl Wash Solution 1 to the column, centrifuging for 1 min at 14,000 rpm and then discarding the flow-through. Afterwards, the column was washed twice by applying 500 μl Wash Solution 2 to the column, centrifuging for 1 min at 14,000 rpm and then discarding the flow-through. The column was centrifuged at full speed for 1 min to thoroughly remove the wash buffer. Finally, the spin column was placed in a new 2-ml Collection Tube and 50 μl Elution Solution was applied to the column. The column was then centrifuged for 1 min at 14,000 rpm to elute the RNA.To a 1.5 ml microfuge tube with 50 mg of plant tissue fine powder, 400 μl of Binding Buffer was added and mixed by lightly vortexing. After incubation for 10 min at room temperature, 100 μl of isopropanol was added and lightly vortexed for about 5 s, and then spun down for 1 min. The liquid was transferred into the binding column and centrifuged for 1 min at 8,000 rpm. Following centrifugation, the binding column was transferred to a new 2-ml collection tube and the column was washed by adding 500 μl of W1 buffer to the column, and centrifuging for 1 min at 8,000 rpm and then washed again by adding 500 μl of W2 buffer, and centrifuging for 1 min at 8,000 rpm. The column was spun down once more at 13,000 rpm for 1 min to completely remove W2 buffer. The binding column was transferred to a 1.5 ml collection tube, and 50 μl of Elution Buffer (pre-warmed to 60 °C) was added to the column. After standing for 1 min, the column was centrifuged at 8,000 rpm for 1 min to elute the RNA.600 μl Lysis Buffer C was added to a 2-ml centrifuge tube containing 50 mg of plant tissue fine powder. The tube was vortexed vigorously and incubated at 56 °C for 5 min. The lysate was then transferred to a filter column placed in a 2-ml collection tube, and centrifuged for 2 min at 14,000 rpm. The supernatant was then transferred to a new microfuge tube and an equal volume of ethanol (96-100 %) was added and mixed immediately by votexing. 600 μl of the clarified lysate with ethanol was applied onto a spin column placed in a 2-ml collection tube and centrifuged for 1 min 14,000 rpm. After discarding the flow through, the column was resembled. The remaining lysate was then loaded onto the column and re-centrifuged for 1 min at 14,000 rpm. The column was washed three times by applying 400 μl of Wash Solution A to the column, centrifuging for 1 min and then discarding the flow-through. The column was centrifuged for 2 min to thoroughly dry the resin. Finally, the spin column was placed in a new 1.5 ml tube and 50 μl Elution Solution A was applied to the column. The column was then centrifuged for 2 min at 200 rpm, followed by 1 min spin at 14,000 rpm to elute the RNA.The purity and concentration of the total RNA preparations were assessed with a NanoDrop spectrophotometer  at the wavelengths of 230, 260, and 280 nm. RNA integrity was verified based on the 28S and 18S rRNA bands after electrophoresis on 1.5 % formaldehyde–agarose gel, stained with ethidium bromide and visualization with UV light. The integrity and size distribution of purified RNAs were also evaluated with a 2100 Bioanalyzer  equipped with an RNA Nano chip. Small RNAs and miRNAs from the total RNA samples isolated from young grapevine leaves with the five commercial kits were analyzed by the Agilent Bioanalyzer equipped with Small RNA Analysis kit  and the yields of small RNAs and miRNAs were calculated using the software Bio sizing version B.02.08. SI648 (SR2).Total RNA from grape leaves isolated with kits from Sigma, Bioneer and Norgen were used in RT-PCR and RT-qPCR. Synthesis of cDNA was performed using the High-capacity cDNA Reverse Transcription kit (Life Technologies). The reaction mix (20 μl) included 500 ng of total RNA, 2.0 μl 10X RT Buffer, 0.8 μl 25X dNTP Mix (100 mM), 2.0 μl 10X RT Random Primers, 1.0 μl Multiscribe™ Reverse Transcriptase (50U/μl). The mix was incubated for 10 min at 25 °C, then 120 min at 37 °C, followed by incubation for 5 min at 85 °C, and then stored at 4 °C for immediate use or −20 °C for later use. PCRs were performed on a PTC-2000 Peltier Thermal Cycler (MJ Research). 25 μl of PCR reactions contained 2.5 μl of 10X PCR buffer, 0.5 μl of dNTPs (10 mM), 0.5 μl of forward primer and reverse primer (10 μM each) , 0.6 μl of F/R primer mix (5 μM each), 5 μl of cDNA diluted 5X in water and 1.9 μl of water. Cycling conditions included an initial denaturation at 95 °C for 10 min, then 40 cycles at 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, followed by melting curve analysis to confirm the specificity of PCR amplification. The primers and RT-qPCR conditions for miRNA 156a and 159a were described by Wu et al. [q) values were calculated using the threshold cycle method [qPCRs were performed on StepOnePlus (Applied Biosystems) in the Genomics Facility of University of Guelph. 15 μl of reaction mix contained 7.5 μl of u et al. . The quae method .


