A decrease in pulmonary surfactant has been suggested to contribute to the lung dysfunction associated with pulmonary inflammation. A number of studies have implicated surfactant clearance as a possible mechanism for altered pool sizes. The objective of the current study was to specifically investigate the mechanisms of surfactant clearance in a rodent model of acute pulmonary inflammation.Inflammation was induced by intrapulmonary instillation of lipopolysaccharide (LPS: 100 μg/kg). Lipid clearance was assessed at 18 and 72 hours post-LPS instillation by intratracheal administration of radiolabel surfactant-like liposomes 2 hours prior to isolation and analysis of inflammatory cells and type II cells.At both 18 and 72 hours after LPS instillation there was significantly less radioactivity recovered in the lavage fluid compared to respective control groups (p < 0.05). At both time points, the number of cells recovered by lavage and their associated radioactivity was greater compared to control groups (p < 0.01). There was no difference in recovery of radioactivity by isolated type II cells or other cells obtained from enzymatic digestion of lung tissue.These results show that increased clearance of surfactant lipids in our model of acute pulmonary inflammation is primarily due to the inflammatory cells recruited to the airspace and not increased uptake by alveolar type II cells. Pulmonary surfactant is a phospholipid-protein complex that lines the inner surface of the lung and is essential for normal pulmonary function. Surfactant acts to promote lung stability by reducing surface tension within the lung, while also protecting against inhaled pathogens. Surfactant is composed of approximately 90% lipids and 10% proteins by weight. The lipid component is primarily phospholipids with phosphatidylcholine (PC) being the most abundant, and the protein component comprised of four surfactant-associated proteins designated SP-A, SP-B, SP-C and SP-D . CollectAlterations of the pulmonary surfactant system, including decreased total surfactant levels, have been implicated in the pathophysiology of acute lung injury. Multiple studies of patients with a variety of lung diseases have shown that surfactant levels are decreased in the inflamed, injured, or infected lung -5. In agex vivo to LPS resulted in the presence of giant lamellar bodies within the type II cells PC was obtained from DuPont New England Nuclear . Elastase for type II cell isolations was purchased from Worthington Biochemicals . Dulbecco's PBS, DMEM, and fetal bovine serum (FBS) were obtained from Life Technologies . Low-endotoxin BSA, O26:B6 Escherichia coli LPS, Rat IgG and all other chemicals were purchased from Sigma . Chloroform and methanol were obtained from EM Science .Dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), dipalmitoylphosphatidylglycerol (DPPG) and cholesterol were purchased from Avanti Polar Lipids . L-α-dipalmitoyl [2-palmitoyl-9,10-3H-DPPC (12 μCi/mg phospholipid). The lipids were dried under nitrogen, reconstituted in 0.15 M saline and extruded from a French Press cell under 900 psi. This resulted in small unilamellar liposomes at a concentration of approximately 1 mg lipid/ml.Small unilamellar liposomes were prepared with a lipid composition similar to pulmonary surfactant: 52% DPPC, 26% egg PC, 15% DPPG and 7% cholesterol by weight with trace amounts of E. coli LPS) was suspended in 300 μl of sterile 0.15 M saline. Control animals received an equal volume (300 μl) of sterile 0.15 M saline. For instillation, animals were anesthetized with halothane such that they remained unconscious throughout the entire instillation procedure and had no cough reflex upon intubation. Animals were placed on a board at a 45° angle, intubated with an 18-gauge blunt ended catheter and either sterile saline or LPS suspension was instilled followed by five 1 ml boluses of air to facilitate the distribution of the instilled fluid. Two hours prior to killing, liposomes (100 μg lipid) were intratracheally instilled in all groups following the identical instillation procedure. Separate groups of both control and LPS animals were sacrificed at 18 or 72 hours after saline or LPS instillation.Male pathogen-free Sprague Dawley rats  were used for the current study. For LPS animals, endotoxin . The lung from the first animal was carefully removed from the thoracic cavity and placed between saline soaked gauze pads while the second lung was perfused and removed in identical fashion. The time for lung perfusion and removal was approximately 10 minutes. After lungs were isolated from both a control and LPS animal, they were lavaged simultaneously with eight 10-ml volumes of EGTA buffer . For each animal the individual lavages were collected and combined to make up the bronchoalveolar lavage fluid (BALF), which was immediately stored on ice. After the lavage procedure, individual lobes were dissected away from the major airways. The lung tissue was then cut into 5 mm pieces in 5 ml calcium buffer and subsequently homogenized with a Polytron PT-MR-2100. After complete homogenization, total lung tissue volume was diluted to 40 ml with calcium buffer and stored on ice. A small aliquot (2 ml) of the total BALF was removed and the remainder was centrifuged at 250 g for 10 minutes at 4°C to generate a pellet that was primarily BALF cells. The cell pellet was subsequently suspended in 10 ml of PBS. Lavage cell numbers were determined by a hemocytometer, viability determined by trypan blue exclusion and cell differential determined by Hemacolor staining of cytospins that were prepared using a Shandon Cytospin 2 centrifuge. Liposome-association in total BALF, cell-free BALF, BALF cells and homogenized lung tissue was determined by scintillation counting.One control animal and one LPS animal were investigated simultaneously, thus all procedures were done in parallel. At 18 or 72 hours after saline or LPS instillation, animals were anesthetized with 0.3 mg of sodium pentobarbital and 700 U of heparin. After loss of toe pinch reflex, the trachea was cannulated and the rat was exsanguinated via transection of the descending aorta. The chest cavity was subsequently opened and the lungs perfused through the pulmonary artery with 40–50 ml of a calcium buffer  and once with elastase solution . The lungs were then filled with the elastase solution and suspended in warmed saline (37°C) for 20 minutes. Lung tissue was removed from the major airways, cut into 5-mm pieces and chopped 200 times with sharp scissors in 5 ml of the calcium solution along with 2 mg of DNase. The tissue suspension was added to a flask with 4 ml of FBS and 35 ml of calcium buffer and shaken vigorously for 2 min in a 37°C water bath. The resultant tissue suspension was strained through gauze, and then decreasing sizes of nylon mesh . The cell suspension was centrifuged at 250 g for 10 min at 4°C, the resulting cellular pellet was suspended in 20 ml of warm DMEM (37°C) and incubated on two IgG coated Petri dishes (150 × 15 mm) for 30 minutes at 37°C. After the incubation period, nonadherent cells were removed from the plate, washed once with PBS, suspended in calcium buffer and used for determination of radiolabel recovery associated with Type II cells. The remaining adherent cells were gently scraped off the plate in 5 ml of DMEM and transferred into a polypropylene tube; this population of cells is referred to as "plate cells" and consisted primarily of macrophages and/or neutrophils as described in detail below. The cells were washed once and suspended in calcium buffer for determination of associated radioactivity. A small aliquot from the isolated type II cells and plate cells was saved for differential cell count and viability. Cell purity was determined by counting a minimum of 250 cells from random fields after staining by the Papanicolaou method [A separate cohort of animals was required for type II cell isolation studies. As with the whole lung studies, type II cell isolation was completed for one control and one LPS rat in parallel for both 18- and 72-hour time points after instillation of saline or LPS. The type II cell isolation procedure was described previously with minor modifications of the original protocol ,23. The u method .All data reported are means ± standard error (SE). Repetitions (n) used to calculate means ± SE were from independent experiments, not from replicates within an experiment. An analysis of variance (ANOVA) was used to determine differences between all experimental groups at a specific time point, followed by a Tukey post-hoc test for multiple comparisons. Significance was accepted when p < 0.05.Table Table Figure Figure Additional controls were performed in which animals (n = 2) received radiolabel liposomes followed by whole lung lavage and lung tissue homogenization 5 minutes after the instillation procedure. Mean total lung radiolabel recovery for this control group was 77% suggesting that ~25% of the radiolabel liposomes were lost in the instillation procedure , or possibly were adhered to the airway epithelium. The distribution of the recovered liposomal radioactivity within the 5-minute control lungs was 91% associated with the BALF, 2% associated with BALF cells, and 7% associated with lung tissue.6 to 21.6 × 106 cells with a mean of 17.1 × 106. The purity of this cell fraction averaged greater than 80% type II cells and viability was greater than 95%. Plate cell recovery from the control group ranged from 1.8 × 106 to 4.7 × 106 cells with a mean value of 3.2 × 106. This cell fraction was predominately macrophages and the viability was greater than 90%. For the LPS group at the 18-hour time point, type II cell recovery ranged from 10.6 × 106 to 20.5 × 106 cells and a mean of 16.3 × 106 cells, with a purity greater than 85% type II cells and viability greater than 95%. Plate cells from the LPS 18 group ranged from 2.7 × 106 to 13 × 106 cells with a mean of 6.6 × 106 cells. This cell population consisted primarily of neutrophils and macrophages and viability was greater than 90%. There were no significant differences in cell numbers obtained from control and LPS animals 18 hours after saline or LPS instillation.Table Figure 