A symmetric pocket-like entity, composed of two L-shaped RNA units, encircles the peptide synthesis site within the contemporary ribosome. This entity was suggested to be the vestige of a dimeric proto-ribosome, which could have formed spontaneously in the prebiotic world, catalyzing non-coded peptide bond formation and elongation. This structural element, beyond offering the initial step in the evolution of translation, is hypothesized here to be linked to the origin of life. By catalyzing the production of random peptide chains, the proto-ribosome could have enabled the formation of primary enzymes, launching a process of co-evolution of the translation apparatus and the proteins, thus presenting an alternative to the RNA world hypothesis. In translation, the complicated process by which genetic information is translated into proteins, amino acids are polymerized into polypeptide chains that in turn fold into proteins. This takes place at the ribosome, a large, complex, universal ribonucleoprotein catalytic machine, composed of a large and a small subunit (50S and 30S in prokaryotes). The ribosome central task, peptide bond formation, occurs in the active site of the large subunit, the Peptidyl Transferase Center (PTC), which, at least in prokaryotes, is composed solely of RNA. Being a ribozyme can increase the likelihood that the ribosome remains active under protein-related stress conditions, enabling it to reproduce defective proteins.i.e. with the tRNA’s stems and elbow and with the various elongation factors. This zone, which forms a pocket-like RNA element . The dimer of the two core units, together with the A-, P- sites, emerging from far ends of H74 and H90 for P-, A- core units, respectively , assemblHere the structure of the dimeric proto-ribosome is discussed in details and a possible relevance to the origin of life is suggested.2.The dimeric proto-ribosome was a pocket assembled from two L-shaped RNA core units  Figure . Within The structure of the ribosomal core unit can be analyzed by comparing it to the known structures of other L-shaped molecules. The secondary structures of the core unit , the tecE.coli ribosomes . Although no two of the sequences gave identical 2D structure, the calculated structures were consistently arranged as a stem-elbow-stem fold. The average free energy of the 3′:5′ elbow form was in the range Δ G=26 ± 3 kcal/mole, indicating the self-folding and stability of the obtained structures [i.e. temperature of 37 °C and ionic conditions fixed at [Na+]=1 M, with no divalent ions. If the prebiotic conditions were similar to the Mfold default ones, the 3′:5′ elbow form may have been the abundant one. Under much different environmental conditions, however, a tRNA-like structure could have been the more stable fold of the core unit.Secondary structures of short RNA sequences, similar in size to the tRNA structure, can be reliably predicted by free energy minimization  and can A 3D comparison of the L-shaped structures reveals that while the core unit elbow angle is stabilized at about 105° , the elbThe core unit and the tRNA have comparable size and outline . In partin vitro . These results support the notion that self-folding of oligonucleotides and self-assembly of core units into pocket-like entities are energetically favorable processes that could have occurred spontaneously in a primordial world, producing the dimeric proto-ribosomes.Experimental work conducted in Ada Yonath’s group, mainly by Chen Davidovich, indicated that some short RNA sequences resembling those observed in the current ribosome are capable of forming dimers spontaneously. Moreover, the variation in the stems length of the ribosomal core units was found to hamper dimerization, suggesting that the dimers have a ‘pocket-like’ structure [E. coli, for instance, only 38% of the symmetrical mates have identical nucleotides type, relative to 25% expected from random sequences. The minor sequential match could have been affected to some extent by later mutations, but it still points to an inherent limited sequence specificity required from the unit core monomer and thus- from the dimeric proto-ribosome.Consistent with the expectation for a continuous evolutionary path from the proto-ribosome towards the modern ribosome, the dimeric proto-ribosome is assumed to have accommodated the reacting amino acids in the same manner as the modern ribosome does , thus al3.Two prerequisites are obligatory for the prebiotic formation of the dimeric proto-ribosome: the primordial existence of amino acids and of oligonucleotides of about 35 mer , or This simple apparatus was probably the smallest possible dimeric proto-ribosome, as indicated by association of the core units into dimers through the tips of their stems . The looThe dimeric proto-ribosome was implicated in catalyzing peptide bond formation and achieving elongation rates exceeding those of the non-catalyzed reactions, but it may have also been involved in an additional task. In-vitro catalysis of protein folding was identified in domain V of 23S rRNA . The nucl- to d- amino acids due to the enhanced steric hindrance exerted on the d conformation [l- over d- amino acids in the prebiotic era as well, pointing at a possible relevance to origin of chirality in proteins.The hypothesis that the proto-ribosome was a dimeric pocket-like structure whose vestige is still embedded at the active site of the modern ribosome ,3 offersormation ,45. As ti.e. the dependence of proteins on RNA for their formation on the one hand, and the dependence of RNA replication on proteins in the present form of life, led to the suggestion of an RNA world, which preceded life as we know it [Above all- the current hypothesis may offer an alternative way to disentangle the egg and chicken paradox concerned with the origin of life. The paradox,  know it –48, wherThe capability of the dimeric proto-ribosome to spontaneously emerge from random oligonucleotides of sufficient length relies on two characteristics: the energetic down-hill processes of formation (folding and dimerization) and the rather limited sequence specificity required from the oligonucleotides involved in the production of the dimeric proto-ribosome.Consequently, if oligonucleotides of sufficient length existed in the prebiotic era, the proto-ribosomes would form spontaneously, possessing, according to their sequence, diverse peptide synthesis efficiencies. They would have produced a variety of random peptides, whose sequence depended on the distribution of the various amino acids in their vicinity. Such peptide chains would have accumulated in larger amounts in the vicinity of “efficient” RNA dimers, increasing the probability that some of the randomly produced peptidyl chains, which exceeded 30 residues in length, being therefore sufficiently long to allow stable folding , will foAn RNA-protein preliminary system would have naturally evolved into an RNA-protein world without the need to shift from an RNA world into an RNP world, as the RNA world hypothesis entails.The limited specificity of the dimeric proto-ribosome sequence increases the probability of randomly obtaining the suggested proto-ribosome, compared to a fixed-sequence ribozyme of the same size, and allows tolerance to the rather poor RNA copying abilities that the initial replicase (ribozyme or enzyme) would have probably had.The chemical versatility and efficiency of enzymes should have been beneficial in promoting the emergence of life.The primary molecular system suggested here to initiate the transition from the inanimate world towards life, i.e. the dimeric proto-ribosomes and the initial enzymes, might offer some advantages over the RNA world hypothesis:Further speculation could maintain that there may have been more than a single proto-ribosome involved in the emergence of life, as some sequence variations could have existed among equally efficient proto-ribosomes, allowing more than one proto-ribosome to be retained by evolution, possibly correlated with the later phylogenetic diversification.The suggested chemical prebiotic process, originating from an oligonucleotide, proceeding via a self-folded core unit that is more stable then the oligonucleotide, into a self-assembled dimer which is more stable than the single core unit, is an energetically downhill process and could have therefore occurred without any enzymatic aid. The process, however, is suggested to produce proto-ribosome pockets with different levels of protein synthesis efficiency, depending on their different sequences, thus having varying chance of survival. This is a distinctive application of Darwin’s principle of variation and selection, a principle that is usually applied to living organisms. In the scenario suggested here, the sequence variation emerged from the randomness of the RNA chains building the proto-ribosomes rather than from mutations, and was therefore a prebiotic phenomenon. Selection of the fittest proto-ribosome, which led to its survival, would have already been a life process. The appearance of the dimeric proto-ribosome may have therefore been the transition between thermodynamic selection, applicable to chemical reactions, and Darwinian selection, applicable to living organisms, thereby initializing the emergence of life.4.The dimeric proto-ribosome, which was probably the minimal one feasible for this kind of apparatus, could have offered a fitted template for the favorable positioning of small substrates, allowed primitive elongation and maybe assisted polypeptide folding, so catalyzing all the aspects of non-coded prebiotic protein synthesis. Limited variations in the sequences of equally efficient proto-ribosomes may have been possible in principle, potentially correlated with later evolved phylogenetic diversification. By catalyzing random polypeptide chains, suggested to result in the emergence of the primal enzymes, these proto-ribosomes may have initiated the co-evolution of the translation apparatus and the proteins, possibly rendering surplus the need for an RNA world. This apparatus, on the border between matter and living organisms, could have therefore been the starting point of our contemporary forms of life.


Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents. Candida sp. is the fourth most common pathogen isolated from blood cultures Cryptococcus sp., Aspergillus sp., Fusarium sp., and zygomycetes, have an unacceptably high morbidity and mortality Members of the fungal kingdom are found in almost all habitats and exist as saprobes, commensals, and pathogens. Approximately 11,000 plant diseases have been attributed to fungi in over 120 genera The diverse nature of fungi can be attributed to such factors as increased capacity to utilize a wide range of carbon and nitrogen sources, the capability for rapid growth, and the ability to adapt to otherwise harsh environments. Fungi are constantly bombarded by toxic compounds from external sources. These compounds can be synthesized by other microorganisms to impede the fungus for a competitive advantage to limited resources, synthesized by hosts as a defense mechanism, or may be present in the environment. Regardless of the source, these toxic compounds force the fungus to evolve mechanisms in order to survive. One common method to overcome these antifungal compounds is active efflux, which prevents any intracellular build-up of the compound and, therefore, renders the fungus resistant or tolerant to the otherwise toxic compound.As research in mycology progresses it is becoming evident that transporters are important factors in pathogenicity. Several transporters have been described as being involved in fungal pathogenicity, including a range of cellular processes such as calcium entry, vescicle transport, stress tolerance, dimorphic switching, capsule synthesis, iron acquition, and virulence factor enzyme activity Aspergillus nidulans and the closely related human pathogen Aspergillus fumigatus both have 45 ABC transporters encoded in their genomes, and A. nidulans has more MFS transporters than A. fumigatus , of which three families are involved in efflux of toxic compounds. Members of these three families, ABCB, ABCC, and ABCG, also referred to as the multi-drug resistance (MDR), multi-drug resistance–associated protein (MRP), and the pleiotropic drug resistance (PDR) families, respectively, have been extensively studied in 6 design of half ABC transporters, which dimerize to form a fully functional protein 6 half transporters, creating one functional protein (The conserved architecture of ABC transporters is composed of a nucleotide-binding domain (NBD) followed or preceded by six transmembrane-spanning helices creating a transmembrane domain (TMD). These two domains together represent the NBD-TMD protein  [21],[22+ antiporter (14 spanner) (DHA14) and the drug:H+ antiporter (12-spanner) (DHA12), are involved in efflux of toxic compounds The MFS transporters are smaller in size compared to the ABC transporters since they do not require an NBD; however, they still contain 12 or 14 transmembrane-spanning helices . TransloVirulence-associated efflux pumps in plant pathogenic fungi can be classified into two categories. The first class of transporters is responsible for secretion of virulence factors or compounds produced by the fungus. Several fungi synthesize and secrete low molecular weight compounds that are not required for growth. These compounds, termed secondary metabolites, are bioactive and are sometimes involved in virulence Cochliobolus and Alternaria species Cochliobolus carbonum produces a cyclic tetrapeptide HST, termed HC-toxin, which functions as an inhibitor of histone deacetylases in several organisms and is a virulence factor on maize TOXA and TOXB, postulated to be responsible for efflux of HC-toxin into the host plant cell Cercospora species produces a host-nonselective toxin, cercosporin, which is also synthesized by a secondary metabolite gene cluster. Cercosporin is exported out of the fungal cell by the cercosporin facilitator protein (CFP) in C. kikuchii and by CTB4 in C. nicotinaeMagnaporthe grisea produces a secondary metabolite via the PKS-NRPS fusion protein termed ACE1 and the surrounding accessory proteins. This cluster contains a gene encoding an MFS transporter (MFS1), although it is not involved in efflux of the ACE1 metabolite as it has a deletion of a single base pair resulting in an early stop codon Phytotoxins produced by fungi that are involved in virulence on a specific host plant, and therefore expand host range, are termed host-specific toxins (HSTs), and while not all plant pathogens produce HSTs, they are important virulence factors for members of the atrD in Aspergillus nidulans, although not in a cluster, produce less penicillin than the wild-type, suggesting this transporter is involved in efflux of the compound Efflux pumps in secondary metabolite gene clusters that are responsible for synthesis of mycotoxins are less well understood. The expression of MFS transporters in mycotoxin secondary metabolite clusters appears not to be controlled by the same transcription factors as the rest of the secondary metabolite cluster but, instead, is induced by the accumulation of the synthesized metabolite Nectria haematococca MPVI and the pea phytoalexin pisatin suggested that a fungal efflux mechanism is involved in the tolerance to this plant-derived defense compound and, therefore, may be a pathogenicity factor M. grisea, the causative agent of rice blast disease The second class of transporters involved in plant virulence is responsible for efflux of molecules produced by the host plant. As a defense mechanism, plants produce antimicrobial compounds. These low molecular weight compounds, termed phytoalexins or phytoanticipins depending on when they are synthesized, have diverse chemical structures. Over 25 years ago, studies conducted on Botrytis cinerea and GpABC1 in Gibberella pulicaris provide tolerance to the host plant's phytoalexins, resveratrol and rishitin, respectively BcatrB mutant is reduced in pathogenicity on grape leaves GpABC1 mutant is essentially nonpathogenic on potato N. haematococca is able to confer an increase in pathogenicity on pea when placed in a nonpathogenic pea isolate PEP5 for pea pathogenicity, is induced upon exposure to the pea phytoalexin pisatin Since that time a number of ABC transporters have been identified as virulence factors on host plants. Most of these transporters are members of the PDR family of ABC transporters . A numbeB. cinerea and Mycospharella graminicola, as well as the model filamentous fungus A. nidulans, have been reviewed recently B. cinerea is also able to provide resistance to the fungicides fenpiclonil and fludioxonil in addition to resveratrol, as referenced above Fungicide resistance is also due to ABC and MFS transporters. Several transporters in the agriculturally relevant fungi C. albicans, has a total of 21 ABC transporters and 85 MFS transporters encoded within its genome , and SNQ2 in C. glabrataS. cerevisiae. The PDR family is one of the largest families of ABC transporters in fungi and is overrepresented when their numbers are compared to other organisms, i.e., there are five and nine ABCG/PDR transporters in humans and Caenorhabditis elegans, respectively A number of transporters that confer resistance to members of the antifungal class of triazoles have been described. These include the ABC transporters CDR1 and CDR2 in CDR2 retain a wild-type level of resistance to fluconazole; however, in a double mutant (ΔCDR1/ΔCDR2) the strain is more susceptible to fluconazole than either single mutant CDR2 genes, and further phylogenetic analysis suggested that as many as 33 codon changes between the two alleles may be selectively advantageous Of the transporters involved in fluconazole resistance in medically important fungi, CDR1 and CDR2 are the most studied. Despite their high degree of amino acid similarity, there are functional differences between the two. Mutants of r) and FLU1 in C. albicansMDR1 have been implicated in fluconazole resistance in C. dubliniensisC. tropicalis+ translocation -Cys(6) finger transcription factor TAC1 CDR1 and CDR2, the transcript levels did not increase trans-acting factors of the CDR genes C. glabrata (CgPDR1 and CgPDR3) have been demonstrated to bind the substrate, promoting the expression of the efflux pumps under its control MDR1FLU1 in clinically resistant isolates is not increased, and therefore FLU1 is not believed to contribute significantly to resistance to fluconazole Pathogenic isolates are able to develop resistance to prescribed antifungal treatment rapidly CDR1/CDR2 overexpressing C. albicans isolates require the TAC1 fluconazole-resistant transcription factor to be homozygous, as heterogygous isolates retain wild-type levels of fluconazole susceptibility MRR1 increases fluconazole resistance CDR1, CDR2, and MRR1 overexpression have been identified, but their role in clinical resistance is less understood. These transcription factors include CaNdt80 in CDR1 overexpression MDR1 overexpression CDR1 transcripts in fluconazole-resistant C. albicans isolates that results in an increase in the number of CDR1 transporters A major difference in the mutations of the major transcription factors that confer fluconazole resistance is the heritability of the resistance phenotype. The S. cerevisiae and C. albicans have relatively few ABC and MFS transporters compared to filamentous fungi, and the scope of research already conducted on these important yeast superfamilies, it appears there is much more to be discovered in other fungi.The role of efflux pumps, in particular ABC and MFS transporters, in fungal virulence cannot be understated. These transporters contribute to pathogenesis by 1) transporting HSTs and mycotoxins outside of the cell, 2) removing host-derived compounds that would otherwise inhibit the fungus, and 3) providing resistance to clinical antifungals. This concept is further supported by the vast amount of substrate redundancy already evidenced with the few transporters that have been extensively studied. Considering The redundant nature of the transporters creates difficulty in characterization of the efflux mechanisms, as single deletion mutants sometimes retain a wild-type phenotype, and it is only through multiple mutations of transporters or expression in heterologous systems that the function of the transporters becomes apparent. Although a daunting endeavor, understanding all of the contributing efflux factors of a given antimicrobial will provide a means to circumvent efflux transporters.