Investigators from University Children's Hospitals in Bern, Zurich, Aarau, and multiple other centers in Switzerland evaluated prospectively the epidemiology, manifestations, and treatment of all full-term neonates with neonatal arterial ischemic stroke (NAIS) and born 2000-2010. Investigators from University Children's Hospitals in Bern, Zurich, Aarau, and multiple other centers in Switzerland evaluated prospectively the epidemiology, manifestations, and treatment of all full-term neonates with neonatal arterial ischemic stroke (NAIS) and born 2000-2010. The NAIS incidence in Switzerland was 13 per 100,000 live births. Median age was 2.0 days (range 1-26 days); median birth weight 3380 g (range 2370-4520 g). Of 100 neonates (67 boys) with NAIS all but 3 (97%) presented with seizures, and 50 (52%) had seizures as the only presenting symptom. Increased or decreased tone abnormalities were present in 32% and movement abnormalities in 11%; 81% had unilateral (80% left-sided) infarcts and 19% had bilateral lesions. The anterior circulation only  was affected in 89%. Risk factors for NAIS were maternal risk conditions (32%), birth complications (68%), and neonatal comorbidities (54%). Genetic testing abnormalities included factor V Leiden mutation in 5%, heterozygous prothrombin mutation in 11%, and heterozygous methylene tetrahydrofolate reductase mutation in 36%. Seventeen percent received antithrombotic and antiplatelet therapy without serious side effects. At aged 2 years follow-up, 39% were diagnosed with cerebral palsy, 7 (9%) were treated for epilepsy (4 had infantile spasms), and 31% had delayed motor development. Children with normal mental performance at 2 years after birth may develop deficits later in life. [COMMENTARY. A comparison of the incidence of NAIS in this population-based, prospective study with that in 9 previously published studies, 5 of which were population-based and 4 were hospital-based , the incNeonatal seizures are most commonly the clinical finding that triggers assessment in neonates with stroke. In children with NAIS without early-onset seizures, perinatal stroke is recognized retrospectively, with emerging hemiparesis or late-onset seizures presenting >14 days after the stroke. Risk factors for perinatal stroke include hereditary or acquired thrombophilia and environmental factors .


Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1, localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3′UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1. One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA.The germ cell lineage in  Xenopus oogenesis, selected RNAs are localized and retained within the vegetal cortex at two distinct time periods. Most RNAs essential to forming the germline localize very early in oogenesis within a macroscopic structure called the mitochondrial cloud (MC) or Balbiani body [nanos RNA whose product is essential to the preservation of the germline in many diverse species including Drosophila, Xenopus, and mouse [Localization of specific RNAs to subcellular domains is one mechanism by which cells restrict protein synthesis in time and space. During ani body ,5,6,7,8.ani body ,10,11,12nd mouse ,16,17,18VegT and a TGFβ family member Vg1. The products of these genes are required for specifying and patterning the endoderm and mesoderm [Vg1 remains uniformly distributed in the cytoplasm while nanos RNA accumulates in the MC remains an unanswered question. The selection process for the different localization pathways is not well understood but is essential for the creation of the future germline and primary germ layers.Another set of RNAs, previously distributed throughout stage I oocytes, is actively localized via a microtubule kinesin-dependent mechanism at later oocyte stages ,20,21. Rmesoderm ,23,24,25nanos RNA form particles that disperse evenly throughout the ooplasm in stage I oocytes. Over time, these particles became progressively immobilized, but only within the MC where they form larger aggregates reminiscent of germinal granule formation [Time-lapse Confocal microscopy and FRAP (Fluorescence Recovery After Photobleaching) analysis show that injected fluorescently labeled ormation . Identiformation , virtualormation ,28. Clusormation ,30,31.Vg1 and VegT are directed to the vegetal pole by a 340-nt localization signal (LS) in their 3′UTR. In vitro and in vivo UV crosslinking analyses reveal six proteins that interact directly with both the Vg1 and VegT-LS [Vg1-LS mutants in E2 and VM1 sites fail to bind their respective proteins in vitro and fail to localize in vivo [Vg1 RNA and co-localize with it at the vegetal cortex [Vg1 in the nucleus while Prrp and Xstau were recruited to the RNP complex only in the cytoplasm [ VegT-LS ,31,32. W VegT-LS ,36,37,38 in vivo ,38,39,40l cortex ,41,42. Rl cortex ,44. Morenanos-3′UTR has two different localization signals. The 240 nt mitochondrial cloud localization signal (MCLS) is both required and sufficient to direct nanos into the MC [nanos into germinal granules, an event that requires the prior functioning of the MCLS [nanos MCLS was shown to bind directly to Vg1RBP/Vera and hnRNP I in vitro, consistent with its ability to use the late pathway after injections into late staged oocytes [nanos association/entrapment event does not involve Vg1RBP/Vera, a protein implicated in linking Vg1 RNA to the ER [nanos, but not Vg1 RNA must exist in the stage I oocyte.The o the MC . In addithe MCLS . The nan oocytes ,46. Howeo the ER . How thehermes/rbpms that localize using both early and late pathways [The nature of the RNA-protein interactions operating in the early pathway that mediate the steps of RNA selection, entrapment, and translational regulation remain unknown. Complicating our understanding of these processes are RNAs such as pathways . Here wepathways  and latepathways . Functiopathways  and cellpathways , but