6 to 40 × 106 cells with a mean of 28.4 × 106. The purity of this cell fraction averaged 75% type II cells and viability was greater than 95%. The number of cells obtained from the plate group ranged from 1.8 × 106to 15.9 × 106 cells with a mean value of 6.2 × 106 with the primary cell type being macrophages with a small percentage of neutrophils and type II cells. Viability for the plate cells were greater than 90%. For the LPS group at the 72-hour time point, type II cell recovery ranged from 19.2 × 106 to 64.6 × 106 cells and a mean of 33.2 × 106 cells, purity greater than 80% type II cells and viability greater than 95%. Plate cells from the LPS 18-hour group ranged from 3.3 × 106 to 16.2 × 106 cells with a mean of 7.8 × 106 cells. This cell population consisted primarily of macrophages and viability was greater than 90%. There were no significant differences in cell recoveries between the control and LPS groups at the 72-hour time point. Of note, there were a greater number of cells recovered from both 72-hour groups compared to both 18-hour groups.Table Figure In the present study, we evaluated the clearance of surfactant-like liposomes at 18 and 72 hours following the intrapulmonary instillation of LPS. At both time points, radiolabel liposomes were instilled two hours prior to killing and the distribution within the lung was determined. Radioactivity was measured in cell-free BALF, isolated BALF cells, whole lung tissue, isolated type II cells and remaining tissue-associated cells. At both time points there was a significant increase in clearance of exogenous liposomes from the airspace and a small decrease in alveolar surfactant phospholipid levels in the LPS groups compared to control groups that received vehicle only. This corresponded with increased radiolabel associated with isolated BALF cells and no difference associated with lung tissue at either time point after LPS instillation compared to controls. There was no difference in liposomal radioactivity associated with isolated type II cells or other tissue cells between both LPS groups and their respective control groups, which was in agreement with whole lung measurements. These data suggest that cells recruited into the airspace as a consequence of the LPS-induced inflammatory response can significantly contribute to increased clearance of surfactant-like liposomes from the airspace Figs. .in vitro [in vitro measurements of lipid uptake, they estimated that neutrophils could account for up to 48% of the observed clearance and could significantly impact surfactant homeostasis. Data from the current study supports this idea by demonstrating that in vivo, neutrophils can indeed contribute to the clearance of surfactant lipids during pulmonary inflammation.Eighteen hours after LPS instillation there was an infiltration of neutrophils into the airspace which resulted in a 10-fold increase in BALF cells. At this time point there was a doubling of total liposomal radioactivity associated with these inflammatory cells compared to alveolar cells recovered from control animals. Neutrophils recruited into alveolar spaces during infection are essential for host defense by phagocytosis and killing of bacteria and other infectious agents. Previous studies have documented that surfactant can in fact modulate neutrophil functions, as surfactant proteins A and D can enhance neutrophil uptake of bacteria . Althougin vitro . Based oin vitro to take up and degrade surfactant lipids and have also been shown in vivo to account for approximately 20% of surfactant lipid clearance in normal lungs [in vitro than macrophages isolated from normal lungs [in vivo data presented in the current study support the theory that activated alveolar macrophages can impact clearance of surfactant lipids.At the 72-hour time point after LPS instillation there was an accumulation of macrophages in the alveolar space that resulted in a 5-fold increase in BALF cells compared to control animals. There was a doubling of radiolabeled phospholipids associated with these alveolar cells and no increase in the amount of radiolabeled phospholipids associated with whole lung tissue. This resulted in a significant increased in clearance of total liposomal radioactivity from the BALF, 72 hours after LPS instillation. Alveolar macrophages have been shown al lungs ,25. Macral lungs , suggestin vitro data had predicted that inflammatory cells (neutrophils and macrophages) could account for a 6 to 13 fold increase in lipid clearance [in vitro study. Although the present in vivo data demonstrated that the inflammatory cells can indeed contribute to increased lipid clearance as suggested previously, the absolute value was considerably less than that predicted from the in vitro studies. Although we do not have an explanation for this quantitative difference between the in vivo and in vitro observations, it is possible that the process of cell isolation in the in vitro study resulted in their activation for subsequent lipid uptake. Alternatively, in vivo, multiple factors can impact total surfactant pool size in addition to clearance by inflammatory cells.Of note, previous learance . In the in vivo, surfactant clearance by type II cells was similar at both 18 and 72 hours after intrapulmonary LPS instillation compared to control groups killed at the same time points . Similar to type II cells, surfactant clearance from these tissue-associated cells isolated from the two LPS groups were similar to their respective control groups. In addition, the clearance attributed to type II cells and tissue-associated cells was similar at both time points for the LPS and control groups. This observation is in agreement with a study by Gurel et al., that documented that alveolar type II cells and tissue macrophages contributed equally to the alveolar clearance of phospholipid in adult mice .in vivo clearance of surfactant lipids by type II cells is unaltered and that the increased clearance of surfactant lipids is primarily due to either neutrophils or macrophages that are recruited to the alveolar space.In conclusion, we demonstrated that after intrapulmonary LPS administration in adult rats, the JLM conceived, designed and performed all aspects of the study, and was the primary participant in its writing. JRW aided in conception and design of study, and participated in its writing.


In the 25 years since they were first discovered, introns have puzzled molecular biologists because of their uncertain function and mysterious origin. Introns are non-coding DNA sequences that reside inside a gene, splitting it into discrete units called exons. The resulting disruption of coding sequence continuity would wreak havoc in protein assembly if eukaryotic cells did not dispose of introns in messenger RNAs—the intermediates in the decoding of gene sequences to produce protein chains—in a now well-described process known as splicing.At first glance, introns may seem like pesky parasites for which eukaryotes have cleverly evolved bypass mechanisms. But introns may also benefit their hosts. Evolutionary advantages of introns include the possibility to create new genes by cutting and pasting exons from existing genes or to diversify the protein output of a single gene by splicing the exons together in different ways. Thus, balancing intron gains and losses clearly has important evolutionary implications for a host.Saccharomyces cerevisiae averages less than one intron per gene, whereas mammalian genes routinely have 10 or more. Whether these differences reflect different propensities for gaining or losing introns is the subject of ongoing debates.Yet different organisms strike that balance differently. The budding yeast Organisms with low intron density display a bias for insertions at the beginning (5′ end) rather than the end (3′ end) of genes. A popular hypothesis is that in these organisms, genes lose their introns through a process that rewrites genomic DNA using as template the messenger RNAs purged of intron sequences. This process might preferentially remove 3′ introns because it relies on an enzyme called reverse transcriptase that can be primed to read RNAs starting at their 3′ end. The hypothesis has gained experimental support in yeast. It also presents the advantage of potentially explaining intron paucity and 5′ position bias in one stroke. In a new study, Cydney Nielsen and her colleagues present evidence that challenges this model.Neurospora crassa, Magnoporthe grisea, Fusarium gramineum, and Aspergillus nidulans) form an evolutionary tree with branching points estimated at 200, 230, and 330 million years ago. While they diverged from yeast some 500 million years ago, they share with yeast a low intron density (one to two introns per gene) and a 5′ position bias. The authors' approach is to tally intron gains and losses during the evolution of these four species and then plot their positions along the genes' length.They address intron dynamics with a genome-wide survey of intron distribution among four Ascomycete fungi with recently completed genome sequences. The four fungi (They identify 3,450 gene regions that are clearly conserved in all four species and harbor an intron in at least one of them. To distinguish intron gains from losses, they rely on a simple parsimony principle, which they refine with additional probability analyses. In brief, an intron present in only one species counts as a gain; an intron absent from one species but present in its closest relative and in a cousin counts as a loss.Nielsen and colleagues record between 150 and 350 intron losses in each lineage. Surprisingly, losses do not occur preferentially at the genes' 3′ end. The authors conclude that while a 3′ reverse transcriptase-based mechanism might be a factor, it cannot be the sole reason for the introns' 5′ bias. The other surprising result is that intron gains occur at almost the same rate as losses in all lineages. Intron gains therefore play an important role in the evolution of even intron-poor genomes. Clearly, intron distribution in fungi owes to forces more complex than simple 3′ intron elimination, forces that the authors propose may also shape evolution of other eukaryotic genomes.


Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes.AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system.Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment. E. coli, and probably most of its homologues, is involved in repair of alkylation damage in DNA and RNA. It repairs 1-methyladenine and 3-methylcytosine by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Recently the protein was identified as a member of the 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenase superfamily [2+ and 2-oxoglutarate, which is subsequently converted into succinate, CO2 and formaldehyde [The purpose of this study has been to identify domains with homology to AlkB in viral genomes, in order to get a better understanding of distribution and possible function of such domains. The AlkB protein of aldehyde .The 2OG-FeII oxygenase superfamily is widespread in Eukaryotes and bacteria , and is AlkB-like genes are widespread in most types of organisms except Archaea. However, whereas bacteria normally have just one or at most two AlkB homologues , multiceA sequence alignment of known ABHs identifies very few residues as totally conserved, basically just a HxD motif, a H and a RxxxxxR motif. These residues are also conserved in the more general 2OG-FeII oxygenase superfamily as described above, except for the final R. The first three residues (HxD and H) are involved in Fe(II)-coordination, whereas the first R is involved in 2OG-coordination. The final R is most likely involved in AlkB-specific substrate binding.E. coli AlkB and the human AlkB homologue hABH3 may be involved in RNA repair. When expressed in E. coli both AlkB and hABH3 reactivate methylated RNA bacteriophage MS2 in vivo. This illustrates that direct repair may be an important mechanism for maintenance of RNA in living cells [In addition to DNA repair, it has been shown that ng cells . RNA repng cells . Howeverng cells . Consequng cells .AlkB homologues have also been found in plant viruses. It has been suggested that methylation may be used in host-mediated inactivation of viral RNAs, and that AlkB homologues in some plant viruses may be used to counteract such defence mechanisms . Howeverin silico mapping of viruses in which such domains have been found, as well as related viruses.The research project reported here has focused on a better understanding of the distribution and potential function of putative AlkB homology domains by using The general mapping strategy of the project was to identify viral genomes with AlkB homology domains, identify common features of these genomes, and subsequently find additional genomes with similar features, but without AlkB homology domains. This data set could then be used to analyse the properties and distribution of AlkB-like domains in viruses, as a basis for generating hypotheses about the evolution and function of these domains.The PSI-Blast search for viruses in the NCBI nr protein sequence database was initiated with ALKB_ECOLI (NCBI gi113638), restricted to residues 110 to 210 and using the default inclusion threshold of 0.005 on E-values. The chosen residue range corresponds to the most conserved region in AlkB homologues [Allexi, Ampelo, Carla, Fovea, Mandari, Potex, Tricho and Vitiviruses, all of which are known to infect plants [The PSI-Blast search converged after 4 iterations, and included 43 hits below the 0.005 inclusion threshold, from 22 different ssRNA positive-strand viruses. The AlkB homologues were found in viruses belonging to t plants .Grapevine leafroll-associated virus 3 . The search for RdRp was initiated with residue range 1361–1798 from Soil-borne cereal mosaic virus . These sequences were chosen based on the output from the previous AlkB search. This gave a library of protein sequences with either AlkB, MT, HEL or RdRp domains, the general composition of which is illustrated in Figure In all of these viruses the AlkB domain is a part of the replicase polyprotein, which normally consists of a viral methyltransferase domain (MT), a viral helicase domain (HEL) and a RNA-dependent RNA polymerase domain (RdRp). Therefore separate PSI-Blast searches for the individual components of the replicase polyprotein were also initiated. All searches were done with PSI-Blast using the default inclusion threshold . The searches for MT and HEL domains were initiated using residue ranges 449–841 and 1938–2178 respectively from Viral_helicase1 and RNA_dep_RNApol2 in all sequences, corresponding to HEL and RdRp domains, respectively. In addition Vmethyltransf and 2OG-FeII_Oxy, corresponding to MT and 2OG-FeII oxygenase (AlkB) domains, were identified in several sequences. However, for sequences from Flexiviridae and Tymoviridae there was no clear identification of any MT domain by Pfam, although they had been retrieved by PSI-Blast in a search for MT domains. Therefore HMMER was used to build a Pfam type profile for these sequences. A PSI-Blast search was initiated using residues 1–500 of Potato virus M . Twelve representative sequences were selected from the search output, covering Carla, Fovea, Potex, Allexi, Capillo and Maculavirus. Subsequences representing the conserved region according to the PSI-Blast alignment, corresponding to residues 35–378 of the query sequence, were aligned using ClustalX, and a Pfam type profile was generated and calibrated using tools from the HMMER package. The resulting profile was able to identify putative methyltransferase domains in all Flexiviridae and Tymoviridae sequences in the data set.The library of protein sequences was screened for known domains in Pfam. This identified Pfam domains Peptidase_C21, C23, C33, C34, C35 and C41, A1pp and OTU – were also identified in subsets of sequences. A1pp is a member of the Appr-1-p processing enzyme family, and the domain is found in a number of otherwise unrelated proteins, including non-structural proteins of several types of ssRNA viruses. OTU is a member of a family of cysteine proteases that are homologous to the ovarian tumour (otu) gene in Drosophila. Members of this family are found in Eukaryotes, viruses and pathogenic bacteria.Other Pfam domains – Closteroviridae, Flexiviridae and Tymoviridae was then used as input for building a phylogenetic tree with MEGA2. The final tree is shown in Figure The MT, HEL and RdRp domains identified by Pfam as described above were extracted from the library sequences, aligned by ClustalX, and combined into a new alignment consisting of only these domain regions. This turned out to be necessary in order to get robust alignments. The intervening regions between the conserved domains are extremely variable in these sequences, and this tended to confuse alignment programs in the sense that conserved regions were not correctly aligned. The combined sequence alignment of domains from A second alignment was generated from all sequences with AlkB-like domains, using only the regions corresponding to MT, AlkB, HEL and RdRp Pfam domains. The domains were aligned individually, and the combined alignment was used as input for MEGA2. However, this data set did not give a reliable phylogeny (data not shown), and the separate domains of this alignment were therefore analysed individually and compared. This analysis is summarised in Table The alignment of the AlkB domain seemed to be of comparable quality to the other alignments. In fact the AlkB domain had the highest average pairwise sequence identity, as seen in Table 2 = 0.95), MT vs. HEL (r2 = 0.87) and HEL vs. RdRp (r2 = 0.81). The plot of the AlkB domain vs. these three domains for the same set of sequences shows a very low degree of correlation for AlkB vs. RdRp (r2 = 0.10), AlkB vs. MT (r2 = 0.12) and AlkB vs. HEL (r2 = 0.16).The degree of co-evolution was analysed by computing pairwise distances between sequence regions in the alignment of MT, AlkB, HEL and RdRp domains described above. In Figure As mentioned above the genome organisation of these replicase polyprotein sequences seems to be very flexible. In order to analyse domain organisation the location of identified Pfam domains were plotted for a number of sequences, as shown in Figure Xanthomonas, X. axonopodis pv citri and X. campestris pv campestris. Xanthomonas attacks plants such as citrus, beans, grapevine, rice and cotton [Xylella fastidiosa. This bacterium infects a great variety of plants, including grapevine, citrus, periwinkle, almond, oleander and coffee [The results described above may indicate that the AlkB domains have been integrated into the replicase polyprotein relatively recently (see Discussion). In order to test for potential sources selected AlkB domains were compared to non-viral sequences. PSI-Blast was used to search the NCBI nr database, removing all viral hits in the final search report. Most of the remaining top-scoring hits were from bacteria. This included two different strains of d cotton . The sead coffee .-50. Homologues of typical viral domains like the viral peptidases were obviously not found, as all viral database entries were excluded. Very few new similarities were found by these searches. Pepper ringspot virus  showed significant similarity to site-specific DNA-methyltransferase from Nostoc sp (E = 1 × 10-74), as well as other cytosine 5C-specific DNA methylases. Bamboo mosaic virus  showed similarity to aggregation substance Asa1 from Enterococcus faecalis (E = 6 × 10-34). A small number of additional similarities seemed to be caused by biased sequence properties (e.g. proline-rich regions), and were probably not significant. This included matches against mucin and cadherin-like proteins from Homo sapiens and multidomain presynaptic cytomatrix protein (piccolo) from Rattus norvegicus. In general the variable regions seemed to be truly variable, with very little similarity to other proteins, except for the Pfam domains already identified.Pfam searches obviously will only identify known domain types in protein sequences. In order to identify potential similarities in regions that were not recognised by Pfam, systematic PSI-Blast searches were performed, using the polyprotein regions between the MT and HEL domains and searching against the NCBI database of reference sequences , excludiCarlavirus sequences, Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191), is shown in Figure Potato virus M is lacking the AlkB domain whereas Aconitum latent virus is lacking the OTU domain. As seen from the dot plot, short regions of similarity close to the diagonal shows that both domains may have been present in an ancestral sequence. However, this region shows a high degree of sequence variation, and as indicated by the dot plot they are almost exclusively mutations. Non-essential or non-functional domains are probably rapidly lost. In this particular case, none of the typical AlkB motifs seem to be conserved in Potato virus M, indicating that this indeed is a non-functional AlkB domain.As seen in Figures  NCBI gi926090 andVmethyltransf) did not match any of the putative methyltransferase domains of Flexiviridae and Tymoviridae, despite the fact that they had been identified via PSI-Blast searches starting with known methyltransferases. Therefore an additional Pfam-type profile was generated. It is obviously a possibility that these domains in Flexiviridae and Tymoviridae are not methyltransferases, and that they are false positives from PSI-Blast. However, the essential residues of a typical viral methyltransferase motif are conserved in the alignment of these domains (data not shown) [Bamboo mosaic virus, which belongs to Flexiviridae, the residues H68, D122, R125 and Y213 have been identified as putative active site residues with similarity to the Sindbis virus-like methyltransferase [Bamboo mosaic