Author affiliation 1 is incorrect. The correct affiliation is: Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China


Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma  Most tissues are composed of a variety of different cell types and therefore it is difficult to analyse gene expression and the biology of specific cell types based on DNA, RNA or protein preparations from the whole tissue Adult testes are composed of seminiferous tubules containing germ cells in different stages of spermatogenesis, embedded in Sertoli cells, which act as nursing cells ensuring the correct environment for the germ cells. Peritubular myoid cells line the tubules, and androgen-producing Leydig cells are located between the tubules in the connective tissue together with other interstitial cells such as fibroblasts, lymphocytes, Leydig cell precursors, and blood and lymph vessels. Thus, a normal adult testis contains at least 25 distinct cell types and their relative abundance varies according to developmental stage in situ of the testis (CIS), also known as intratubular germ cell neoplasia (ITGCN) is the precursor of the majority of testicular germ cell tumors (TGCTs) Carcinoma When microdissecting cells from frozen, dehydrated tissues, it can be very difficult to distinguish between the different cell types. The commonly used haematoxylin and eosin staining is not sufficient in testis tissue with many different cell types, therefore it is vital to have a specific staining protocol that can mark the cells for microdissection. For our studies of TDS, we needed to identify fetal germ cells, CIS cells and Leydig cells. For fetal germ cells and CIS cells we took advantage of their embryonic stem cell (ESC)-like properties The collection and use of human tissue samples for this project was approved by The Regional Committee for Medical Research Ethics in Denmark (no. KF-01-186 and H-KF-012006-3472). A written informed consent from the patients to use the leftover tissues for research was obtained by an andrologist assisting with the semen cryopreservation or the attending urologist before the surgery. Small samples of human adult testicular tissues containing CIS or seminoma  were collected at the Department of Pathology, Rigshospitalet after careful examination by a pathologist. The rat samples were collected under conditions approved by the Danish Agency for Protection of Experimental Animals and by the inhouse Animal Welfare Committee of the Institute for Food and Veterinary Research, DTU.2 in 1.5 L H2O; Solution II: 400 g 40% formaldehyde and 80 g 98% acetic acid, just before use mix 38 ml Solution I and 12 mL Solution II); or GR fixative . After fixation the tissue samples were embedded in paraffin.Tissue samples from four seminomas were fixed overnight in formalin ; Stieve's fixative  compound  and rapidly frozen. The frozen tissues were cut (10 µm sections) and collected on either superfrost slides  or nuclease and human nucleic acid-free membrane slides  , and fixed immediately.Seminoma samples from four patients were used for evaluation of RNA integrity in frozen and paraffin embedded, formalin, Stieve's or GR fixed tissues. Two 10 µm sections were cut under RNAse free conditions and immediately transferred to lysis buffer (frozen sections) or stored under dry, dark conditions (paraffin embedded tissues) until RNA purification.Cryosections were either stored at −80°C in a slide box, stored in absolute ethanol at −80°C, or the tissue was repeatedly removed from −80°C storage and sections cut from the same tissue samples after 0, 1, 3, 7 and 30 days. The experiment was performed in duplicates on two seminoma samples and two rat testes.We tested eleven different fixatives with regard to RNA quality, morphology and compatibility with the subsequent staining protocols, the fixatives were: 75% ethanol, 100% ethanol, 100% acetone, 100% methanol, 60% isopropanol, metacarn (6∶3∶1 methanol∶ chloroform∶acetic acid) modified metacarn (8∶1 methanol∶acetic acid), Carnoy's (6∶1∶3 ethanol∶acetic acid∶chloroform), Clarke's (3∶1 ethanol∶acetic acid), Meyer's (19∶1 ethanol∶acetic acid), and formalin. Fixation time was 10 min for all fixatives. The experiments were performed on two seminoma samples and two rat testes and the RNA evaluation was performed in duplicates.2], 10 sec haematoxylin and 10 sec wash in diethylpyrocarbonate (DEPC) treated destilled water quickly followed by dehydration in ethanol .Mayer's haematoxylin : 10 sec incubation buffer , 10 sec in DEPC water, followed by dehydration in ethanol as described above.Alkaline phosphatase with NBT BCIP 2 , 0.05 M TRIS-HCl buffer pH 8.7], 10 sec DEPC water and dehydration in ethanol as described above.Alkaline phosphatase with Kiel ORO RNA degradation after the different staining protocols was assessed in two seminoma samples that were sectioned and stained with haematoxylin, Kiel, NBT BCIP or ORO and two rat testes that were stained with haematoxylin and ORO. The RNA quality of sections from the four samples was examined immediately before and after the respective staining protocols and 4 h, 24 h and 72 hours after staining. All samples were run in duplicates.We used several different kits for testing the RNA quality of frozen versus paraffin embedded tissues. Frozen tissues: Nucleospin RNA II  and Rneasy Mini Kit ; paraffin embedded tissues High Pure RNA Paraffin Kit , Absolutely RNA FFPE Kit , Pure Link FFPE RNA Isolation Kit  and Rneasy FFPE Kit (Qiagen). In addition both frozen and paraffin embedded tissues were purified using TRIzol reagent according to the manufacturer , followed by precipitation with an equal amount of isopropanol, 0.1 volume 3 M sodium acetate (pH 5.2) and resuspension in RNAse free water. Furthermore, the samples were DNase treated using the RNAqueous-Micro Kit  according to the provided protocol.RNA from single frozen sections was purified using the Ambion RNAqueous micro kit (Ambion) according to the protocol provided by the manufacturer. The RNA quality was determined by the Bioanalyzer Picokit (Agilent Technologies).The statistical significance was evaluated by Student's t-test. All analyses were two-tailed and a p value less than 0.01 and 0.05 was considered statistically significant.Isolation of RNA from specific cells by LMD is preceded by a series of steps starting already when the tissue is removed from the patient or animal. In principle, all manipulations including removal and collection of tissues, should be performed as fast as possible and the tissue samples should be kept on ice and transferred to −80°C as fast as possible, after thawing and sectioning the tissues should be in aqueous solution for as short time as possible as this is where the RNAses thrive and RNA quality decreases.We tested the quality of RNA isolated from testicular tumor biopsies that were either frozen or fixed in different cross-linking fixatives  and embedded in paraffin .in situ hybridisation (ISH) Previous reports have shown that RNA-extraction from formalin fixed, paraffin embedded tissue results in small amounts of partially degraded and chemically modified RNA When microdissecting single cells, it may be necessary to collect cells from >30 tissue sections. Depending on the pace of RNA degradation, the optimal storage of sections may become an important question. We had a suspicion that repeated thawing of frozen tissues resulted in degradation of the RNA, and were sceptic about storing the sections at −80°C under dry conditions. Therefore we tested different storage conditions for the cryosections: we either stored the ethanol-fixed and dried sections at −80°C in a slide box; transferred them to 100% ethanol at −80°C; or cut fresh sections from frozen tissues that were repeatedly thawed prior to sectioning. Repeated thawing of the tissue markedly compromised the morphology and the RNA integrity was significantly decreased (p<0.05) after 30 days . FreezinThe choice of fixative is based on obtaining the best possible morphology while preserving the RNA quality. We tested the morphology and the RNA quality of frozen sections after fixation in 11 different fixatives and at the same time tested the compatibility with our staining protocols . PreviouWe optimised short staining protocols using Kiel solution, NBT BCIP, ORO and haematoxylin. Because TGCTs and fetal germ cells retain the expression of the “ESC-specific” alkaline phosphatase enzyme visualized by NBT BCIP and Kiel, these staining protocols can be used to stain TGCTs ORO staining is dependent on the presence of lipid droplets in the cytoplasm and both fixation and staining therefore cannot include incubation in solvents that dissolve the droplets. Thus, ORO staining worked well on sections fixed in 60% isopropanol, but formalin fixed sections could also be stained . ORO wasPrevious studies have shown that it is possible to perform reliable gene expression profiling using partially degraded RNA