itspathways .nanos in the MC, concentrating in the germ plasm forming region and within the germinal granules [nanos but not Vg1 or VegT RNA within the nucleus. Hermes/Rbpms binding to nanos 3′UTR requires an unknown component present in stage I but not stage VI oocyte extracts. The UGCAC repeats essential for nanos RNA MC localization are also required for Hermes/Rbpms binding to the MCLS. In addition, Hermes/Rbpms binds the GGLE domain, a region that does not contain UGCAC elements but does contain two VM1 hnRNP I binding sites. Further, we find that the terminal 34 amino acids in Hermes/Rbpms, a region conserved with human RBPMS, is required to form homodimers as well as to bind the nanos 3′UTR.We find that Hermes/Rbpms protein is present in the MC, throughout the cytoplasm and in the nucleus of stage I oocytes. However, unlike other RNA-binding proteins, we confirm that Hermes/Rbpms co-localizes with granules . Importananos RNP particle. Hermes/Rbpms functions as a homodimer in concert with other proteins to facilitate granule formation. We propose that Hermes/Rbpms binds nanos RNA in the nucleus in association with hnRNP I, initiating a series of events that results in nanos but not Vg1 entering the early pathway. We suggest that, by incorporating nanos RNA into a Hermes/Rbpms nuclear particle, nanos RNA is effectively prevented from being translated after it exits the nucleus. To our knowledge, this is the first evidence for a nuclear RNA-binding protein specific for the early pathway.Taken together, our observations show that Hermes/Rbpms is an important component of the l-glutamine, and 5 µg/mL gentamycin).Stage I/II oocytes were obtained after collagenase treatment of surgically removed ovarian tissue as previously described . Myc-HerAfter the two-day culture period, the nuclear and cytoplasmic fractions were manually isolated in OR2 buffer. After injection of 1nl of water, the nucleus emerged from the needle-puncture made in the plasma membrane. Whole oocytes, ooplasms, and nuclear samples were homogenized in YSS buffer  and centrifuged at 16,000 g for 3 min to remove the insoluble materials. Anti-myc antibody  and protein G beads were added to the supernatant. The mixture was incubated at 4 °C overnight. The beads were then washed with YSS buffer four times. The bound RNAs were recovered with proteinase K digestion and analyzed by RT-PCR using the primers: Nanos-F, gaggctacacttgccctttg and Nanos-R, gcccattagtggtgcagaat; Vg1-F, atgcctattgcttctatttgc and Vg1-R, ggtttacgatggtttcactca and VegT-F, caagtaaatgtgagaaaccg and VegT-R, caaatacacacacatttccc). Possible cross contamination between ooplasm and nuclei during isolation procedures was monitored by western blotting with anti-alpha tubulin  and anti-nucleoplasmin  antibodies respectively .Hermes/Rbpms deletion mutants were constructed as follows:in vitro transcription . The RNAs were translated in reticulocyte lysates following the manufacturer’s instructions (Promega).Hermes/Rbpms deletion A (a.a. 101–121 were deleted): the N terminal fragment was amplified with Hermes/Rbpms ATTTAGGTGACACTATAGAgtacaccatgagcggcatcaagtcagaca (forward) and Hermes/Rbpms-DA1 (CTTTGTGTTGGCCTTTGC). The C terminal fragment was amplified with Hermes/Rbpms-DA2 (GCAAAGGCCAACACAAAGCACTTCATTGCACGAGAT) and Hermes/Rbpms TTTTTTTTTTTTTTTTTAACAAAACTGCCGAGACT (reverse 1). The two fragments were then fused by PCR. Hermes/Rbpms deletion B (a.a. 122–161 were deleted): the N terminal fragment was amplified with Hermes/Rbpms ATTTAGGTGACACTATAGAgtacaccatgagcggcatcaagtcagaca (forward) and Hermes/Rbpms-DB1 (TGCGCCAAGTGCTGGGTG). The C terminal fragment was amplified with Hermes/Rbpms-DB2 (CACCCAGCACTTGGCGCAGCTTTCACATACCCTGCT) and Hermes/Rbpms TTTTTTTTTTTTTTTTTAACAAAACTGCCGAGACT (reverse 1). The two fragments were then fused by PCR. Hermes/Rbpms deletion C (a.a. 164–198 were deleted) was amplified with Hermes/Rbpms ATTTAGGTGACACTATAGAgtacaccatgagcggcatcaagtcagaca (forward) and Hermes/Rbpms TTTTTTTTTTTTTTTTTAAGCAGCATGTGGAATGGC (reverse 2). The resulting PCR products from Hermes/Rbpms deletion mutants were used as templates for et al., 2007 [Myc-Hermes/Rbpms: plasmid pCS2+6Xmyc Hermes/Rbpms was designed as detailed in Song l., 2007 . hnRNP Il., 2007   and GST-Hermes/Rbpms (AGCTATCTCGAGTTAACAAAACTGCCGAGA (reverse). The PCR product was digested with EcoRI and XhoI and cloned into pGEX-5x-1.et al., 2004 [nanos-3UTR was injected 20 h before fixation and antibody staining.Sense RNAs were transcribed from NotI-linearized plasmids with SP6 RNA polymerase and the mMessage mMachine kit . For fluorescent probes, 600 µM Alexa 488-UTP or tetramethylrhodamine-UTP  was used exactly as described in Chang 35S-Methionine labeled Hermes proteins were synthesized in rabbit reticulocyte lysates (Promega) using capped in vitro transcribed RNA following the manufacturer’s protocol. For radiolabeling, 20 µCi of 35S-methionine was included . Samples were immunoprecipitated under denaturing conditions with anti-myc antibody and analyzed by SDS-PAGE. The bound protein was quantitated using a PhosphorImager™  with the ImageQuant software package .RNA was transcribed with mMessage Machine (Ambion) and purified by LiCl precipitation. 