virus has methyltransferase activity, as it catalyses the transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP or guanylylimidodiphosphate (GIDP). The corresponding sequence positions are almost completely conserved in the alignment of Flexiviridae and Tymoviridae N-terminal domains. This is most likely significant, as only 7 positions in total are completely conserved in this alignment, which means that the majority of the conserved positions are known to be essential for methyltransferase activity. Work e.g. by Hataya et al. seems to support the assumption that this sequence region is a methyltransferase domain [As described above the Pfam methyltransferase motif (t shown) . In Bambnsferase , and it e domain . It there domain .2+ coordinating H, D and H residues at alignment positions 19, 21 and 91 of Figure Based on the bioinformatic evidence generated here, it seems reasonable to assume that the viral AlkB domains identified by Pfam are functional. All the essential residues found in 2-oxoglutarate- and Fe(II)-dependent oxygenases are conserved, in particular the putative FeFlexiviridae, and in particular to a subset of the Flexiviridae consisting of Viti, Capillo, Tricho, Fovea and Carlavirus. This subset is well separated from the remaining Flexiviridae in the phylogenetic analysis. The split seems to be robust from bootstrap analysis, therefore this family will be discussed here as two subfamilies, Flexiviridae 1 and 2. The same split was observed by Adams et al. in their recent analysis of the Flexiviridae family [Flexiviridae 2. The remaining AlkB domains are found in Flexiviridae 1 (5) and Closteroviridae (2). In general, all the Flexiviridae 2 sequences have at least one extra domain in addition to MT, HEL and RdRp: either AlkB, OTU-like cysteine protease or a peptidase. Most other plant viruses that are included in this survey do not have additional domains, except for Tymoviridae where a peptidase domain seems to be common. For the remaining plant virus families included here (excluding Tymoviridae and Flexiviridae 2), only 14% seem to have additional domains.The Pfam searches show that AlkB domains are found only in a subset of the viral genomes. This subset is phylogenetically consistent  may have been the main driving force for spreading the AlkB domain to new genomes. In the first case a large number of individual integrations could have lead to the present situation. If horizontal gene transfer was the main driving force, the initial number of integrations might have been quite small. It is not easy to differentiate between these two situations.If we assume that AlkB domains have been integrated relatively recently, then either Flexiviridae 2 polyproteins . This property may possibly facilitate the insertion of such domains into the viral genome.The map of Pfam motifs in the variable region between the MT and HEL domains in s Figure  shows thX. fastidiosa and campestris. It is therefore a reasonable possibility that AlkB domains in plant viruses have originated from bacterial mRNA. It is also possible that the mRNA originated from other vectors or from the host itself, but at the present time this is not easily verified or disproved because of the limited number of insect and plant genomes that have been sequenced.There are many groups of organisms that can act as vectors and spread viruses, including bacteria, fungi, nematodes, arthropods and arachnids. The plant viruses may have acquired the AlkB domain either from the vector or from the host itself. As already mentioned, searching with viral AlkB domains in protein sequence databases resulted mainly in bacterial sequences, including the plant pathogens Cucumber mosaic virus [de novo methylation of nearly all cytosine residues within the region of sequence identity between RNA and DNA [It has previously been suggested that the viral AlkB domain may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system of the host . PTGS isic virus . Althougic virus . This RN and DNA . Both RNAs shown here, only a subset of plant viruses have the AlkB domain. However, other viruses may be utilising naturally occurring AlkB proteins in the host. Viruses have to rely on a number of host proteins in order to replicate . In someFlexiviridae. However, the source of these viruses points at another common feature. As seen from the table given in However, there is an alternative hypothesis with respect to the AlkB integration that also has to be considered. As discussed above, the AlkB domain seems to have been integrated relatively recently in viruses found at very different geographical locations, and the only obvious connection seems to be that most viruses belong to a subset of the It could be argued that a more active PTGS system in these plants would give a similar effect. However, in that case we would expect to see more ancient integrations of AlkB domains. It could also be argued that the presence of AlkB domains may be an artefact caused by promiscuous viral domains picking up available mRNA sequences during cultivation of viruses in the laboratory. However, given the large number of different laboratories involved, and the number of different hosts used (data not shown), this seems to be a very unlikely explanation.The hypothesis that environmental compounds, in particular pesticides, may have provoked the integration of AlkB domains into the viral genomes depends upon a high mutation rate and frequent integrations of non-viral domains. The integrations have to be recent, not only in relative terms, compared to other domains in the same genome, but also in absolute terms, compared to the progress of modern agriculture. The integrations also have to be frequent, in the sense that it is likely that integration could have happened several times, in different biotopes.-3 substitutions per site per year [-3 non-synonymous substitutions per site per year, which is considered to be a "moderate" ssRNA mutation rate [-3. In that case, the MT, HEL and RdRp trees shown in Ampelovirus sequences have not been included, as they seem to have diverged from the remaining AlkB-containing viruses at a much earlier stage. If we believe that the AlkB integrations happened after the divergence of most sequence included here, as indicated by the lack of co-evolution in Figure It is difficult to estimate mutation rates in RNA viruses. They evolve very rapidly, and it is often difficult to assign reliable phylogenies. However, recent studies indicate that most ssRNA viruses have a mutation rate close to 10per year , e.g. thion rate . If we aPestivirus may be relevant in this context. There are two biotopes of the pestiviruses, cytopathogenic (cp) and noncytopatogenic (noncp). The host is infected by the noncp form which is converted into the cp form by integration of a fragment of a cellular gene into the viral genome [Although it is generally accepted that viruses frequently use recombination to acquire functionality , it is ll genome . This inl genome , althougl genome . Integral genome . This shde novo integration vs. recombination can be achieved by a closer investigation of the other variable domains, e.g. by looking at how they correlate with the evolution of the AlkB domains.This study has focused on the AlkB domain, mainly as an attempt to get a better understanding of potential functions associated with this domain. However, it is likely that additional information about integration patterns and the relative importance of We believe that the viral AlkB-like domains are conventional repair domains targeted towards the viral RNA. The integration of AlkB domains into viral genomes may have been provoked by environmental methylating agents, e.g. the introduction of DNA/RNA-methylating pesticides in farming. The hypothesis  that theFlexiviridae and Tymoviridae was generated from a ClustalX alignment, using hmmbuild and hmmcalibrate from the HMMER package. Visualisation of motif positions in viral sequences was generated directly from the HMMER output files using a local tool as an interface to the GNU [The NCBI nr protein sequence database was searched with PSI-Blast , with th the GNU  groff so the GNU . Dot plo the GNU .MT – Methyl transferase; HEL – Helicase; RdRp – RNA-dependent RNA polymerase; ssRNA – Single-stranded RNA; PTGS – Post-transcriptional gene silencing; 2OG – 2-oxoglutarate; (h)ABH – (human) AlkB homologue; OTU – Ovarian tumour-like protein; NJ – Neighbour-joining; ML – Maximum likelihood; SJA – Strict joint assertions.MSB carried out all PSI-Blast searches, generated local (sub)sequence databases, and drafted the initial manuscript. FD conceived the study, carried out HMMER/Pfam searches, and estimated evolutionary distances. Both authors participated on sequence alignment, phylogenetic analysis and writing of the manuscript. Both authors have read and approved the final manuscript.Full listing (with GI numbers) of viral sequences and domains included in the analysis.Click here for fileIndividual NJ and ML trees for relevant domains .Click here for file


The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei.We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 μm – wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data.Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments.The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus. Interphase nuclei are structurally highly organized with chromosomes restricted to defined territories. The movKinetic analysis of nuclear bodies requires spatio-temporal microscopic imaging of live cells generating a huge amount of data that is only difficult or impossible to analyze in a standardized way without computational support. The present developments of an Open Microscopy Environment (OME) aims at providing a standardized informatics solution for the storage, management and analysis of light microscopic image data . For quaIn the present study we describe a combined computational and experimental approach to study the dynamic behaviour of nuclear body-like particles formed by GFP-NLS-vimentin  in respoXenopus laevis GFP-NLS-vimentin and synthetic particles (polystyrene microspheres) within the nucleoplasm. GFP-NLS-vimentin is deposited at 37°C within the nucleus of stably transfected SW13 cells in multiple discrete bodies (8 – 40). On average the cells contain bodies that are nearly 1 μm in diameter as observed in the light microscope . Additionally, the artificial microspheres have to undergo tedious processing steps such as sonification and centrifugation prior to injection to avoid the formation of aggregates.For the analysis of complex data derived from spatio-temporal imaging of trafficking particles we developed a proprietary image processing platform, TIKAL (see Methods). The platform allows to directly and easily handle complex microscopic data and to dynamically interact with the data set throughout the whole quantitative data analysis steps. The image processing pipeline is initiated by image pre-processing steps including noise reduction followed by object segmentation (for details see Methods).In many cases, cells move and also change their morphology during the observation period. Global movements include translocation and rotation, whereas morphological changes are either caused by global changes in size (affine transformation) or by local deformations. Since any of these transformations overlay the actual movement of nuclear particles within the cell, we corrected for them by rigid transformations (translocation and rotation), affine transformations  and by thin-plate spline models   Figure , Table 1In summary kinetic changes were most prominent for the directed motion mode. ATP depletion decreased directed motion about 30 % relative to the control group. Treatments with cytochalasin D and nocodazole even showed a 70 % decrease in directed motion relative to the control group. The most striking effect was encountered by treating the cells with sorbitol. Simple diffusion and directed motion were totally abolished whereas the number of particles exhibiting confined diffusion increased by a factor of 7 relative to the control group.In this study we developed comprehensive bioinformatics tools to analyze the kinetic behaviour of small particles in the cell nucleus. For this purpose, fast time-lapse confocal laser scanning microscopy was used to record fluorescent particles in their chromatin environment. Automated image processing algorithms such as image registration and single particle tracking were instrumental to analyze the resulting complex data sets in a most efficient way. Usually, sophisticated image processing methods are widely not accessible for cell biology laboratories working with multi-dimensional data sets. A qualitative, interactive analysis of complex processes in living cells can yield interesting results. However, a quantitative insight into the underlying mechanisms can only be achieved by a rigorous computational analysis. While computational systems have been provided for estimating diffusion and binding constants based on photobleaching experiments of populations of small proteins ,31-33, iWe visualized the different kinetic behaviours of nucleus-injected 100 nm polystyrene microspheres. Furthermore, a stably transfected cell line expressing GFP-NLS-vimentin, which forms nuclear particles in the same size range as microspheres, was used. The majority of nuclear particles moved with obstructed diffusion within distinct corralled regions. This kind of movement was essentially found also for microinjected polystyrene beads. The obstructed diffusion behaviour supports the notion that these particles can diffuse within corrals restricted by dense chromatin regions. Upon chromatin remodelling distinct less dense chromatin regions are formed and enable the particle to move to an adjacent corralled region. We were able to quantitatively assess this phenomenon by measuring the chromatin intensity around an individual particle. Our data show that chromatin intensity decreases prior to a global velocity increase of the particle. Therefore we conclude that the particles do not actively push their way through the chromatin. Moreover, the chromatin itself is able to support or induce the movement of individual particles. The ability of the particle to move from one corral to the next is restricted and regulated by the surrounding chromatin remodelling activities. However, whether local chromatin regions can actively influence the destination of small nuclear particle movement has to be resolved in future investigations. With the present assay we cannot discern whether changes in the velocity of a body simply correlate with the entry of a body into a domain or whether the changes are caused by interaction between a body and the surrounding chromatin domain surfaces.The addition of cellular inhibitors caused significant changes in the diffusional behaviour of nuclear particles. In all treatments a reduction of active transport processes were observed. This suggests that the coordination of nuclear processes such as chromatin remodelling is not solely dependent on single factors such as ATP, i.e. ATP consuming enzymes. Moreover, chromatin regions in interphase nuclei apparently move in a diffusional way , while oThe phenomenon of energy dependent nuclear body movement has been also described in other studies where an anomalous diffusion behaviour and an ATP- and transcription-dependent association of Cajal bodies with chromatin was reported . FurtherA further interesting observation was the reversible formation of chromatin dense regions upon energy depletion. The general effect of this reorganization seems to influence the mobility distribution pattern of the particles only slightly. However, we could observe a significant decrease in directed motion of up to 30 % upon inhibition of energy-depended processes.Furthermore, in order to evaluate the degree of nuclear particle movement with respect to cytoplasmic dynamics, we used the vimentin system to analyze cytoplasmic particle mobility. Mostly active transport processes were observed. Two possible explanations for this phenomenon are suitable. Vimentin, which belongs to the group of intermediate filaments, forms crossbridges to other cellular structures. Since the SW13 cells lack endogenous expression of intermediate filament proteins such as cytokeratins and vimentin, possible interactions with these cytoplasmic intermediate filaments can be omitted. Specific interactions with dynein have been described ,38. HencFrom our data we conclude that the NLS-vimentin system is very suitable for further studies of nuclear architecture. Though we obtained the same results with microinjection assays, the GFP-NLS-vimentin system has significant advantages such as the higher expression efficiency and the fact that the experiments can be performed in a cell system with normal proliferation characteristics.We presented a novel image analysis platform TIKAL that for the first time allows the 4-D tracking of nuclear particles on the background of moving and shape changing objects. TIKAL is complementary to other software systems designed for diffusion studies based on photobleaching experiments. Applying TIKAL we were able to analyze the dynamics of nuclear bodies under various different conditions and thus demonstrated that local chromatin remodelling accounts to a large extent for changes in the dynamics of individual nuclear bodies.Xenopus laevi GFP-NLS-vimentin expression plasmid has been described previously  2> and plotted as <sion (α) ,26 was d). The program consists of a graphical user interface for easy access of the underlying algorithms. The software is written in C/C++ and can be deployed both on Linux and Windows systems. The main components of the program are separated into several main modules, namely a filter, registration, tracking, visualization and data handling module. The filter module contains 2-D and 3-D image processing algorithms such as gamma correction, median, gaussian, anisotropic diffusion filters [TIKAL is an in-house developed platform for multi – purpose image processing . With this platform he was able to perform data analysis and data evaluation. Further, microinjection of cells and nuclei and their respective imaging was carried out by CPB. In additions he assisted MR with imaging and cell culture of the vimentin transfected SW13 cells.MR performed the transfection, culturing, imaging and inhibitor treatment of SW13 cells. Additionally she contributed to the interactive 4-D tracking analysis. CA was involved in planning of this study at an earlier stage. HH supervised the experimental part, whereas RE was responsible for the entire setup and planning of the computational part of this study.


We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold. The SARS RNA genome contains a unique structure that resembles a portion of ribosomal RNA; this may allow the virus to hijack its hosts protein synthesis machinery The virus that causes SARS, like other pathogenic coronaviruses and astroviruses, possesses a linear plus-sense strand RNA genome that has a 5′ methylated cap and 3′ poly-A tail. The viral replicase is translated directly from the genomic sense-strand RNA, and it then creates a full-length complementary (minus-sense strand) copy of the genomic RNA, as well as a nested set of shorter, subgenomic mRNAs having common 3′ UTRs. These 3′ UTRs all share with the genomic SARS RNA a 32-nucleotide element, immediately upstream of the 3′ poly-A tail  2+ ions in the native structure (see cis-[(NH3)2Cl2Pt(IV)]2+ and [Ru(NH3)6]3+ metal complexes that were introduced for heavy atom isomorphous replacement phasing. These highly structured and rigorously conserved features allow us to suggest that SARS pathogenesis might be inhibited by a drug designed to bind to s2m and disrupt one of these structures.G(11), A(12), and A(33), despite their extrusion from the helical base-pair stack, form a well-defined structure that is highly ordered, judging by electron density in the initial map as well as the comparatively low temperature factors these residues have in the refined structure. They conspire with the remaining residues in the asymmetric bubble and the helical region above it to form a rather wide tunnel whose channel runs approximately perpendicular to the main helical axis. The phosphates of G(11) and A(12) are turned inward, creating a negatively charged environment within the tunnel cavity. Consequently, the tunnel forms a binding site for two [Mg modification patterns A of the The intricate three-dimensional structure of the SARS s2m RNA, along with its rigorous sequence conservation, is compelling prima facie evidence for its biological importance in coronaviruses and astroviruses. The structure by itself, however, does not indicate what the function of this motif must be. Hence, comparison of this unique fold with those of known RNA structures is of particular value for formulating testable hypotheses regarding potential biological functions of the s2m RNA. In addition, identification of novel and rigorously conserved tertiary structures that are unique to the viral RNA is of critical importance for future rational design of anti-viral therapeutic agents that specifically target SARS and other coronaviruses and astroviruses.The s2m RNA sequence we crystallized was originally identified from the genomic sense strand within a rigorously conserved region of the 3′ UTR of the RNA. However, because RNA replication and transcription take place via a full-length negative-strand RNA intermediate, it is formally possible that the conserved sequence instead corresponds to a conserved structure at the 5′ end of the anti-sense RNA. We believe this to be improbable because of the energetically unfavorable tertiary structures that would be required to form from the sequence complement. For example, the variant of the energetically stable and rather common GNRA loop structure (GAGUA) would have to be replaced with an energetically unstable and rare CUCAU loop. Similar arguments apply to the other non-Watson–Crick regions of the structure.Crystal packing interactions may potentially distort RNA structures. This effect is sometimes observed for small stem-loop sequences, which often crystallize as duplex dimers rather than as monomeric hairpins. The s2m RNA structure is sufficiently large, and apparently contains enough stabilizing secondary and tertiary interactions, to offset any energetic advantage that might come from crystallizing as a duplex. In addition, the 73% solvent content of the s2m RNA crystals ensures that most of the crystallized RNA is solvent-exposed, rather than involved in extensive packing interactions. At least three inter-molecular contacts are required to form a crystal. The most extensive contact is the base of residue G(11); it stacks upon that of its 2-fold symmetry mate C. It is Crystallographic temperature factors provide direct physical evidence for the relative flexibility or mobility of various regions of a macromolecule. Therefore, based on our chemical probing data, analysis of crystal packing interactions, and consideration of the crystallographic temperature factors, along with the ability to rationalize the sequence conservation pattern and intolerance for nucleotide insertions or deletions based on the structure, we conclude that the crystal structure of s2m is likely to be a close