RNA is increasingly recognized for its significant functions in biological systems and has recently become an important molecular target for therapeutics development. Aminoglycosides, a large class of clinically significant antibiotics, exert their biological functions by binding to prokaryotic ribosomal RNA (rRNA) and interfering with protein translation, resulting in bacterial cell death. They are also known to bind to viral mRNAs such as HIV-1 RRE and TAR. Consequently, aminoglycosides are accepted as the single most important model in understanding the principles that govern small molecule-RNA recognition, which is essential for the development of novel antibacterial, antiviral or even anti-oncogenic agents. This review outlines the chemical structures and mechanisms of molecular recognition and antibacterial activity of aminoglycosides and various aminoglycoside mimics that have recently been devised to improve biological efficacy, binding affinity and selectivity, or to circumvent bacterial resistance. RNA performs pivotal biological functions when organisms, including bacterial and viral pathogens, replicate. Developing small molecules that can interact with RNA and interrupt undesired cellular activities is a promising new path for drug design.Antisense and RNAi strategies have been widely recognized as a powerful tool to control RNA genes and regulate development of various organisms and cellular functions of interest.RNAs can fold into complex three-dimensional structures such as loops, bulges and pseudo-knots, and consequently, they present a unique challenge for custom design of the RNA binding molecules. Although some progress has been made in recent years in understanding how small molecules recognize RNA,Since they have been shown to selectively bind to a variety of RNA targets, aminoglycosidesmyo-inosa-1,3-diamine is actually derived from D-glucose-6-phosphate biosynthetically, the numbering system is based on streptamine’s biogenic precursor myo-inositol as described by Fletcher.15Clinically significant aminoglycosides include both naturally occurring drugs and some semi-synthetic derivatives, all of which have a highly-conserved aminocyclitol ring ring II, , a centr1 substituent and are typically not used alone as drugs, leaving apramycin being the only 4-monosubstituted compound that is actually used pharmaceutically. Apramycin is unique in that its ring I is bicyclic.Neamine, paromamine, and apramycin fall under the 4-monosubstituted aminoglycosides. Paromamine and neamine differ only in the RRibostamycin, butirosin B, neomycin B, paromomycin, and lividomycin A belong to 4,5-disubstituted aminoglycosides. These compounds can be further divided into three groups based on the additional ring III and its substituents. Lividomycin A is the only aminoglycoside in this category that contains a fifth ring.4,6-disubstituted aminoglycosides includes kanamycin A, kanamycin B, kanamycin C, tobramycin, and amikacin in one category and gentamicin C1, gentamicin C2, gentamicin C1A, and geneticin in another. The major differences between these two subclasses is the variation of an extra R group attached to the 6’ position of ring I as well as the ring III connected to the 2-DOS.Aminoglycosides have been recognized to have binding capabilities with many different functional RNAs such as the prokaryotic ribosomal A-site,The binding of the aminoglycosides to these target RNAs is mediated through two different interactions: (1) hydrogen bonding between amino and hydroxyl functional groups of aminoglycosides and RNA bases,Experiments have shown that aminoglycoside binding is somewhat dependent on the size of an asymmetric interior loop of the target RNAs rather than its sequence.Despite the convincing facts that aminoglycosides recognize RNAs in a promiscuous manner, studies suggest that there are some sequence-dependent elements in the recognition of target RNAs. A wide variety of RNAs, such as A-site rRNA, ribozymes, and HIV-1 RRE and TAR, are known to bind to various aminoglycosides. These RNAs contain 5′-GU-3′ or 5′-GG-3′ base steps in common next to the nucleotides in bulges or in noncanonical stem structures. A similar binding pattern was observed when A-site RNA sequence was treated with aminoglycosides of various sizes and substitution patterns on ring II. Specifically, the crystal structures of six aminoglycoside antibiotics  and oligonucleotides containing the decoding A site of bacterial ribosome revealed that rings I and II (2-DOS) are essential for recognition of A-site RNA, and interactions between the rings I and II of all aminoglycosides and the target RNA are highly conserved and sequence-specific .11 The tThe sequence-specific recognition pattern of 2-DOS is further supported by the following mutation study. A mutation of G1494A in the target site was deleterious for paromomycin binding because it prevented specific hydrogen bonding of 2-DOS to the RNA.Strikingly, the same binding pattern of 2-DOS was demonstrated when Puglisi and coworkers treated isolated 2-DOS, without the aminosugar subunit attached, with wild type A-site of 16S rRNA .12 The bUniversal existence of the 2-DOS moiety at the central position of almost all clinically important aminoglycosides suggests its crucial role in RNA recognition and biological activity. Therefore, mimicking the 2-DOS structure has been of great interest in preparing novel aminoglycoside analogs. The 2-DOS mimics that have been reported so far can be divided into two different groups based on whether an aminosugar subunit was included in the mimic or not .1) by removing aminosugar rings from the original aminoglycoside structures, leaving only the core 2-DOS as the main constituent of the compound.dK value of 88 μM) for the target RNA A-site and inhibited bacterial translation. This example supports the idea of generating small synthetic compounds that mimic the conformation and function of aminoglycosides without using aminosugar residues.Although most 2-DOS mimics are based on the neamine backbone as the building platform, some use 2-DOS as the sole component, as 2-DOS alone has demonstrated RNA-binding capability without the assistance of aminosugars. Wang et al. demonstrated this example (3) did not show any increase.Following are examples that use 2-DOS as the central scaffold and replace the aminosugar ring I of neamine with other structural motifs. Ding and coworker prepared heterocyclic 2-DOS derivatives 2 based on modeling studies.3 conjugated with non-sugar heterocycles through an amide bond  to mimic the 2-DOS scaffold.2 substituent was replaced by aromatic structures that facilitated better cell membrane penetration. Recently, van Delft and coworkers reported a carbohydrate mimic of the 2-DOS that can be used to prepare conformationally constrained aminoglycosides.383,5-Diamino-piperidinyl triazines (DAPT) 5) by connecting two 2-DOS molecules with various aliphatic and aromatic linkers of different sizes, they were found to bind tightly to RNA hairpin loops of various sizes from 4 to 8 nucleotides, most of which contain 5′-GU-3′ sequence.Aminoglycosides like neomycin show binding affinity to various RNA targets such as the 16S ribosome,These examples show that novel RNA binding molecules may be built solely based on 2-DOS without the assistance of conjugated aminosugar(s), and the intrinsic RNA binding affinity of the 2-DOS may be useful targeting not only helical RNAs whose base pairing is disrupted, but also RNAs of other types of secondary structure such as loops that conventional aminoglycosides do not bind.6, (−)-neamine ent-6, its 5-positional isomers 7, ent-7 as well as 6-positional isomers 8, ent-8.ent-8 was found to be the most effective inhibitor among this series, which allows 1-NH2 and 3-NH2 to face the active site. The isomer ent-6 with both amino groups pointing away from the active site exhibited the least potent activity, while 7 and ent-7 with only one of the amino groups away from active site exhibited less binding affinity.Many other 2-DOS mimics were prepared based on the neamine backbone. To have a better understanding of the effect of stereospecificity of aminoglycosides, Rando and co-workers synthesized and compared the activity of (+)-neamine 9 and 10 exhibited moderate antibacterial activity against Gram-positive Staphylococcus aureus and retained this activity against aminoglycoside-resistant strains.11 and 12, designed based on 3-(aminomethyl)-piperidine,In an effort to mimic the unique spatial arrangement of the two amino functional groups in 2-DOS, the Hermann group prepared two series of compounds by replacing the 2-DOS in neamine with azepane- and piperidine-based structures. Azepane-glycosides 9–12 showed moderate antibacterial activities, the acyclic 2-DOS mimics 13 that have increased flexibility have a detrimental effect on their RNA binding efficacy.46While conformationally-restricted azepane- and 3-(aminomethyl)-piperidine-based compounds 14 demonstrated a high affinity for the oncogenic Bcr-Abl and PAX3-FKHR single-stranded mRNA breakpoint and a capability to regulate gene expression.15 in an attempt to inhibit interactions between HIV-1 RRE and Rev protein. But, the inhibition activity was only slightly better than neomycin B.16 were reported with modifications at 6′-OH and 1-NH2 positions.2 was adopted from another aminoglycoside butirosin. This compound 16 was found to be especially interesting because it appears to be uncompromised by aminoglycoside-resistant enzymes and show a considerable increase in antibacterial activity. The crystal structure of the complex between 16 and A-site of the ribosome suggested that existence the AHB substituent on the 1-NH2 hinders proper complex formation with key aminoglycoside-modifying enzymes.Since Wong and coworkers found that the 5-position is sensitive for aminoglycoside-RNA recognition, small libraries of neamine mimetics were reported with various substituents at the 5′-OH position. Polyamine and 6-aminoquinoline analogs A number of synthetic aminosugars and aminoglycosides have been prepared in an effort to accomplish several goals including: mimicking the core aminosugar rings for enhanced antibacterial activity, limiting the overall structural flexibility of the aminoglycosides, and addressing recognition of the conventional aminoglycosides by the aminoglycoside-modifying enzymes in resistant strains . Below a2 and −OH functional groups at various positions of ring I, II and IV of aminoglycosides to find optimal substitution patterns for aminosugars. Wong and coworkers prepared a series of compounds by placing −NH2 and −OH functional groups at various