0.2–0.5 µg of RNA was oxidized with 1 mg/mL of sodium periodate in 0.1 M NaAc (pH 5.0) at room temperature for one hour. The oxidized RNA was then immobilized on adipic acid dehydrazide agarose (AADA) beads  in 0.1 M NaAc (pH 5.0) at 4 °C over night. The RNA-AADA beads were washed with 1x TPB buffer .in vitro translated Myc-Hermes/Rbpms labeled with 35S-methionine for 5 min at room temperature. The mixture was then precleared for 30 min with AADA agarose beads prewashed with 1x TPB buffer at room temperature. After centrifugation at 16,000 g for 3 min, the supernatant was mixed with RNA-AADA beads at room temperature for 5 min. The beads were then washed three times with 1x TPB buffer. The radio-labeled bound proteins were analyzed by SDS-PAGE and visualized using a PhosphorImager™.Stage I/II oocytes were homogenized in the presence of 1x protease inhibitor (Roche), DTT (0.3 mM) and heparin (5 mg/mL). Insoluble materials were removed by centrifugation at 16,000 g for 3 min. The supernatant was mixed with Xenopus oocyte two-hybrid cDNA library cloned in pGAD10 was obtained from Clontech. Two hundred micrograms of plasmid DNA from each library was transformed into competent yeast L40-coat/WT cells as described by Clontech. The Hermes/Rbpms coding region was used as bait and amplified with pAS1-Hermes/Rbpms AGCTAGGCCATGG AGagcggcatcaagtcagac (forward) and pAS1-Hermes/Rbpms AGTCAGGTCG ACTTAACAAAACTGCCGAGA (reverse). The PCR product was cloned into the pAS1 vector (from Dr. S. Elledge) using NcoI and SalI. Hermes/Rbpms was fused to the C terminus of Gal4 DNA binding domain by PCR.We identified proteins interacting with Hermes/Rbpms using the Two-Hybrid System 3 from Clontech following their detailed protocol . System 3 uses yeast strain AH109, which includes four reporter genes  to help reduce false positives. High stringency conditions were used plating yeast on SD/-Ade/-His/-Leu/-Trp/X-a-gal plus 10mM 3-AT following the procedures presented by Gietz and Schiestl . A Xenopet al., 2004 [et al., 2004 [® 568-conjugated goat anti–rabbit  at 1:100 [et al., 2003 [Oocytes were fixed and processed exactly as described in Chang l., 2004  for immul., 2004 . Anti- Vat 1:100 . Rat monl., 2003   RNA and Hermes/Rbpms protein were identified by in situ hybridization as described in Song et al., 2007 [et al., 2002 [All hybridization and post-hybridization steps, embedding, and sectioning were done as described in Kloc al. 2001 . Nanos  of the University of Miami. All activities are in compliance with federal, state, and institutional regulations. The University was granted full accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) in February 2005 and received its current re-accreditation in 22 October 2013. In addition, University of Miami is licensed by the U.S. Department of Agriculture (USDA) and has filed a Letter of Assurance with the Office of Laboratory Animal Welfare (OLAW), U.S. Department of Health and Human Services (DHHS).et al., 2004 [nanos RNA are predominantly localized within the germinal granules . These findings strongly suggested another protein(s) is required for Hermes/Rbpms to efficiently associate with the nanos 3′UTR [nanos. Importantly, Hermes/Rbpms did not bind the Vg1 localization signal (LS), suggesting a level of discrimination between these two RNAs at the level of Hermes/Rbpms protein binding  just downstream of the MCLS directs the MCLS . The rednanos RNA and whether that region was required for Hermes/Rbpms to form homodimers. Hermes/Rbpms protein has an N terminal RRM domain required but not sufficient for binding polyA+ RNAs [nanos binding. Each deletion mutant as well as wild-type Hermes/Rbpms was first translated in vitro and shown to translate equally well by western blotting  made from Xenopus oocyte mRNA containing 2.5 × 106 independent clones with an average insert size of 1.5 kb in a yeast-two-hybrid assay. The known RNA-binding protein hnRNP I was identified from approximately 700 × 103 clones . Thus, we were unable to use this approach to extend our analysis in vivo.We attempted to visually capture Hermes/Rbpms-nanos RNA associated with Hermes/Rbpms while the late pathway RNA Vg1 did not. These results suggested that Hermes/Rbpms could discriminate between endogenous RNAs that employ different localization pathways. To determine if such discrimination occurs in vivo, myc-Hermes/Rbpms RNA was injected into stage I oocytes. The oocytes were cultured for two days to allow translation to occur  Hermes/Rbpms co-localizes with nanos RNA within the MC and is a component of germinal granules  for sorting RNAs into their correct locations remains poorly understood. Early recognition by RNA-binding proteins within the nucleus is one attractive mechanism for initiating an essential sorting step in RNA localization. In this report, we show that Hermes/Rbpms recognizes ranules  Thethe MCLS B; (4) ThVg1 RNA have provided support that the late RNA localization pathway initiates in the nucleus [Vg1 RNA in the nucleus. Interestingly, once the Vg1 RNP exits the nucleus and enters the cytoplasm, additional proteins are now added and Vg1RBP/Vera and hnRNP I are no longer in direct contact. Staufen is one of the additional proteins found in the cytoplasmic Vg1RNP complex. Staufen likely initiates association with the motor protein kinesin and thus promotes active transport to the vegetal pole [Vg1 active transport to the vegetal cortex using the late pathway.Studies on  nucleus ,60. Thostal pole ,42. TherVg1 studies and those reported here. Nanos RNA passes through five cellular compartments before localizing into germinal granules: the nucleus, cytoplasm, MC, ER, and germ plasm. None of these steps require active transport. Each cellular location could involve remodeling, that is proteins added, subtracted, or modified. Hermes/Rbpms association with nanos RNA, but not with Vg1 or VegT RNAs within the nucleus suggests that Hermes/Rbpms binding might initiate a sorting pathway that terminates with nanos/Hermes/Rbpms within the germinal granules.There are parallels to be drawn between the nanos pulled down Hermes/Rbpms only when the reticulocyte lysate was supplemented with a protein extract from stage I oocytes. These results strongly suggested other proteins were required for Hermes/Rbpms binding to the nanos 3′UTR [nanos RNP? In vitro UV-crosslinking experiments show a specific interaction between hnRNP I and nanos RNA, however an association in vivo remains unclear [nanos would be temporary  and could be a good candidate for one of the factors required for Hermes/Rbpms nanos RNA binding. Their analysis