representation of the structure that forms in solution and in the context of the SARS virus RNA genome.Escherichia coli and Thermus thermophilus. However, if we relax the sequence constraints and focus attention upon the conformation of the RNA backbone, we find that the phosphodiester backbone fold accompanying the 90° kink in s2m RNA mimics that found in the 530 stem-loop of 16S ribosomal RNA [The several unique features and unanticipated tertiary contacts we identified in the SARS s2m RNA crystal structure allowed us to reexamine genomic sequences and previously determined RNA tertiary structures for similar motifs with additional constraints imposed by knowledge of the tertiary structure. Our analysis of the human genome, other animal and viral genomes, and the currently available database of RNA three-dimensional structures revealed that the s2m element is found only in astroviruses and coronaviruses; no cellular homologs are immediately apparent. The G(11) to A(33) tertiary contact in the s2m RNA is homologous to the G to A contact in Domain III of the 23S ribosomal RNA, but the context of the interaction in the ribosome is completely different, and the sequence is not conserved between omal RNA  , 100 mM Mg(OAc)2, and 20% MPD. Data from a native crystal diffracting to 2.7-Å resolution, and 3.0-Å cis-(NH3)2(Cl)2Pt(IV)–derivative single-wavelength anomalous dispersion data, were collected at Beamline 9.1 at Stanford Synchrotron Radiation Laboratory on a 3 × 3 CCD detector using 0.98-Å wavelength X rays and crystals that were cryoprotected in the reservoir solution spiked with 12% glycerol and maintained at 100 K. The native and platinum derivative data were processed using CCP4's MOSFLM and reduced and scaled within CCP4 version 5.0 [122 but was unambiguous in P6522, permitting the hand of the space group to be determined. A 47-nucleotide poly-C model was built into the SIRAS map using O, the actual nucleotide-sequence register was then confirmed by inspecting the electron density, and residues 1–47 were built in using O [Crystals of a 48-nucleotide T7 RNA transcript containing the conserved s2m RNA element were obtained via hanging-drop vapor diffusion by equilibrating a solution containing equal volumes of the RNA sample and the reservoir solution against 1-ml of the reservoir solution. The RNA sample solution contained 4.5 mg/ml s2m RNA dissolved in 30 mM Tris (pH 7.6), 100 mM NaCl, and 60 mM MgClsion 5.0 ,33. A sision 5.0 . The ini using O . The pho using O , and the using O . All cry using O ,39 and b using O , and wer using O  and RNAB using O ,43. Tran using O . Primer  using O  using a http://www.rcsb.org/pdb/) under accession number 1XJR and are also available with other supplementary materials at http://www.chemistry.ucsc.edu/%7Ewgscott/sars.Coordinates, native and derivative amplitudes, and experimental phases have been deposited in the RCSB Protein Data Bank (The RCSB Protein Data Bank accession number for the SARS s2m RNA structure reported here is 1XJR. The RCSB Protein Data Bank accession numbers for the other protein and RNA structures discussed in this paper are as follows: the 30S ribosome (1J5E), the 30S ribosome in which prokaryotic IF-1 has been added (1HR0), the eukaryotic analog of prokaryotic IF-1 (1D7Q), and the crystal structure of nsp9 (1QZ8 and 1UW7).


Clusters or runs of purines on the mRNA synonymous strand have been found in many different organisms including orthopoxviruses. The purine bias that is exhibited by these clusters can be observed using a purine skew and in the case of poxviruses, these skews can be used to help determine the coding strand of a particular segment of the genome. Combined with previous findings that minor ORFs have lower than average aspartate and glutamate composition and higher than average serine composition, purine content can be used to predict the likelihood of a poxvirus ORF being a "real gene".Using purine skews and a "quality" measure designed to incorporate previous findings about minor ORFs, we have found that in our training case (vaccinia virus strain Copenhagen), 59 of 65 minor  ORFs were correctly classified as being minor. Of the 201 major  vaccinia ORFs, 192 were correctly classified as being major. Performing a similar analysis with the entomopoxvirus amsacta moorei (AMEV), it was found that 4 major ORFs were incorrectly classified as minor and 9 minor ORFs were incorrectly classified as major. The purine abundance observed for major ORFs in vaccinia virus was found to stem primarily from the first codon position with both the second and third codon positions containing roughly equal amounts of purines and pyrimidines.Purine skews and a "quality" measure can be used to predict functional ORFs and purine skews in particular can be used to determine which of two overlapping ORFs is most likely to be the real gene if neither of the two ORFs has orthologs in other poxviruses. In poxviruses, predicting which ORFs are likely to be expressed (genes) without the use of biochemical analysis usually involves simply choosing a minimum ORF length cut-off and excluding all ORFs that are smaller than the cut-off. Analysis may be extended to include manual inspection of each predicted ORF for the presence of promoter consensus sequences. Excluding ORFs that are smaller in size than the cut-off, however, risks missing genes that are unusually short; during annotation of vaccinia virus strain Copenhagen (VACV-COP) at least three recently verified genes (ranging from 162 bp – 231 bp) were not included in the initial annotation of the complete genome; these genes, VACV-COP A2.5L  and G5.5Poxvirus genes are transcribed from both DNA strands and so far have never been shown to overlap more than a few nucleotides. Despite this knowledge, some poxvirus genomes have been liberally annotated so as to include all ORFs above a certain size, irrespective of whether they overlap larger well-characterized genes. Thus, the current GenBank file for VACV-COP contains 202 major  ORFs and 64 minor  ORFs ,11. The In this paper, it is shown that for the AT rich poxviruses, the purine skews can be used to help predict the synonymous (coding) strand, particularly in regions where smaller ORFs overlap each other on opposite strands of the genome and neither have orthologs in other poxvirus genomes. Furthermore, it is shown that the majority of minor ORFs found in VACV-COP are unlikely to be functional genes and that based on purine content, two of the three genes initially excluded from the annotation of the vaccinia virus genome due to their small size, fit our definition of a major ORF.Figure When the purine skew Figure  slopes iIt was previously shown that minor ORFs in VACV-COP tend to have higher than average serine content as well as lower than average aspartate and glutamate content . Based oPlotting the results of this equation, we found that of the 266 ORFs originally predicted in VACV-COP, 6 ORFs  were incorrectly classified as being major and 9 ORFs  were incorrectly classified as being minor.It was found that the majority of incorrectly classified major ORFs were misclassified because they are small membrane proteins that had a lower aspartate and glutamate content than other major ORFs and that the majority of incorrectly classified minor ORFs were misclassified because they have a lower serine and higher purine percentage compared to other minor ORFs despite the fact that all but one minor ORF (VACV-COP A ORF T) overlap a major ORF on the opposite strand Table . There wA similar analysis was repeated for the genome of amsacta moorei (AMEV), an extremely AT-rich (82%) entomopoxvirus . The AMEIt was found that there were 51 ORFs that had a positive "quality" value and are therefore considered minor. Of these 51 ORFs, 41 ORFs further fit our definition of a minor ORF as they overlapped another larger ORF on the opposite strand and 4 major ORFs  were incorrectly classified as minor even though they each have orthologs in other poxviruses and are therefore major Table . The remThere were three ORFs that had been classified as major  yet overlapped a larger gene on the opposite or same DNA strand Table . Two of For the analyses shown in figures Thus far we have shown that purine skews can be used to predict the coding strand of poxvirus genomes and that major ORFs in VACV-COP and in AMEV usually contain greater than 50% and 49% purines, respectively. In order to explain this purine richness in genes, the purine (R) to pyrimidine (Y) ratio (R:Y) was calculated for each codon position of each coding and non-coding ORF in VACV-COP. A Student's T-test was used to compare the mean R/Y ratio values for the coding (genes) and non-coding ORFs at each codon position; means were considered statistically different when the p-value was less than 0.05. At the first nucleotide position in the codon, both VACV-COP major and minor ORFs are rich in purines but the major ORFs (genes) have significantly (p < 0.05) higher levels of purines at this position . A second method that can be used in conjunction with purine skews is to calculate the "quality" of each predicted ORF using information from amino acid composition and purine content. For a given ORF, if the results of this calculation are negative the ORF is predicted to be a functional gene, and if the results of the calculation are positive, the ORF is predicted to be minor.By comparing purine to pyrimidine (R/Y) ratios at each codon position of major and minor vaccinia virus ORFs, it was found that the purine abundance seen for major ORFs stems primarily from the first codon position with both the second and third codon positions containing equal amounts of purines and pyrimidines.The software used to create the purine skews (DNAGrapher) and the VOCs database are both available for public use via the web ,15.Purine skews were created using the DNA Grapher feature in VOCs . The DNAThe 202 major ORFs (genes) of VACV-COP were ordered in ascending order according to their start positions on the genome and then plotted according to which strand they are located using Microsoft Excel. The first gene was plotted at position 0 of the y-axis of the graph and a value of either -1 or +1 was added to the next gene on the genome depending on if it was on the bottom or top strand respectively.The analysis of VACV-COP ORFs was performed by plotting the results of the following equation: Ser%-Asp%-Glu%+(50%-AG%) where Ser% is serine percentage, Asp% is aspartate percentage, Glu% is glutamate percentage, AG% is purine percentage and the value of 50% is the average purine content of the VACV-COP genome. The "quality measure" for AMEV ORFs used the following formula: Ser%-Asp%-Glu%+49%-AG%) where the only modification of this formula from VACV-COP was the value of 49% which reflects the average purine content of the AMEV genome. The amino acid composition and purine data was obtained from the VOCs database which is available on the internet as a Java Web Start program %-AG% whe.The results of the equation for each ORF were tabulated, sorted in ascending order and assigned a rank from 1 being the ORF with the most negative value to either 266 in the case of VACV-COP or 292 in the case of AMEV being the ORF with the most positive value. These results of the calculation were plotted on the y-axis and the rank of each ORF was plotted on the x-axis using Microsoft Excel.To analyse the ratio of purines to pyrimidines at each codon position, the total number of each nucleotide at each codon position was first calculated using the codontree program with the BC=A option  selected ,18. OnceMD performed all analyses and wrote the manuscript. CU conceived of, and supervised the study, and edited the manuscript.