positions of the ring I in neamine and found that 2′,6′-diamino substitutions on ring I to be the most effective A-site binder.17 showed activity against E. coli, P. aeruginosa, as well as S. aureus. However, its MIC was less than that of neamine, which can be explained by the loss of the highly conserved hydrogen bonds and electrostatic interactions observed in crystal structures. This suggests that neamine is the most promising pseudodisaccharide core that should be kept for further development.One of the aminosugar mimics was achieved by −NHSimilar approaches have been performed on the ring III of tobramycin by replacing the ring with various mono- or diaminosugars.5218 have been developed for the discovery of small molecules using a 1,3-hydroxyamine motif.18a by substituting R1- and R2-positions with highly basic amino acids and amine-containing linear chains, repectively.2- or R3-positions to make compounds like 18b, they were only minimally active against various target RNAs.19 suggest that a high degree of diversity can be achieved by conjugating only a small set of different SDAs through amide bond.Some aminosugar mimetics did not include the 2-DOS core in its modified structures. A library of compounds such as 20 as a novel aminolgycoside analogue by connecting four aminosugar rings using linear alkyl linker.Carlomagno and coworkers reported a neooligoaminodeoxysaccharide 21 demonstrated up to 87 times stronger binding affinity to HIV RRE mRNA than neamine. Neomycin B was also conjugated with acridine at its 5″-position on the ring III via a short linker. Conjugate 22 was one of the strongest competitive inhibitors of HIV rev-RRE interaction, which came at the expense of losing binding selectivity to RRE.60Most aminoglycoside mimics have been prepared by adding extra functionality onto the original aminoglycoside structures without removing aminosugar substituents. A number of intercalating agents were included in the extra functionality with the anticipation that aromatic compounds may have an intercalating capability to the RNA targets that aminoglycosides typically bind. Hamasaki and coworkers conjugated acridine, anthracene and other similar aromatic structures to the ring I of neamine.23) was reported to inhibit the viral replication 100 times more potently than neomycin B by targeting HIV RRE.64Guadinylation of multiple amino groups of aminoglycosides shows similar effects as intercalating agents. Guanidylated neomycin B  to reduce the number of available conformations, thus changing target selectivity.27 based on neamine.The lack of high RNA target selectivity of aminoglycosides is partially attributed to their conformational flexibility that allow adjusting to different shapes of RNAs. Tor and coworkers prepared conformationally-constrained neomycin and paromomycin derivative Arya and coworkers reported an oligonucleotide-neomycin conjugate for sequence-specific targeting of RNA. The nucleotide-neomycin conjugate demonstrated enhanced duplex formation for its target RNA α-sarcin loop.68While most aminoglycoside mimetics employed single point modification on one of the rings of aminoglycosides, Houston and coworkers reported multisite modification on the structure of neomycin B.The increasing bacterial resistance to clinically important aminoglycosides catalyzed the search for novel aminoglycoside mimics. Among several different resistance mechanisms, bacterial inactivation of aminoglycosides by intracellular aminoglycoside-modifying enzymes is the most significant source of resistance development.2 groups of the ring I and 3-NH2 of the ring II are prone targets for acetylation.According to recent studies mainly focused on tobramycin, kanamycin A and B, amikacin, gentamicin, and geneticin, differences in ring III did not seem to alter the interaction of the drug with target rRNA, but subtle variations of ring I significantly influenced binding. Ring II is also affected by the enzymes. The 2′-and 6′-NHOne way to meet the challenge presented by aminoglycoside resistance is to discover new aminoglycoside analogs that are less prone to enzymatic modification. Following are some examples of the aminoglycoside mimics that are designed rationally to fight against aminoglycoside resistance or that are found to be effective against resistant bacterial strains , many of9 and 10 demonstrated moderate antibacterial activity against resistant strains as well as S. aureus.Various azepane mimics of the 2-DOS including 16) with substitutions at 1-NH2 and 6-OH revealed potential structural aspect that may be used to target resistant strains.2 position may cause steric clash with the aminoglycoside-binding loop of the aminoglycoside phosphotransferase APH(3′)-IIIa and prohibits proper complex formation.The ring II mimic of neamine  hydration to give geminaldiol, (2) phosphorylation of the hydroxy group by APH(3′), then (3) self regeneration of the ketone by dephosphorylation. While the MIC of kanamycin A increased 500 to 1000 fold when treated with resistant E. coli, the self-regenerating analog 28 has shown only 4-fold increase of MIC toward resistant E. coli, which signifies that the self-regenerating strategy is a feasible approach in lowering MIC for resistant strains.Distinct from all of the above examples that are designed to avoid interactions with resistance enzymes, Mobashery and coworkers designed an aminoglycoside that binds to the resistance enzyme APH(3′) but not affected by its enzymatic activity.In addition to being a useful model in understanding the RNA recognition process by small molecules, aminoglycosides have been used as versatile antibiotics for almost six decades due to their enormous therapeutic value. For instance, gentamicins and amikacin are used to treat meningitis, pneumonia, and sepsis. Paromomycin is used for amoebic dysentery762+ and Ca2+ bridges that connect neighboring lipopolysaccharides.Pseudomonas, Acinetobacter, and Enterobacter and some Gram-positive bacteria.Aminoglycosides exert their antibacterial activities by two different mechanisms. The first is by inhibiting the translation of essential proteins for bacterial growth. The binding of aminoglycosides to bacterial 16S rRNA, as shown in Despite the versatile antibacterial application against a broad-spectrum of bacteria, the high level of ototoxicity and nephrotoxicity of the aminoglycosides often limits their use in broader applications. While nephrotoxicity is known to originate from reversible accumulation of amino-glycosides in the renal cortex,2 group located on the ring I of neamine, gentamine, and other aminoglycoside analogs.2, increased the toxicity dramatically, while removal of the relative distant 4′-OH had a limited effect on toxicity. This is also understood by the basicity change of the neighboring amino group. Owada reported that N-acetylated amino-glycosides possess considerably lower toxicity than their free aminocompounds.91Several toxicity elements of aminoglycosides were identified in relation to structures of aminoglycosides. These were determined based on effect of the number of amino groups available in aminoglycosides and compared to the toxicity of clinically used aminoglycosides and their derivatives, Baasov and coworkers pointed out that decreases in the number of amino groups in a given aminoglycoside resulted in lower toxicity.Recently, a significant discovery was reported regarding the toxicity of gentamicin. Sandoval and coworkers isolated each of four congeners  of gentamicin using HPLC from a native gentamicin sample and found that gentamicin C2 does not have nephrotoxicity but retains the native antimicrobial efficacy.The unique and complex three-dimensional structures that RNA forms provide unmet opportunities to design novel therapeutics targeting various RNAs. However, it has been generally believed that the intrinsic flexibility of RNA structures makes the structure-based rational drug design approach, which resulted tremendous success in protein-targeting therapeutic development, less suitable unless the target RNA is locked into a rigid conformation, which is not the case for most mRNAs. Despite the unfavorable view, the examples described in this review demonstrate that the structure-based drug design approach has been instrumental to a great degree in understanding the fundamental principles that govern the interactions between various RNA targets and small organic molecules and to design aminoglycoside mimics with better target selectivity and/or binding affinity. Thus, structure-based drug design will continue to play a significant role in meeting the challenges to overcome bacterial resistance to natural aminoglycoside antibiotics and design novel RNA-binding ligand targeting RNAs other than bacterial sources. The highly conserved sequence-specific recognition pattern of rings I and II of neamine, a common component for many clinically important aminoglycosides, may be put to use through the rational drug design approach to help devise small molecules that recognize specific sequences or sites of RNA. High throughput screening approaches, combined with the rational design, may offer fresh insight into the design of novel ligands, especially in identifying target-specific ligands.In addition to aminoglycosides, riboswitches may serve as another important model in understanding the principles that govern small molecule-RNA recognition. Riboswitches are embedded within 5’-untranslated regions (5’-UTR) of certain bacterial mRNAs and regulate gene expression in response to small cellular metabolites. Since their discovery in 2002,