further suggests that Rbm24b and XveloSV associate with each other in the cytoplasm, but not in the nucleus indicating dynamic remodeling likely does occur.Nijjar and Woodland  have rece stages . Althouge stages . With thnanos RNP after it exits the nucleus.Hermes/Rbpms colocalization with Xvelo within the germ plasm particles is a significant finding . Xvelo iXenopus germinal granules [The question remains as to what function Hermes/Rbpms performs within the germ plasm. Hermes/Rbpms has been studied in a wide range of cell types and tissues ,63,64. Agranules ,65 or mogranules . Hermes/granules .Xenopus MC revealed the presence of NonO (our unpublished results). Hermes/Rbpms containing granules are transported down growing axons. Hermes/Rbpms gain-of-function and loss-of function experiments in vivo in RGCs reveal a complex phenotype consistent with a role in balancing synaptogenesis and axon arborization [In vivo, this mutant failed to form granules and localize properly. Their results are consistent with our findings that the C-terminus is required for Hermes/Rbpms to oligomerize and to bind nanos RNA.In RGCs, Hermes/Rbpms forms RNP granules by interacting with other RNA binding proteins NonO, PSF, and G3BP1. Proteonomic analysis of the rization . In thatnanos RNA  within the MC [nanos RNP particle as it moves to the germ plasm and is incorporated within germinal granules is an important concept. Additional studies will be required to clarify the endogenous nanos RNP particle during its different localization steps that culminate in the germinal granule.Our data are consistent with a model where multiple copies of Hermes/Rbpms specifically bind n the MC ,58. With


The conserved motif consists of three hairpins with one being especially thermodynamically stable. The biological importance of this conserved secondary structure is supported in experiments using antisense oligonucleotides in cell line, which found that disruption of this motif led to inhibition of viral fitness. These results suggest that this conserved motif in the segment 5 (+)RNA might be a candidate for oligonucleotide-based antiviral therapy.Influenza A virus is a threat to humans due to seasonal epidemics and infrequent, but dangerous, pandemics that lead to widespread infection and death. Eight segments of RNA constitute the genome of this virus and they encode greater than eight proteins via alternative splicing of coding (+)RNAs generated from the genomic (-)RNA template strand. RNA is essential in its life cycle. A bioinformatics analysis of segment 5, which encodes nucleoprotein, revealed a conserved structural motif in the (+)RNA. The secondary structure proposed by energy minimization and comparative analysis agrees with structure predicted based on experimental data using a 121 nucleotide  Influenza A virus is a grave threat to human health. In 1918 the Spanish flu (H1N1strain) caused the deaths of over 50 million people . In 1997Orthomyxoviridae and possesses a segmented, negative-sense RNA genomic viral (v)RNA. RNA is used throughout infection and plays roles in every process of viral life cycle. Replication begins with vRNA acting as a template to produce two plus-sense (+)RNAs: a complementary (c)RNA intermediate, which in turn becomes the template for producing more vRNA strands, as well as protein coding mRNA 3+ at concentrations from 5 to 25 mM.The consensus sequence of nts 1051–1171 (+)RNA of segment 5 was synthesized (M121) for 2 at 23°C and pH 7. For enzymatic mapping RNase T1 (cleaves after unpaired G), RNase S1 (cleaves after unpaired nucleotide), and RNase V1 (cleaves after paired nucleotide) were used. Small molecules CMCT (modifies N3 of U and N1 of G when unpaired), DMS (methylates N1 of A and N3 of C when unpaired), and kethoxal (modifies G when unpaired) were used to probe the Watson–Crick face of bases. Lead ion cleavage and SHAPE mapping were used to identify flexible backbone regions.RNA secondary structure was mapped in a buffer containing 100 mM KCl, 5 mM MgClThe results of chemical mapping were used to constrain minimum free energy secondary structure prediction using RNAstructure v. 5.5  Fig 2A)Fig 2A). 2+ cleavages were occurred after 1124 and 1126 nucleotides. There were RNase T1 cuts after G1124 and G1126 and S1 cuts after nucleotides 1124–1128 and no cleavage by RNase V1, which offers good support for this region being unpaired.Chemical and enzymatic mapping are complementary to each other and show two the most flexible regions Figs , 4 and 52+ cleavage was observed after A1090 and there were no enzymes cleavages. Single stranded regions between hairpins undergo strong modification in 1081–1084 by NMIA, DMS and kethoxal. In this region strong Pb2+ cleavages were observed in region 1080–1082. This fragment is more accessible than hairpin 1085–1096. Lead ion cleavage also indicates additional flexible nucleotides: 1096–1098 and 1150, 1151 1154, 1155. These predicted single-stranded regions have only two identified chemical mapping hits  as nts 1171–1132 were not able to be read-out by reverse transcription. Hairpins 1064–1080 and 1103–1147 and helix 1053-1062/1168-1160 are well confirmed in structural analysis. Additionally, mapped by NMIA, DMS and kethoxal nucleotides confirmed bulges: A1111-A1112, C1116, A1120-A1121, G1132, and A1058. After 1110 and 1116 nucleotides strong Pb2+ cleavages were observed, where 1110 is the strongest from all.  and 2,6-diaminopurine modifications at selected positions , 35. In LUCLCGL (symbol NL indicate specific locked nucleic acid (LNA) nucleotide) has binding site at 1105 which is helix region. It is possible, because of G and A rich contest of target, that this probe form triplex instead of disrupting the double helix. Additionally, possible binding sites are in single stranded regions: 1084–1085, 1096, 1099, 1101–1103, 1126. Binding sites at 37°C provide target regions