There is a need for a clear statement of what Evidence-Based Practice (EBP) means, a description of the skills required to practise in an evidence-based manner and a curriculum that outlines the minimum requirements for training health professionals in EBP. This consensus statement is based on current literature and incorporating the experience of delegates attending the 2003 Conference of Evidence-Based Health Care Teachers and Developers ("Signposting the future of EBHC").A variety of definitions of evidence-based practice (EBP) exist. However, definitions are in themselves insufficient to explain the underlying Evidence-Based Practice has evolved in both scope and definition. Evidence-Based Practice (EBP) requires that decisions about health care are based on the best available, current, valid and relevant evidence. These decisions should be made by those receiving care, informed by the tacit and explicit knowledge of those providing care, within the context of available resources.Health care professionals must be able to gain, assess, apply and integrate new knowledge and have the ability to adapt to changing circumstances throughout their professional life. Curricula to deliver these aptitudes need to be grounded in the five-step model of EBP, and informed by ongoing research. Core assessment tools for each of the steps should continue to be developed, validated, and made freely available.All health care professionals need to understand the principles of EBP, recognise EBP in action, implement evidence-based policies, and have a critical attitude to their own practice and to evidence. Without these skills, professionals and organisations will find it difficult to provide 'best practice'. Willing is not enough, we must do" Health care delivered in ignorance of available research evidence, misses important opportunities to benefit patients and may cause significant harm -4. Proviprocesses of EBP and to differentiate between an evidence-based process and evidence-based outcome.A variety of definitions of evidence-based practice (EBP) have been proposed. However, definitions are in themselves insufficient to explain the underlying Towards this goal, we propose three points to clarify and promote the realisation of EBP:1) A clear statement of what EBP means.2) A description of the minimum skill set required to practise in an evidence-based way.3) A curriculum that outlines the minimum standard educational requirements for training health professionals in EBP.This statement was conceived by the delegates of the second international conference of Evidence-Based Health Care Teachers and Developers held in Sicily in September 2003 . In resDuring the last century there has been an exponential growth of research and knowledge ,14. The With this expansion of information, our knowledge should be greater and our practice should be more effective. Unfortunately this is too often not the case . This resome of the uncertainties in this decision process by using the explicit knowledge obtainable from research information. But to do so the research information must be transformed into clinicians' knowledge. Information can be defined as data that has been sorted, analysed, & displayed and communicated through language, graphic displays, or numeric tables. Explicit knowledge is then the meaning people create using this information and its application through action in specific settings [Good practice including effective clinical decision making – step 4 of the EBP process – requires the explicit research evidence and non-research knowledge (tacit knowledge or accumulated wisdom). Clinical decision making is the end point of a process that includes clinical reasoning, problem solving, and awareness of patient and health care context . This prsettings . For exaThe term "Evidence-based medicine" was introduced in the medical literature in 1991 . An origEBP evolved from the application of clinical epidemiology and critical appraisal to explicit decision making within the clinician's daily practice, but this was only one part of the larger process of integration of evidence into practice. Initially there was a paucity of tools and programmes to help health professionals learn evidence-based practice. In response to this need, workshops based on those founded at McMaster by Sackett, Haynes, Guyatt and colleagues were set up around the world. During this period several textbooks on EBP were published accompanied by the development of on-line supportive materials.all knowledge gaps led to early recognition of practical limitations and disenfranchisement amongst some practitioners [Clinical Evidence [The initial focus on critical appraisal led to debate on the practicality of the use of evidence within patient care. In particular, the unrealistic expectation that evidence should be tracked down and critically appraised for itioners . The groitioners . In respitioners , the eviEvidence  and secoEvidence  have beeEvidence , though The five steps of EBP were first described in 1992  and most1. Translation of uncertainty to an answerable question 2. Systematic retrieval of best evidence available 3. Critical appraisal of evidence for validity, clinical relevance, and applicability 4. Application of results in practice 5. Evaluation of performance This five-step model forms the basis for both clinical practice and teaching EBP, for as Rosenberg and Donald observed, "an immediate attraction of evidence-based medicine is that it integrates medical education with clinical practice" .Different practitioners at different levels of responsibility within evidence-based organisations will require different skills for EBP and different types of evidence. It is a minimum requirement that all practitioners understand the principles of EBP, implement evidence-based policies, and have a critical attitude to their own practice and to evidence. Without these skills and attitidues, health care professionals will find it difficult to provide 'best practice'. Teachers, commissioners, and those in positions of leadership will require appraisal skills that come with higher training and continued use .The wider knowledge and use of these skills will help health professionals meet some of Hurd's list of desired educational outcomes  in being• distinguish evidence from propaganda (advertisement)• probability from certainty• data from assertions• rational belief from superstitions• science from folkloreEvidence-based practitioners need additional skills to supplement traditional knowledge. Health care graduates should "be able to gain, assess, apply and integrate new knowledge and have the ability to adapt to changing circumstances throughout their professional life" . Observaconscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients" [Learning has three components: knowledge, skills and attitudes. It is said that "attitudes are caught, not taught" . Attitudatients" . This pratients"  and is aatients"  where prThe curriculum framework for EBP should consider the importance of all steps shown in Table The term 'EBM' has evolved into a larger phenomenon, as increasing numbers of practitioners in various disciplines recognise the importance of evidence to inform all types of health care decisions. Furthermore, greater patient choice and complexity of care mean that many professionals practise as a team. In recognition of the importance of a united commitment to the principles of 'best practice', we propose that the term 'evidence-based practice' (EBP) be used to describe all aspects of this discipline.To ensure that future health care users can be assured of receiving 'best practice' regardless of the type or location of the care received, we make the following recommendations for education:1. The professions and their colleges should incorporate the necessary knowledge, skills and attitudes of EBP into their training and registration requirements.2. Curricula to deliver these competencies should be grounded in the "five-step model" Table .3. Further research into the most effective and efficient methods for teaching each step should be fostered, and linked with ongoing systematic reviews on each step.4. Core assessment tools for each of the steps should be developed, validated, and made freely available internationally.all components.5. Courses that claim to teach EBP should have effective methods for teaching and evaluating Evidence-Based Practice (EBP) requires that decisions about health care are based on the best available, current, valid and relevant evidence. These decisions should be made by those receiving care, informed by the tacit and explicit knowledge of those providing care, within the context of available resources.Finally, EBP requires a health care infrastructure committed to best practice, and able to provide full and rapid access to electronic databases at the point of care delivery. We believe that without the skills and resources for all the relevant components of this framework, the practice of a health care professional, or a health care organisation, cannot be said to provide their users with evidence-based care.1. This consensus statement is from an international working group representing both organisations and individual teachers and developers of evidence-based practice.2. Evidence-Based Practice (EBP) requires that decisions about health care are based on the best available, current, valid and relevant evidence. These decisions should be made by those receiving care, informed by the tacit and explicit knowledge of those providing care, within the context of available resources.3. All health care professionals need to understand the principles of EBP, recognise it in action, implement evidence-based policies, and have a critical attitude to their own practice and to evidence. Without these skills professionals will find it difficult to provide 'best practice'.4. The teaching of EBP should, as far as possible, be integrated into the clinical setting and routine care so that students not only learn the principles and skills, but learn how to incorporate these skills with their own life-long learning and patient care.All of the authors have received honoraria for teaching EBP FP is a consultant of Lilly Deutschland GmbH. The International Conferences of EBHC Teachers and Developers do not accept sponsorship from health technologies  manufacturers.MD, WS & PG wrote the original draft. AC, JM, KH, FP, AB & JO contributed to the concept and all revised drafts of the statement.The pre-publication history for this paper can be accessed here:


Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation  compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate . The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% . In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture. Vitamin A is essential for reproduction, and deficiencies and excesses may result in embryonic loss and/or congenital defects . Retinoltrans and 9-cis retinoic acid (RA) are natural cellular metabolites of retinol and mediate biological activity through interaction with nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR), respectively. Ligand-bound RARs and RXRs influence transcription by interacting with response elements in the promoter regions of retinoid-regulated genes [Retinol is transported systemically and intercellularly bound to retinol-binding protein (RBP). Cellular retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins function in intracellular vitamin A transport, metabolism and homeostasis . All-traed genes . Within ed genes . Concented genes . RBP syned genes ,12.cis RA to in vitro oocyte maturation medium affects trophectoderm differentiation, total cell number and inner cell mass-trophoblast cell ratios, following fertilization in cattle oocytes [The cumulus oocyte complex (COC) may be a target for retinol, since the cells that nurture and communicate with the oocyte, contain transcripts and protein for several RARs and RXRs, RBP and retinaldehyde-2 dehydrogenase (RALDH-2) a metabolizing enzyme . Bovine  oocytes ,16. TogeThe mechanism by which retinol or retinoic acid administration influences oocyte maturation and positively impacts early embryonic development is not known and is the subject of much investigation. Retinoic acid may influence oocyte maturation through its effects on FSH or LH receptor expression as demonstrated in porcine  and rat 2 and reduced O2 conditions. Results suggest beneficial effects of retinol administration during maturation especially to less competent oocytes, and improved development of embryos cultured under atmospheric oxygen conditions, indicating protection from oxidative stress.In the present study, we investigated the effects of retinol administration to in vitro matured oocytes, and cultured bovine embryos under atmospheric O3, 12 mM HEPES, and supplemented with 2 mM glutamine, 2% fetal bovine serum , and penicillin/streptomycin . Oocyte maturation medium (OMM) consisted of bicarbonate buffered TCM-199 supplemented with 50 μg/mL of gentamycin, purchased from Specialty Media, 5 μg/mL of FSH purchased from Vetrepharm Canada, Inc. , 0.3 μg/mL of luteinizing hormone (LH) that was generously provided by the USDA, Beltsville, MD, 10% FBS, 0.2 μM sodium pyruvate and 2 mM glutamine. Modified Tyrode's Albumin Lactate Pyruvate (TALP) media used in sperm preparation (SP-TALP), removal of cumulus cells from oocytes (HEPES-TALP) and in vitro fertilization (IVF-TALP) were prepared as described by Parrish et al. [All chemicals were purchased from Sigma Chemical Company, St. Louis, MO unless otherwise noted. Bovine oocyte collection medium (OCM) was composed of modified M199, 4.2 mM NaHCOh et al. . In vitrh et al.  supplemetrans retinol was dissolved in 100% ethanol, appropriate dilutions made, and aliquots were stored at -80°C until use. Retinol was prepared fresh each month and checked on a spectrophotometer for accuracy. The concentration of ethanol during maturation or culture was less than 0.1%.All-2, ambient air, and saturated humidity.Ovaries from mature, cycling cattle were obtained from an abbatoir and pooled. Cumulus oocyte complexes (COCs) were quickly harvested by slicing follicles (2–5 mm) with a sterile surgical blade, and collecting them in OCM. Intact COCs with homogeneous ooplasm and two or more layers of cumulus cells were selected, washed, and approximately 50 were transferred to 500 μl of pre-equilibrated OMM, and matured for 22–23 hours in a 38.5°C incubator with an atmosphere of 5.0% COg. The pellet was removed and resuspended in SP-TALP and centrifuged for 8 minutes at 460 g. After removal of the supernatant, the sperm sample was reconstituted in 500 μL of IVF-TALP for a final concentration of 1 × 106 spermatozoa/mL. The plate was incubated for 22 hours at 38.5°C in an atmosphere of 5.0% CO2 and ambient air with saturated humidity.Fertilization (Day 0) was performed with combined semen from two bulls of proven fertility prepared according to the method by Parrish and coworkers . Briefly2, 7% O2 and 88% N2 (first and second experiments) with saturated humidity. The mSOF medium was changed every 48 hours. Cleavage was assessed on Day 3 and blastocyst rate was calculated on Day 8.Approximately 18 hours after fertilization putative zygotes were denuded of cumulus cells by vortexing in 500 μl of HEPES-TALP for four minutes (Day 1). Putative zygotes (approximately 35–40) were cultured in 500 μL of mSOF for eight days in a 38.5°C incubator in an atmosphere of 5% COtrans retinol  and embryos were allowed to develop under low oxygen conditions. In the second experiment all-trans retinol was added only to embryo culture medium  on days 1, 3, 5, and 7, and the embryos developed in a low oxygen atmosphere. In the third experiment embryos were cultured under atmospheric oxygen conditions (air and 5% CO2) and all-trans retinol (0 or 5μM) was added to embryo culture medium on days 1, 3, 5, and 7.In the first experiment maturation medium alone was supplemented with all-Data were analyzed as an incomplete block design , or a randomized block design (experiment 4), blocked on plate using mixed model procedures of SAS . At leasIn the first experiment addition of 5μM retinol during IVM tended to improve (p < 0.07) embryonic development to the blastocyst stage, compared to controls Table . The conFurther analysis of the maturation data Table  revealedFurther experiments were conducted during IVC under both low and atmospheric oxygen tensions. Under low oxygen conditions concentrations of 1, 2, and 5μM retinol were not statistically different from controls, and 10μM was deleterious Table . Prelimicis retinoic acid was shown to improve subsequent blastocyst development but high concentrations were detrimental [In the present study, over 3000 bovine oocytes were used to evaluate effects of retinol supplementation during IVM and IVC on embryonic development to the blastocyst stage. Retinol administration during the maturation period alone resulted in concentration-dependent effects. Whereas the presence of 1μM retinol had no effect on development, 5μM retinol tended to improve blastocyst rate of development, at the p < 0.07 level, compared to controls. At a concentration of 10μM, retinol did not significantly improve embryo development compared to controls. In preliminary studies, higher concentrations (100μM) were observed to be cytotoxic (data not shown). Similarly, exposure of bovine oocytes to low concentrations of 9-rimental .A more striking effect on embryonic development (p < 0.02) was observed by supplementation of 5 μM retinol to groups of oocytes with reduced developmental competence in which development of control oocytes to blastocyst was less than 20%. These results indicate that retinol supplementation during maturation may not benefit oocytes competent to progress, but rather, it improves the viability of oocytes that are developmentally challenged. In support of this, we have shown previously that retinol supplementation during maturation improves developmental competence of bovine oocytes compromised by heat stress .Since most transcription in the oocyte occurs prior to maturation during preovulatory development, in vitro culture deprives oocytes of much of this activity. Meiotic inhibitors have been used as a potential means of investigating regulation of oocyte transcription and mRNA processing in vitro . Treatme2 (air and 5% CO2) but not in an atmosphere of low O2 . The present study, and all previous in vitro studies demonstrating a positive effect of retinoid administered during maturation, were performed in an atmosphere of approximately 20% O2 [Retinol supplementation of embryo culture medium dramatically improved development to the blastocyst stage (p < 0.001) when cultured in an atmosphere of approximately 20% Oy 20% O2 ,16,34, ay 20% O2 .2 has been demonstrated to be beneficial to in vitro survival of embryos from a variety of species [Mammalian cells, including the oocyte and those of the early embryo, have evolved several mechanisms to protect against ROS damage and maintain appropriate balances in REDOX reactions. Antioxidants present in the oocyte, embryo and/or its environment include vitamins A (retinol), C and E, pyruvate, glutathione (GSH), hypotaurine, taurine, and cysteamine . Antioxi species .Retinoids participate in a biological antioxidant network, and have been implicated as important regulators of redox signaling pathways ,24. CaroRetinoids may also protect against oxidative damage by maintaining adequate endogenous levels of antioxidant compounds and enzymes. Glutathione is the major non-protein sulphydryl compound found in mammalian cells responsible for strong basal ROS scavenging activity . MaintenResults from the present study indicate that retinol administration during in vitro maturation particularly improved embryonic development in those oocytes that may have been developmentally compromised. Moreover, retinol addition during in vitro culture, under atmospheric conditions, also improved embryonic development compared to those embryos incubated in a 7% oxygen atmosphere. The mechanisms by which retinoids affect the developmental capacity of oocytes and early embryos may include modulation of expression of growth factors and other developmental genes, improving mRNA quality, and direct and/or indirect affects on antioxidant defense mechanisms.TL Performed the experiments under low oxygen conditions, helped to coordinate experiments, and drafted the manuscript.DE Performed the experiments under high oxygen conditions and helped to coordinate experiments.JE Coordinated and assisted in the experimental design.JG Conceived of and coordinated the experiments and drafted the manuscript.