PLoS Biology by Harm van Bakel, Timothy Hughes, and colleagues shows that most dark matter transcripts are likely to be by-products of transcription of known genes and that many of the rest of them are likely not messages of great import, but simple background noise.But a new study published in this issue of The earlier reports detected dark matter transcripts using “tiling arrays,” DNA microarrays embedded with probe sequences drawn from regularly spaced regions across the genome. These reports indicated that a quarter or more of all transcripts created by the nucleus arose from DNA completely outside the boundaries of known genes. The nature and function of these transcripts was unclear, but while some likely arose from spillover transcription of known genes, some from novel genes, and some from erroneous activity of RNA polymerase, it seemed logical, given their apparent numbers, that many others were RNAs with new and unknown function. But data from arrays are prone to false positives—since a probe may bind to a less-than-perfect match when no perfect match is available—leading skeptics to wonder whether many of the signals arising from dark matter are really there at all.More recently, it has become practical to sample the sequence of large numbers of RNA transcripts, a technique unavailable even a few years ago when dark matter transcripts were first discovered. So, the authors began their study by comparing results in both mouse and human tissues from tiling arrays to those from exhaustive RNA sequencing. They showed convincingly that the sequencing data identified transcription from many fewer non-gene regions, suggesting that much of the tiling data arose from false-positive noise, rather than actual unique RNA sequence signal.So, if exhaustive sequencing is the right tool for exploring the dark matter of the genome, what does it reveal? First, transcripts from dark matter make up only 12% of all polyadenylated transcripts; the rest arise from exons of well-known genes. In fact, after excluding introns and a couple of other well-described categories of transcripts, only about 2% of all transcripts are left unexplained. Many of these turn out to be fragments transcribed from the tail end of genes, possibly arising as a result of alternative termination sites or when RNA polymerase fails to disengage after transcribing the gene proper. Three quarters of the unexplained RNA sequences arose from within 10 kb of either side of known genes, although these regions make up less than 20% of the intergenic regions. Most of the rest of the unexplained sequences resembled a random distribution in both location and copy number. Similar conclusions were reached by analyzing non-polyadenylated transcripts.There were nonetheless some intriguing real signals from the vast regions outside of known genes. About 1% of all RNA transcripts, amounting to several thousand distinct sequences, occurred in high enough copy number and, in many cases, in regions conserved between mammals to suggest they were products of active transcription. These predominantly arose in open chromatin—DNA that is unpacked and accessible to RNA polymerase. But, whether the chromatin was open in order to transcribe these sequences, or whether they were transcribed because the chromatin is open for other reasons, is unknown. Neither is there any known function for the transcripts, leaving open the possibility that these, too, are a byproduct of other, more directed RNA polymerase activities.The emerging picture of RNA polymerase is of an inherently imprecise, not to say promiscuous, copyist, one whose output includes some mistakes along with lots of valuable product. In this view, most dark matter transcripts are not signals emerging from a hidden universe within the genome, but instead simply the noise emitted by a busy machine.van Bakel H, Nislow C, Blencowe BJ, Hughes TR (2010) Most “Dark Matter” Transcripts Are Associated With Known Genes. doi:10.1371/journal.pbio.1000371


Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active.Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with  E. coli RNA polymerase, respectively, and of the equivalent largest subunits of eukaryotic RNA polymerases I, II and III. These subunits interact to form the entry and exit channels for the DNA template, the catalytic center for RNA polymerization and the exit channel for the RNA transcript A. thaliana genome sequence, which led to the identification of two genes for an atypical fourth class of largest subunit and two genes for an atypical fourth class of second-largest subunit in addition to the canonical Pol I, II and III subunits The largest and second-largest subunits of eukaryotic multi-subunit nuclear RNA polymerases are homologs of the β′ and β subunits of For purposes of subunit nomenclature, nuclear RNA polymerases I, II and III in Arabidopsis are designated NRPA, NRPB and NRPC and their largest subunits are NRPA1, NRPB1 and NRPC1. Extending this convention to the atypical polymerases, their largest subunits have been designated either NRPD1a and NRPD1b NRPD1 (NRPD1a), NRPE1 (NRPD1b) and NRPD2/NRPE2 genes are not essential for viability de novo DNA methyltransferase, DRM2 The In a previous report, we showed that column fractions enriched for Arabidopsis NRPD2/NRPE2, and therefore presumably containing Pol IV and Pol V complexes, lack detectable promoter-independent RNA polymerase activity using sheared template DNA whereas activity was readily detected in fractions enriched for Pol I, II and III in vivo. We show that the Metal A and Metal B sites are required for the biological functions of Pol IV and Pol V including siRNA production, RNA-directed DNA methylation and transposon silencing. Additionally, the active sites are required for the distinctive punctate nuclear localization patterns observed for Pol IV and Pol V We hypothesized that if RNA Polymerases IV and V function as RNA polymerases, their Metal A and Metal B consensus sequences should be essential for their known biological activities. To test this hypothesis, we conducted site-directed mutagenesis of the Metal A and Metal B motifs within the NRPD1, NRPE1 and NRPD2/NRPE2 subunits, stably incorporated the engineered genes into transgenic plants that were defective for the corresponding endogenous genes and tested for the restoration of Pol IV and Pol V functions S. cerevisiae Pol II, implying that these amino acids are critically important for polymerase structure and function. Interestingly, numerous amino acids that are invariant among the canonical polymerases  are substituted by other amino acids in Pol IV and Pol V {Herr, 2005 #2694}{Onodera, 2005 #2695}. In S. cerevisiae Rpb1 and Rpb2 subunit structures in the context of a yeast Pol II elongation complex crystal structure. Interestingly, a large proportion of the “invariant” amino acids that have been substituted in NRPD1, NRPE1 (NRPD1b) and NRPD2/NRPE2 cluster in the vicinity of the catalytic center. In particular, sequences surrounding the Metal A binding site, bridge helix, cleft and funnel domains of NRPD1 and NRPE1 and the hybrid binding region of NRPD2 Pol IV and Pol V are rapidly-evolving enzymes, with Arabidopsis NRPD1 (formerly NRPD1a) and NRPE1 (formerly NRPD1b) having amino acid substitution rates 20 times greater than the NRPB1 subunit of Pol II, and NRPD2/NRPE2 having a substitution rate 10 times greater than the Pol II NRPB2 subunit E. coli, chloroplast, archaeal (Pyrococcus), viral, and eukaryotic Pol I, II and III polymerases. This sequence motif is part of an extended sequence, YNADFDGDEMN that is conserved in eukaryotic Pol I, II and III and archaeal polymerases. However, despite having apparently evolved from a duplicated Pol II largest subunit, the NRPD1 subunit of Pol IV has only the core DFDGD sequence that includes the three magnesium-coordinating aspartates. In the NRPE1 (NRPD1b) subunit of Pol V, this core sequence consensus is extended by only one amino acid: the alanine preceding the first aspartate (ADFDGD). Importantly, the consensus sequence DxDGD occurs at the active sites of single-subunit RNA-dependent RNA polymerases, such as Arabidopsis RDR2 and RDR6 or Neurospora QDE-1. Therefore, the conservation of the minimal DFDGD sequence in NRPD1 and NRPE1 is consistent with the hypothesis that these subunits have minimal Metal A sites. Likewise, the NRPD2 subunit utilized by both Pol IV and Pol V contains the core ED motif of the Metal B site as part of an extended G(Y/F)NQEDS motif also present in the second-largest subunit of Pol II. Collectively, these observations suggest that Pol IV and Pol V have Metal A and Metal B sites at their presumptive active sites.Multiple sequence alignment in the vicinity of the Metal A and Metal B sites of RNA polymerase largest and second-largest subunits illustrates the sequence divergence that has occurred in Pol IV and Pol V subunits relative to other RNA polymerases . ImmediaNRPD1 transgenes were introduced into the nrpd1a-3 null mutant, NRPE1 (NRPD1b) transgenes were introduced into the nrpd1b-11 null mutant and NRPD2 transgenes were introduced into the nrpd2a-2 nrpd2b-1 double mutant. Note that the NRPD2b gene is a pseudogene due to a frameshift mutation, such that the double mutant is used only as a precaution. The double mutant is hereafter referred to simply as nrpd2. Six or more independent transformants for each transgene construct were analyzed to determine the ability of the transgenes to genetically rescue their respective null mutants and all lines for a given construct were found to display the same phenotypes. The active site mutant transgenic lines are abbreviated as DDD-AAA-FLAGNRPD1, DDD-AAA-FLAGNRPE1 or ED-AA-FLAGNRPD2 in To address whether the presumptive active sites of Pol IV and Pol V are required for their functions, we performed site-directed mutagenesis to change the acidic residues of the Metal A and Metal B sites to alanines. Three amino acid substitutions were performed in the