for potentially clinically useful antisense oligonucleotides.The results of hybridizations of M121 to isoenergetic microarrays for three temperatures  are collected in in vitro construct are shown by blue letters. The binding site for the reverse transcription primer was chosen to be an appropriate distance to motif and enable to read out nts 1051–1171. In general, reactivity is in agreement with proposed secondary structure: e.g. hairpins 1064–1080 and 1103–1147 RNA is at nts 1150–1156, which are unreactive in the (+)RNA. No alternative folds of the studied region (including pseudoknot) could explain these differences; they may be due to a tertiary interaction with another region in the (+)RNA, for example, the nearby region (nts 991–997) is complementary to 1150–1156.The structure of M121 was checked in context of entire segment 5 (+)RNA sequence [A/VietNam/1203/2004(H5N1)]. The results of chemical mapping of nts 1051–1171 using NMIA, DMS, CMCT and kethoxal is presented on 103–1147 . Also, m103–1147 . The helInfluenza RNAs were previously targeted with oligonucleotides as potential antiviral agents –43. SeveThe importance of the M121 structural motif was investigated using an antisense oligonucleotide inhibition assay with a single-cycle infectious influenza A virus (sciIAV) [strain A/California/04_NYICE_E3/2009 (H1N1)] in MDCK-HA cells. The chosen strain differs slightly  and LUCULCAULUUGAAGCCAU (3A) targeted the long hairpin and internal loop of M121   (CAUUCUCAUUU (1A) slightly inhibit virus: FFU/ml decrease by 1.64 times, which gives 0.16 LOG10 FFU/ml difference.2’-O-Methylated oligonucleotides with LNA modifications: ference) . This haCCUGG/3’UGAGAAC5’AC). Targeting nts 1148–1158 with 2’-O-methyl oligonucleotides: 1B or 2B , respectively, did not yield significant effects and only moderate inhibition for both 1B and 2B were observed. This is in agreement with in vitro mapping results, which show partial protection of the region from chemicals and lack of accessibility by microarray. Additionally, RNase H cleavage in presence of DNA oligonucleotide TCAAGAGTG complementary to 1150–1158 resulted in only weak cutting after U1155. It is likely that nts 1151–1157 is involved in weak tertiary interactions.For (+)RNA5 of A/California/04_NYICE_E3/2009 the long distance interactions between regions: 1151-1157/991-997 is possible, but weaker: there are two mismatches (AUCACUUUCUU (1C) complementary to region 1067–1077 did not influence the virus replication, suggesting that this hairpin is less important to the virus or that it is less accessible to the oligonucleotide.2’-O-Methyl oligonucleotide Presenting here approach leading from finding conserved secondary structure motifs in virus RNA to application of antisense oligonucleotides disturbing the motif possible function would be useful in therapeutics development.in vitro RNA construct comprising an influenza A virus consensus sequence and also an entire segment 5 (+)RNA (strain A/VietNam/1203/2004 (H5N1)). The conserved motif consists of three hairpins with one being especially thermodynamically stable. The biological importance of this conserved secondary structure is supported in experiments using antisense oligonucleotides in cell line, which found that disruption of this motif led to inhibition of viral fitness. These results suggest that this conserved motif in the segment 5 (+)RNA might be a candidate for oligonucleotide-based antiviral therapy.A bioinformatics analysis of the viral genome segment 5, which encodes nucleoprotein, revealed a conserved structural motif in the (+)RNA. The secondary structure proposed by energy minimization and comparative analysis agrees with structure predicted based on experimental data: using a 121 nucleotide


RNA silencing is a biochemical mechanism that regulates gene expression by post-transcriptionally activating a sequence-specific RNA degradation by three different pathways: (i) small interfering RNA (siRNA) silencing of exogenous mRNA; (ii) micro RNA (miRNA) silencing of endogenous mRNAs, and (iii) associated with DNA methylation and suppression of transcription .These processes share three basic biochemical phases: (i) formation of double-stranded (ds)RNA; (ii) processing of dsRNA into small dsRNA molecules (sRNA); and (iii) targeting of single-stranded small RNA to sequence-specific DNA or RNA molecules. Although several mechanisms can generate dsRNA, the sRNA processing and effector phases have a common biochemical core.One key-component of this system is small RNA molecules of 21 to 26 nt called siRNAs  which originated from longer double-stranded RNA (dsRNAs) cleaved by a specific RNase endonuclease called DICER with distinctive dsRNA binding, RNA helicase, RNase III and PAZ (Piwi/Argonaute/Zwille) domains. The siRNA strands are further relaxed (unwound) and one strand is incorporated in a RISC complex (RNA-induced silencing complex) which contains a member of the Argonaute (Ago) protein family, guiding this complex to an mRNA with a complementary sequence, which is then cleaved, leading to gene silencing.RNAi can be triggered by double-stranded or partially self-complementary hairpin RNA formation. In addition to playing a powerful role in creating loss of function mutations in plants, RNAi biological functions include also the regulation of endogenous gene expressions, such as micro RNA (miRNA), heterochromatin formation, transposon repression and defence against viral infection ,3.Several strategies have been employed for genetically engineering resistance to viruses in transgenic plants, including the expression of coat protein genes, the expression of truncated defective genes and antisense RNA. Now that we better understand the mechanisms of RNA silencing (RNAi) and its biological functions, it is possible to look back on initial experiments from a new perspective. It is now known that plants naturally process