largest subunits of Pol IV and Pol V: for NRPD1 these were D447A, D449A and D451A and for NRPE1 (NRPD1b) they were D449A, D451A and D453A. For NRPD2/NRPE2, E785A and D786A mutations were introduced . Full-leAtCopia or AtSN1 retrotransposons  mutants, in agreement with prior studies NRPD1-FLAG, NRPE1-FLAG or NRPD2-FLAG transgenes in their respective mutant backgrounds, siRNA production is restored, albeit to lower than wild-type levels in the case of the NRPD1 transgene. A delay in flowering time observed in the nrpd1 mutant, and other mutants affecting the siRNA-directed DNA methylation pathway, is also not fully restored by the NRPD1 transgene  and Pol V (NRPE1/NRPD1b) largest subunits are required for small RNA biogenesis or accumulation. Trace siRNA signals were detected in the ED-AA-FLAGNRPD2 transgenic plants but not in the nrpd2 mutant parental line  and nrpd2/nrpe2 mutants, similar losses of CNN or CG methylation occur relative to wild-type (WT) controls. In these mutant backgrounds, methylation is restored to wild-type levels by the corresponding wild-type transgenes . However, the equivalent transgenes bearing the active site mutations  fail to rescue the defects in DNA methylation caused by the nrpd1a-3, nrpd1b-11 (nrpe1) and nrpd2/nrpe2 mutations.The requirement for the presumptive Pol IV and Pol V active sites in RNA-directed DNA methylation at 5S rRNA gene repeats was tested by Southern blot analysis using the methylation sensitive restriction endonucleases, nd HpaII . In thisAtSN1 retrotransposons are subjected to siRNA-directed DNA methylation in a Pol IV and Pol V-dependent manner AtSN1 methylation using chop-PCR  plants, AtSN1 elements are methylated, rendering them resistant to HaeIII digestion  or nrpd2/nrpe2 mutants, methylation is lost, resulting in HaeIII susceptibility and the loss of PCR product. Whereas wild-type NRPD1-FLAG, NRPE1-FLAG and NRPD2-FLAG transgenes rescue their respective null mutants and restore DNA methylation at the AtSN1 loci, the corresponding active site mutants fail to do so  plants but are apparent in nrpd1a-3, nrpd1b-11 (nrpe1) or nrpd2 mutants. Transforming these mutants with the NRPD1-FLAG, NRPE1-FLAG or NRPD2-FLAG transgenes, respectively, restores AtSN1 and solo LTR silencing. However, the active site mutant versions of the transgenes fail to restore AtSN1 or solo LTR silencing in the mutant backgrounds.Consistent with the losses in mulation  and DNA nsposons , silenciin vivo function, as shown by their failure to genetically rescue their corresponding null mutants, the proteins are expressed at the same levels as their wild-type counterparts and the mutated largest subunits assemble with the NRPD2/NRPE2 subunit, as shown by co-immunoprecipitation and immunoblotting .Although RNA polymerase activity has not yet been demonstrated In the vicinity of the Pol IV and Pol V active sites, numerous amino acids that are invariant in Pol I, II and III are missing or replaced by other amino acids. Many of these amino acids occur in regions that influence the predicted template channel, including the bridge helix of the largest subunit, a highly conserved structure from bacterial to eukaryotic polymerases over which the template strand passes en route to the active site nrpd2 null mutants transformed with the NRPD2 active site mutant. One explanation for this observation may be that the second-largest subunit's contribution to magnesium ion binding at the Metal B site is slightly less critical than the magnesium binding coordinated by the largest subunit. Consistent with this interpretation, single amino acid substitutions in the Metal B site of an archaeal RNA polymerase were shown to substantially decrease, but not completely abrogate, transcriptional activity ED-AA-FLAGNRPD2 lines are apparently not sufficient for rescue of RNA-directed DNA methylation at 5S rRNA genes or AtSN1 retroelements or for restoration of AtSN1 or solo LTR silencing.Accumulation of 24 nt siRNAs requires the Metal A consensus sequences of NRPD1 and NRPE1 . InteresNRPD1-FLAG transgene did not fully rescue delayed flowering time in the nrpd1a-3 mutant background to that of wild-type plants , nor did the transgene fully rescue siRNA levels  and thus never localized to chromatin. Although we cannot rule out this possibility, Arabidopsis thaliana mutants nrpd1a-3, nrpd2a-2 nrpd2b-1 (abbreviated as nrpd2a/2b) and nrpd1b-11 were described previously Agrobacterium tumefaciens-mediated, multi-kb insertions that disrupt the genes Zea mays NRPD2 (AAY45706), Arabidopsis RDR2 (NP_192851), Arabidopsis RDR6 (NP_190519) and Neurospora crassa QDE-1 (CAB42634). NRPD1 , NRPE1  and NRPD2  sequences from poplar were identified using the Poplulus trichocarpa unmasked genome assembly v1.1 by JGI and the tBLASTn tool with Arabidopsis protein queries. Sequences were aligned using ClustalW2 and colored using BOXSHADE.GenBank sequences for largest and second-largest RNA polymerase subunit alignments were those described previously   and NRPE1 (NRPD1b)  and to mutate the Metal B site of NRPD2a . Nucleotides 910-2232 of the NRPD1a genomic sequence were PCR amplified from pENTR-NRPD1a with NRPD1a active site-F and NRPD1a active site-R primers  genomic clone was mutated with primers NRPD1b DDD/AAA-F, NRPD1b mut-F, NRPD1b DDD/AAA-R and NRPD1b mut-R (see Site-directed ligase independent mutagenesis (SLIM) mers see . The resut-R see . The pDOut-R see . Proper NRPD1 (NRPD1a) and NRPE1 (NRPD1b) genomic sequences and generation of NRPD1-FLAG and NRPE1 (NRPD1b)-FLAG transgenic lines that rescue the nrpd1a-3 or nrpd1b-11 null mutants, respectively was described previously NRPD2a genomic sequence, including 1310 bp upstream of the translation start site, was amplified by PCR from A. thaliana (ecotype Col-0) genomic DNA using NRPD2a BP-F and NRPD2a BP-R primers  probes were prepared according to 5′TGATTGAGCCGCGCCAATATCcctgtctc3′.RNA was isolated from 300 mg of inflorescence tissue using the mirVana miRNA isolation kit (Ambion). RNA samples (9.5 µg each) were resolved by gel electrophoresis, transferred to nylon membrane and hybridized to radioactive probes as described previously HaeIII or HpaII-digested genomic DNA isolated from leaves of 3 to 4-week old plants. Digested DNA was subjected to agarose gel electrophoresis and transferred to uncharged nylon membranes. The 5S rRNA gene probe, labeled with a32P-dCTP, was generated by random priming of a full-length 5S gene repeat amplified by PCR from clone pCT4.2 AtSN1 DNA methylation assay involving PCR amplification of undigested or HaeIII-digested genomic DNA was performed as described previously Southern blot analysis was performed using 250 ng of AtSN1 RT-F and AtSN1 RT-R primers were used to amplify AtSN1 transcripts from the cDNA with GoTaq Green (Promega) and samples were analyzed by agarose gel electrophoresis.RNA (∼1 µg) isolated from 3 to 4-week old leaf tissue was treated with RQ1 DNase (Promega) and used to generate random-primed cDNA using degenerate dN6 primers (NEB) and Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Immunoprecipitation and immunoblot detection of Pol IV and Pol Vproteins was performed using 4.0 g of 3-week old leaf tissue from T3 generation plants, as described previously Table S1DNA oligonucleotides used in this study(0.07 MB DOC)Click here for additional data file.Table S2Positions of amino acids that are invariant among Arabidopsis Pol I, II and III and yeast Pol II but have diverged in Arabidopsis Pol IV and Pol V largest and second-largest subunits. The table lists amino acids, numbered according to the PDB:1R9T crystal structure for yeast Pol II, and the changes at these positions in NRPD1, NRPE1 or NRPD2. These are the amino acids highlighted in (0.18 MB DOC)Click here for additional data file.Figure S1http://www.ebi.ac.uk/Tools/clustalw2/index.html) in conjunction with final editing by hand. Alignments were colored using BOXSHADE v3.21 (http://www.ch.embnet.org/software/BOX_form.html). DNA-dependent RNA polymerase conserved domains A to H are underlined and designated to the right of the alignments. Yeast Pol II structural features, according to Cramer et al (2001), are designated below the alignments. Regions that make contact with other RNAP subunits are designated in italics above the alignments. The Metal A site is designated with asterisks above the alignment.Multiple alignment of A. thaliana RNAP Largest Subunits and the Yeast Pol II Largest Subunit. Full-length protein sequences for A. thaliana NRPA1 (At3g57660), NRPB1 (At4g35800), NRPC1 (At5g60040), NRPD1 (At1g63020), NRPE1 (At2g40030) and S. cerevisiae Rpb1 were aligned using ClustalW2 ((0.18 MB DOC)Click here for additional data file.Figure S2Flowering time control is dependent upon the Pol IV and Pol V active sites. nrpd1a, nrpe1/nrpd1b and nrpd2 mutants, or transgenic lines generated by transforming these mutants with wild-type or active site mutant versions of NRPD1, NRPE1/NRPD1b or NRPD2a full-length transgenes, were grown side-by-side under short day conditions (8 hours light/16 hours dark). The positions of pots were changed every 4–6 days according to a randomized plot design. The total number of rosette leaves for each plant was counted when the bolt  achieved a height of 5 cm. The histograms show the average number of leaves at flowering+/−the standard error of the mean. Asterisks denote mean values that are significantly different (p<0.05) from the wild-type  control population as determined by using the Student t-Test; a double asterisk denotes a value that is significantly different from both the WT and nrpd1a-3 controls. The number of individual plants analyzed for each genotype is denoted by the numeric value inside each vertical bar. As expected, based on prior studies (0.45 MB TIF)Click here for additional data file.