viral RNAs to generate small sequences of a pathogen's genetic material that can be specifically used against that pathogen through the RNA-induced silencing complex. It was recognized that an RNAsilencing  mechanism was responsible for the resistance against RNA viruses, and that it depends on the formation of double-strand RNA (dsRNA) whose antisense strand is complementary to the transcript of a targeted gene. This discovery led to the introduction in transgenic plants of constructs to produce intracellular generation of siRNA-like species to induce targeted gene silencing and virus resistance.RNA silencing has been an important tool to generate plants resistant to a large range of viruses. RNA sense- or antisense-mediated strategies resulted in a maximum resistance frequency of 20%, but often far lower frequencies were obtained. In addition, not all viral genes used in transgenic constructs rendered resistant plants. The use of inverted repeat constructs, resulting in dsRNA transcripts, rendered a very efficient system in which much higher frequency of transformed lines will display efficient gene knockdown or virus resistance. The probable reason is that the dsRNAs fed directly into the silencing pathway at the level of the RNase III-like enzyme Dicer, and therefore there is no reliance on the action of plant-encoded RNA-dependent RNA polymerase proteins. So far, most examples of RNAi-mediated virus resistance are related to RNA plant viruses.Bean golden mosaic virus (BGMV), transmitted by the whitefly Bemisia tabaci Gen., is one of the most limiting diseases for bean cultivation in the American continent. Since highly resistant bean varieties are unavailable, control practices have focused on controlling the vector with high-toxicity insecticides, with cost-benefit ratio and environmental concerns. In Brazil, the golden mosaic causes annual losses in bean production ranging from 90,000 to 280,000 tons. In addition, there are approximately 180,000 hectares unsuitable for bean cropping in the dry season due to the high prevalence of BGMVGolden mosaic, caused by the rep viral gene, which encodes the only protein strictly essential for viral genome replication [B. tabaci pressure . In contrast, non-transgenic varieties have shown up to 100% infection, presenting characteristic severe symptoms of the golden mosaic disease.The concept of using RNAi constructs was explored to generate genetically modified (GM) bean lines resistant to the BGMV by silencing the lication . Two linIn 2011, a transgenic bean event  resistant to the BGMV was approved for cultivation and consumption in Brazil. Biosafety evaluations were carried out taking into account the protocol proposed by the Brazilian Biosafety Committee (CTNBio) to demonstrate the safety of transgenic bean event to the environment and human health. Analyses have suggested no differences between the transgenic event and its non-transgenic counterpart.


The recent review of Matthew D. Krasowski on ‘Therapeutic Drug Monitoring of the Newer Anti-Epilepsy Medications’ is a useful foundation of comparative interpretations on our current knowledge about therapeutic drug monitoring. Within the review, the statement that therapeutic drug monitoring has a minimal role in the therapeutic use of eslicarbazepine acetate due to its relatively predictable pharmacokinetics reflects the existing body of evidence although some information such as eslicarbazepine acetate’s chemical structure, proportions of its metabolites, their pharmacokinetics and chiral method of plasma level measurement need to be revised. These critical characteristics differentiate the novel compound from former dibenzazepines such as carbamazepine and oxcarbazepine in its clinical effects and needs for therapeutic drug monitoring. Pharmaceuticals [a S.A. located in São Mamede do Coronado, Portugal.We read with interest the recent review of Krasowski on ‘Therapeutic Drug Monitoring of the Newer Anti-Epilepsy Medications’, published in the June 2010 edition of ®) summary of product characteristics (SPC) approved by the European Medicinal Agency (EMA) [The comparative information depicted was useful in terms of providing comprehensive knowledge on TDM needs for AEDs in general, and very much in line with the data published in peer reviewed journals, non-published data on file in our records, and also the publicly available ESL (Zebinixcy (EMA) ,3. The sHowever, some of the ESL related information in the tables and figures were not in agreement with the referred text on page 1915, nor did the chemical structure in ‘Figure 1’ of the same page precisely represent the compound. In fact, the correct structure of ESL differs from that of other dibenzazepines in an acetate group that requires the addition of a methyl group to the existing drawing . As seenAn additional note in the same column of the ‘Table 2’ highlights the propensity of auto-induction with chronic dosing as a characteristic of ESL that favors the use of TDM. However, this highlight in ‘Table 2’ is not in line with our current knowledge that indicates ESL as a compound not susceptible to cause metabolic autoinduction, which was also defined paradoxically within the text of the same review on p. 1915 .max) of 2-3 hours, which is not in line with the data presented in ‘Table 1’ on page 1913 [According to the SPC and published research, ESL displays a bioavailability exceeding 90% and a time to peak concentration  has been developed as an enantioselective method for the detection of ESL and its main active metabolites such as eslicarbazepine is true . As addiWe believe that with the inclusion of some revisions and additional information, this comprehensive review will provide the clinicians with accurate guidance and useful hints on therapeutic plasma level monitoring of antiepileptic compounds, and will close some of the information gaps in this field.