Cardiovascular magnetic resonance (CMR) allows non-invasive phase contrast measurements of flow through planes transecting large vessels. However, some clinically valuable applications are highly sensitive to errors caused by small offsets of measured velocities if these are not adequately corrected, for example by the use of static tissue or static phantom correction of the offset error. We studied the severity of uncorrected velocity offset errors across sites and CMR systems.In a multi-centre, multi-vendor study, breath-hold through-plane retrospectively ECG-gated phase contrast acquisitions, as are used clinically for aortic and pulmonary flow measurement, were applied to static gelatin phantoms in twelve 1.5 T CMR systems, using a velocity encoding range of 150 cm/s. No post-processing corrections of offsets were implemented. The greatest uncorrected velocity offset, taken as an average over a 'great vessel' region (30 mm diameter) located up to 70 mm in-plane distance from the magnet isocenter, ranged from 0.4 cm/s to 4.9 cm/s. It averaged 2.7 cm/s over all the planes and systems. By theoretical calculation, a velocity offset error of 0.6 cm/s (representing just 0.4% of a 150 cm/s velocity encoding range) is barely acceptable, potentially causing about 5% miscalculation of cardiac output and up to 10% error in shunt measurement.In the absence of hardware or software upgrades able to reduce phase offset errors, all the systems tested appeared to require post-acquisition correction to achieve consistently reliable breath-hold measurements of flow. The effectiveness of offset correction software will still need testing with respect to clinical flow acquisitions. Phase contrast cardiovascular magnetic resonance (CMR)  measuremAs illustrated in Figure The background offset errors in typical cardiac flow applications have been studied previously e.g. -10 and tin-vivo efficacy [in-vivo modality. It should be understood that this study uses uncorrected offset data, revealing how much dependence there is on the correction methods and their routine in-vivo reliability.When baseline offset correction methods have been applied with proof of their efficacy ,10, the The study reported here was initiated by members  of the EuroCMR Working Group of the European Society of Cardiology (ESC). Among them, they had experience of several types of commercially available CMR system, and they shared concerns regarding possible inaccuracies in derived flow measurements. They agreed that background phase offset errors were, amongst other possible problems, likely to be the principal cause for concern. Therefore, the purpose of this study was to use static gel phantoms to investigate whether their concerns were justified, potentially motivating further optimisation of CMR velocity mapping for clinical flow measurements. The ability of CMR to rapidly and non-invasively measure flow through planes transecting the large vessels is unique, clinically valuable and worth optimising.To eliminate the possibility of convection or motion-induced currents within fluid phantoms, an aqueous solution of gelatine was set within 10-15 litre uniform plastic tanks, with sufficient dimensions at all sites to enable measurement over the regions specified below. To reduce T1 for improved signal to noise ratio of the gelatine, 5 mmol/l of Gd-DTPA was added. These methods were adopted because the background offsets being studied were potentially very small.The study was limited to 1.5 T as this is currently the most widely-used main field strength for CMR. Automatic correction of concomitant gradient terms  was emplTo ensure consistent test protocols for each type of scanner, four of the investigators  set up an acquisition protocol for each type of system, and transferred this protocol to the other sites of that type. This protocol included the slice orientations described below.temporally interpolated cine phases was reconstructed by the Siemens and GE machines, but without consequences for the true flow acquisition.We aimed to use similar phase-contrast sequence parameters for each of the three scanner types. The following sequence parameters were reproduced for each type . All cine phase-contrast acquisitions were by retrospectively gated pulse sequences, where the phase-encode was updated by each detected ECG R-wave. The continuous gradient activity of this approach, with no silent gap while waiting for the next R-wave, has the advantage of a more stable background offset during the cardiac cycle , as wellSystems used were as follows:i.e. temporal interpolation was not performed during reconstruction).Philips Achieva R2.53 (4 sites). TR5.5 ms, TE2.8 ms, pixel bandwidth 355 Hz/pixel (Fat/Water Shift 0.62). The slice-selective RF pulse used an asymmetric design with a late centre. The background phase-offset correction ("LPC filter") was switched off for this study (see Discussion). Fifteen cardiac phases were reconstructed .Siemens Avanto VB15 TR6.6 ms (4 systems over 3 sites), TE2.8 ms, sampling bandwidth 355 Hz/pixel. The controls for RF pulse and gradient mode, which control the use of faster and stronger RF and gradient pulses, were both set to "Normal" mode in order to achieve TR and TE similar to the other scanners. Twenty-five cardiac phases were reconstructed .GE Signa Excite 14M5 (4 systems over 3 sites). This used symmetric velocity-encoding . The "flow analysis" flag was on, disabling a spatial high-pass filter used for phase-contrast angiography background suppression. (In a slightly different form this filter apparently resembles the Philips approach to background correction). The GE "flow optimization" control resulted in longer TE and TR than the other scanners and was therefore not used. The readout ADC bandwidth was 41.67 kHz (pixel bandwidth 326 Hz/pixel). On the GE, the TR and TE ranged over 5.9-6.0 ms and 2.9-3.0 ms respectively for the oblique slices tested but were reproduced exactly at all 4 sites, as the sequence optimised its timings depending on image plane orientation. Twenty cardiac phases were reconstructed (Each centre acquired the same two planes (3 acquisitions), as defined in the transferred protocol files . The software reported the most extreme mean velocity offset measured in a circular 'great vessel' region, 30 mm diameter , centereTable The data of Table Since the MPA plane results appeared larger than the Ao plane on average, the question arises whether this was due to the larger analysis region used (MPA 70 mm vs Ao 50 mm). Analysis was therefore repeated over a central region of up to 50 mm from isocentre for all three plane orientations, giving 1.6 cm/s, 2.2 cm/s and 2.6 cm/s for the Aortic, MPA (head-foot) and MPA (left-right phase-encode) respectively.in-vivo post-processing correction of velocity offsets for the breath-hold protocol that was used. Referring to the 0.6 cm/s threshold of acceptability defined in the Introduction, the velocity offsets actually measured in the current study were on average four times greater than 0.6 cm/s, which could measure the 15% regurgitant fraction example given above as anywhere between 5% and 25%. In some clinical applications, for example in straightforward measurements of peak jet velocity for the assessment of a stenosis, such offsets remain insignificant. But in other situations, the effect may be magnified further, for example if a great vessel is significantly dilated, if stroke volume is reduced, or the R-R interval is increased due to bradycardia.In 35 of the 36 results, the uncorrected offset exceeded a 0.6 cm/s limit of acceptability which we explained in the Introduction. This implies that all 12 scanners would need to rely on accurate From Figure Velocity offsets caused by Maxwell (concomitant) gradients in phase-contrast imaging are calculable allowing automatic correction during reconstruction . The remThe methods reported here, which aimed to implement comparable phase contrast acquisitions across CMR sites and systems, turned out to be more challenging than had been anticipated. An initial attempt to invite data collection from different sites resulted in unacceptably wide variations of acquisition parameters, in spite of the careful work undertaken by colleagues at several sites. It became clear that the phantom studies would require pre-set acquisition protocols, transferred digitally between systems. This necessitated cross-system planning and collaboration, which brought to light important variables between systems. Certain variables precluded complete standardisation of acquisition parameters, using clinically available software . A further limitation of the study lies in the analysis method of searching for the largest offset in regions displaced in in-plane directions, while the main pulmonary artery tends to have an anterior offset. Taking that example, slices acquired through the isocenter of the magnet may not be optimal for MPA flow measurement. The couch move facility should in this case have been used to relocate the vessel region rather than the centre of the slice. We nevertheless believe that the results recorded give a reasonable indication of the offsets that might be expected in clinical practice.This study was not designed to find out if any particular plane had greater offsets, but used 45° oblique 'aortic' and 'MPA' planes to test orientations that might typically be used in acquisitions for cardiac output, shunt or regurgitant flow measurement. The apparently larger offset in most MPA planes compared to the aortic planes was not necessarily regarded as representative as orientations might vary in clinical practice, so the significance of this was not tested. Although most of the results have negative polarity, this was not investigated further. Opposite polarities from the same scanner have been reported previously .Two obvious questions arise from this work. First, how could a phase-contrast protocol be optimised to minimise the background error in clinical routine? Due to the variability between different scanner types general advice cannot be given beyond the following. Most current CMR systems support automatic couch positioning for flow imaging, aiming to bring the vessel of interest for flow measurement into the isocenter plane  as possible to minimise velocity offset errors. However, implementation of this facility varies between systems and it is important to be aware that the vessel region of interest, not necessarily the centre of the slice, should be positioned with zero offset along the head-foot direction. . Beyond this basic step, any further optimisation of sequence waveforms to minimise background offsets is highly sensitive to residual errors in the correction of eddy currents. The term "residual errors" refers to the difference between the eddy current-related field distortion and the pre-calibrated compensation for eddy-currents (known as "pre-emphasis") applied to the gradient waveforms. The accumulation of this residual phase error during the time between velocity encoding and the echo may be positive or negative, leading to positive or negative offsets in the phase-subtraction velocity image. It can be estimated that a velocity offset of 0.6 cm/s in a 200 cm/s VENC scan may arise from a residual error (of time-constant ≈ Te) of ≈ 0.01% of the amplitude of the gradient change made for velocity-encoding, which is extremely challenging for manufacturers to achieve. This is around ten times more demanding of accurate pre-emphasis than balanced SSFP cine imaging, and it remains uncertain whether this could ever be improved reliably so that offset post-processing correction techniques will not be needed.in-vivo since it appears that most systems require them? This study did not use any correction methods which might have appeared unrealistically effective when applied to images from the large uniform, gelatin phantoms. Correction software may be less reliable in-vivo, particularly if there is insufficient stationary tissue in the acquisition plane, or if signal from it is poor. Furthermore, phase-encode wraparound and possible spatial non-linearity of the offset may be problematic. An alternative but more time-consuming approach to offset correction requires identical flow acquisitions using a stationary phantom after a patient study, subtracting the corresponding apparent phantom velocities from the clinical acquisition [in-vivo reliability of correction methods, for example using post-acquisition phantom scans as a temporary gold standard [The second question that arises from the work is, how reliable are velocity offset correction techniques uisition   showed velocity offset values larger than the 0.6 cm/s shown necessary for <10% error in the most sensitive cardiac applications. It is therefore necessary to have a reliable background offset correction method for images acquired by a typical breath-hold flow protocol. The reliability of background velocity offset correction techniques needs to be tested with respect to clinical flow acquisitions.Peter Gatehouse: Research agreement with Siemens.David Firmin: Research agreement with Siemens.John Totman: Research agreement with Philips.Martin Graves: Research support from GE.All other authors: None.PJK, JS, AvR, JS-M, ML forming the EuroCMR Working Group of the European Society of Cardiology (ESC) conceived the study. PG designed the study with the assistance of MPR, MH, MG, JT. All other authors installed the designated test protocol, made appropriate test phantoms and ran the tests.