N-(arylsulfonyl)-4-fluorobenzamides, namely 4-fluoro-N-(2-methylphenylsulfonyl)benzamide, (I), N-(2-chlorophenylsulfonyl)-4-fluorobenzamide, (II), and N-(4-chlorophenylsulfonyl)-4-fluorobenzamide monohydrate, (III), are described and compared with related structures. The conformation of the three molecules is very similar with the aromatic rings being inclined to one another by 82.83 (11) and 85.01 (10)° in the two independent molecules of (I), 89.91 (10)° in (II) and 81.82 (11)° in (III).The crystal structures of three  N-arylsulfonyl-4-fluorobenzamides, namely 4-fluoro-N-(2-methylphenylsulfonyl)benzamide, C14H12FNO3S, (I), N-(2-chlorophenylsulfonyl)-4-fluorobenzamide, C13H9ClFNO3S, (II), and N-(4-chlorophenylsulfonyl)-4-fluorobenzamide monohydrate, C13H9ClFNO3S·H2O, (III), are described and compared with related structures. The asymmetric unit of (I) contains two independent molecules (A and B), while that of (II) contains just one molecule, and that of (III) contains a molecule of water in addition to one main molecule. The dihedral angle between the benzene rings is 82.83 (11)° in molecule A and 85.01 (10)° in molecule B of (I), compared to 89.91 (10)° in (II) and 81.82 (11)° in (III). The crystal structure of (I) features strong N—H⋯O hydrogen bonds between the A and B molecules, resulting in an R44(16) tetrameric unit. These tetrameric units are connected into sheets in the bc plane by various C—H⋯O interactions, and adjacent sheets are further interlinked via C—H⋯πaryl interactions, forming a three-dimensional architecture. The crystal structure is further stabilized by πaryl–πaryl and S=O⋯πaryl interactions. In the crystal of (II), molecules are connected into R22(8) and R22(14) dimers via N—H⋯O hydrogen bonds and C—H⋯O interactions, respectively; the dimers are further interconnected via a weak C=O⋯πaryl interaction, leading to the formation of chains along [1-10]. In the crystal of (III), N—H⋯O and O—H⋯O hydrogen bonds involving both the main molecule and the solvent water molecule results in the formation of sheets parallel to the bc plane. The sheets are further connected by C—H⋯O interactions and weak C—Cl⋯πaryl, C—F⋯πaryl and S=O⋯πaryl interactions, forming a three-dimensional architecture.The crystal structures of three  An intramolecular C14B–-H14B⋯O2B hydrogen bond S(6) ring motif.The asymmetric unit of compound (I)B) Fig. 1, that diB) Fig. 1 containsB) Fig. 1 crystallnt Fig. 1. This isA—H1A⋯O1B and N1B—H1B⋯O1A hydrogen bonds , while in the second level the tetrameric unit has a graph-set motif of R44(16). Adjacent tetramers are connected into sheets in the bc plane  aryl–πaryl interactions  Å; Cg1 and Cg2 are the centroids of the fluorobenzoyl rings of molecules A and B, respectively] and also by weak S1A=O2A⋯πaryl interactions .The crystal structure of (I)s Table 1 between it Fig. 4. The unine Fig. 4 via C6A—s Table 1. AdjacenB) Fig. 5 to form ns Fig. 6  connect these dimers, thus forming a one-dimensional architecture .In the crystal of (II)re Fig. 7b.via bridging water molecules, through strong N1—H1⋯O4, O4—H1O4⋯O1, O4—H2O4⋯O2 and O4—H2O4⋯O3 hydrogen bonds  Å], C11—F1⋯Cg2 [F1⋯Cg2 = 3.8674 (17) Å] and S1=O2⋯Cg1 interactions [O2⋯Cg1 = 3.2039 (17) Å] , forming a complex three-dimensional architecture s Table 3, resultie Figs. 8 and 9 ▸.viz N-(arylsulfonyl)-4-(substituted)benzamides, gave 14 hits. These fourteen compounds along with the three title compounds, (I)–(III), are grouped into three series; series 1: N-(2-methylphenylsulfonyl)benzamide, N-(2-methylphenylsulfonyl)-4-(chloro/methyl/nitro/methoxy)benzamides and (I)series 2: N-(2-chlorophenylsulfonyl)benzamide, N-(2-chlorophenylsulfonyl)-4-(chloro/methyl/nitro/methoxy)benzamides and (II)series 3: N-(4-chlorophenylsulfonyl)benzamide, N-(4-chlorophenylsulfonyl)-4-(chloro/methyl/nitro)benzamides and (III)A search of the Cambridge Structural Database benzamide -4-nitrobenzamide -4-methoxybenzamide -4-chlorobenzamide -4-methylbenzamide -4-fluorobenzamide (I)ortho-methyl group on the benzenesulfonyl ring is syn to the N—H bond in the central –C–SO2--N–C(O)– segment. The values of the dihedral angle between the two aromatic rings in the molecules of series 1 fall in the range 73.9 (1)– 89.4 (1)°, the smallest dihedral angle being in N-(2-methylphenylsulfonyl)benzamide and the largest in N-(2-methylphenylsulfonyl)-4-chlorobenzamide  hydrogen bonds. However, except for compound (I)1 Table 4, the asye Table 4. CompariSeries 2: The asymmetric units of all of the compounds in series 2 – segment. The values of the dihedral angle between the two aromatic rings in the molecules fall in the range 73.3 (1)–89.91 (10)°, which is almost the same as in series 1, the smallest being in N-(2-chlorophenylsulfonyl)benzamide -4-fluorobenzamide (II)N-(2-chlorophenylsulfonyl)-benzamide, N-(2-chlorophenylsulfonyl)-4-chlorobenzamide -4-methylbenzamide  hydrogen bonds, while strong N—H⋯O(S) hydrogen bonds in N-(2-chlorophenylsulfonyl)-4-nitrobenzamide -4-methoxybenzamide -4-fluorobenzamide (II)via C=O⋯πaryl interactions, forming a one-dimensional architecture.2 Table 5 contain Series 3: In series 3, the parent compound N-(4-chlorophenylsulfonyl)benzamide -4-chlorobenzamide -4-methylbenzamide -4-nitrobenzamide -4-fluorobenzamide (III)N-(4-chlorophenylsulfonyl)benzamide and the largest for N-(4-chlorophenylsulfonyl)-4-methylbenzamide (Table 6e Table 6. Except Compounds (I)–(III) were prepared by refluxing a mixture of 4-fluorobenzoic acid, the corresponding substituted benzenesulfonamides and phosphorousoxychloride for 3 h on a water bath. The resultant mixtures were cooled and poured into ice-cold water. The solids obtained were filtered, washed thoroughly with water and then dissolved in sodium bicarbonate solutions. The compounds were later re-precipitated by acidifying the filtered solutions with dilute HCl. They were filtered, dried and recrystallized. [Melting point (m.p.) of (I)Uiso = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms. In the final cycles of refinement, reflections (0 1 1), (0 0 2) and  I, II, III, global. DOI: 1470505, 1470504, 1470503CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: