Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease. Know your enemy. Description of the ‘virus gene chip’ that helped to classify the SARS virus as a novel coronavirus Over the past two decades, technological advances in molecular biology have fuelled progress in the discovery of new pathogens associated with human diseases. The identification of novel viruses such as hepatitis C virus , sin nomWe have previously described a prototype DNA microarray designed for highly parallel viral detection with the potential to detect novel members of known viral families . This miDuring the initial phase of research into the etiology of SARS, an unknown virus was cultured in Vero cells from a patient suffering from SARS . Total nNSP11 gene . A pair of PCR primers was designed to amplify the intervening sequences between the two conserved regions, and a fragment that possessed 89% identity over 37 amino acids to MHV, a murine coronavirus, was obtained  , we wereA key feature of this approach is that direct recovery of hybridized material from the microarray provides a rapid route for obtaining sequences of novel viruses. By contrast, conventional strategies for subsequent sequence identification would require time-consuming steps such as library screening or additional rounds of PCR primer design and synthesis. In the case of SARS, we were able to ascertain within 24 h that a novel coronavirus was present in the unknown sample, and partial genome sequences of this virus were obtained over the next few days without the need for specific primer design. To our knowledge, this is the first demonstration of the feasibility and utility of directly recovering nucleic acid sequences from a hybridized DNA microarray. In light of the continuous threat of emerging infectious diseases, this overall approach will greatly facilitate the rapid identification and characterization of novel viruses.Total nucleic acid was purified using the automated NucliSens extraction system . Following the manufacturer's instructions, 100 μl of each specimen was added to tubes containing 900 μl of prewarmed NucliSens lysis buffer and incubated at 37°C for 30 min with intermittent mixing. Fifty microliters of silica suspension provided in the extraction kit was added to each tube and mixed. The mixtures were then transferred to a nucleic acid extraction cartridge and loaded onto the extractor workstation for processing. Approximately 50 μl of total nucleic acid eluate was recovered.For the culture supernatants, 450 ng of nucleic acid was used as input for the amplification protocol. In parallel, 50 ng of HeLa cell RNA was used as a positive amplification control and water was used for a negative control. Samples were amplified using a random-primer protocol as described by infection-Cy5mock) > 1500 intensity units are listed in DNA microarrays were printed and hybridized essentially as described by PCR primers were designed by aligning the hybridizing oligonucleotides (Oligo IDs 15081544_766 and 12175745_728) to the IBV genome  and selecting stretches of near-identity. Primer-B-amplified material was used as the template for 35 cycles of thermocycling using the following program: 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s.Amplified viral sequences hybridized to individual microarray spots were recovered by scraping a 100 μm area of the microarray using a tungsten wire probe  mounted on a micromanipulator while visualized by fluorescence microscopy (Nikon TE300). Recovered material was PCR amplified using primer-B, cloned into pCR2.1TOPO (Invitrogen), and sequenced. A detailed protocol is available as Primer-B-amplified nucleic acid (see above) was cloned in pCR2.1TOPO, plated on 2xYT/kan plates, and grown overnight at 37°C. White colonies were picked into 384-well plates containing 2xYT/kan plus 8% glycerol and incubated overnight at 37°C. DNA was purified by magnetic bead isolation. DNA sequencing involved adding 3 μl of water to each bead pellet, followed by 3 μl of Big Dye terminator (v3.1) sequencing cocktail, and incubation for 35 cycles of 95°C for 5 s, 50°C for 5 s, and 60°C for 2 min. Reaction products were ethanol precipitated, resuspended in 25 μl of water, and loaded onto the ABI 3730xl sequencer. The resulting sequence reads were trimmed to remove primer sequences from the RT-PCR step and then assembled by Phrap . Resulting contigs were screened by blast to remove any contigs with high human or monkey sequence similarity. The remaining contigs were edited to high quality, making any obvious joins. (Sequences are available as Data S1(91.5 KB DOC)Click here for additional data file.Protocol S1(28 KB DOC)Click here for additional data file.Protocol S2(39.5 KB DOC)Click here for additional data file.Table S1(97 KB DOC)Click here for additional data file.Table S2(2.2 MB XLS)Click here for additional data file.The Gene Expression Omnibus accession number for the array sequence is GSE546.


Normative models of choice in economics and biology usually expect preferences to be consistent across contexts, or “rational” in economic language. Following a large body of literature reporting economically irrational behaviour in humans, breaches of rationality by animals have also been recently described. If proven systematic, these findings would challenge long-standing biological approaches to behavioural theorising, and suggest that cognitive processes similar to those claimed to cause irrationality in humans can also hinder optimality approaches to modelling animal preferences. Critical differences between human and animal experiments have not, however, been sufficiently acknowledged. While humans can be instructed conceptually about the choice problem, animals need to be trained by repeated exposure to all contingencies. This exposure often leads to differences in state between treatments, hence changing choices while preserving rationality. We report experiments with European starlings demonstrating that apparent breaches of rationality can result from state-dependence. We show that adding an inferior alternative to a choice set (a “decoy”) affects choices, an effect previously interpreted as indicating irrationality. However, these effects appear and disappear depending on whether state differences between choice contexts are present or not. These results open the possibility that some expressions of maladaptive behaviour are due to oversights in the migration of ideas between economics and biology, and suggest that key differences between human and nonhuman research must be recognised if ideas are to safely travel between these fields. Decisions made by European starlings in certain choice contexts are shown to depend on the training regimes rather than being inherently irrational behavior The study of animal behaviour has often incorporated concepts from economic theory. This was the case, for instance, with the introduction of game theory to the study of animal conflict . SimilarOne consequence of expecting individuals to behave as if they maximised the expected value of a particular function  is captured in the economic concept of rationality. Since “rationality” is used with very different meanings in different fields see . TransitBreaches of rationality are well documented in observational or experimental studies on human choice , and havAn alternative view  is that A number of psychological mechanisms have been proposed to explain the effect of inferior alternatives on choice and other examples of irrationality. According to them, the observed failure to exhibit consistent preferences across contexts would be attributable to the dependence of the information-processing mechanisms used by individuals, or of the heuristics used for making choices, on the nature of the choice problem and available alternatives . While nIt is worth remembering that consistency of preference is accepted by all parties to be only relevant when constancy in the state of the subjects and in the properties of the options is assumed. A subject that prefers lamb to ice cream before dinner, ice cream to coffee immediately after dinner, and coffee to lamb a few minutes later is not considered to be showing intransitivity or violating any principle of rationality, because she is  changing state between the choices. Similarly, a subject that takes a mango when presented with a basket of many mangoes and only one apple, but takes an apple when faced with equal numbers of both fruits may not be considered irrational, because the value of an option may change when it is the last one, as there is a reputation cost of being impolite and taking the last available fruit of any kind . Many huViolations of rationality by animals have also been reported . If thesAlthough these possibilities make the study of rationality valuable, critical procedural differences between the two fields have not been sufficiently acknowledged. One crucial distinction derives from the fact that, while human subjects can be verbally instructed about the properties of the alternatives, animals must be exposed to the contingencies to experience or learn about them. This difference hinders the comparison of the mechanisms underlying human and animal choices, since repeated exposure to different contexts often affects the organism's state, thus removing the justification for expecting transitivity, regularity, or any other principle of consistency. In the case of foraging research, different contexts can alter the subjects' net rate of intake during training, so that at the time of choice the resulting state differs and it may be unjustified to expect consistency of preferences. The fact that optimal decisions should be contingent upon state  has been(Sturnus vulgaris) between members of a fixed, focal pair of options across different choice contexts. Our basic paradigm is defined in Here we further develop the basis upon which to compare economic rationality between humans and nonhumans, and test whether state-dependent decision-making can be responsible for apparent violations of economic rationality in animal choices. To this end, we compare the foraging preferences of European starlings We tested preference between the focal options under three conditions: (1) treatments differing in energetic states when the decoys are absent; (2) treatments differing in which of the two decoys is present, when the energetic consequences caused by each decoy are not controlled; (3) treatments differing in which decoy is present, when the energetic differences they may cause are abolished by supplementary feeding. Our rationale for this design is that if the effects of the decoys are independent of their energetic consequences , then these effects may indeed be evidence for a comparative cognitive mechanism of valuation, possibly caused by the same types of cognitive biases and heuristics reported in the human literature. However, if the effects of the decoys are abolished by controlling for energetic consequences and are generated by imposing state changes in the absence of decoys, it would be more parsimonious to explain the effects as state-dependent decision-making. Our results strongly favoured the latter hypothesis.p < 0.01 in all cases), confirming that the birds discriminate between these amounts.We started by testing whether the birds could discriminate between the amounts of reward associated with each of the foraging options shown in The proportion of choices made for each focal option in the absence of decoys when energetic state was manipulated experimentally is shown in F1,8 = 12.1, p < 0.008; The critical observation for the present purposes, however, is that the magnitude of the preference between the focal options differed significantly between intake treatments. Specifically, preference for FA over FD was higher in treatment “Low Intake” (the treatment with lower accumulated intake) than in treatment “High Intake” .The stability criteria see  were reaIn this experiment, two groups of six starlings each  were trained with three options  and then allowed to choose between either two  or three  of those options. The two treatments, “High Intake” and “Low Intake”, differed in which of the two decoys  was present during training and in the trinary choices. Within each group, every subject experienced both treatments.F1,4 = 7.4, p = 0.06; F1,4 = 0.2, p = 0.677; Considering the results of the previous experiment, preference for FA over FD should be higher in the treatment with lower accumulated intake (treatment “Low Intake”) for group NC, in which such intake differences were not eliminated. No differences should, however, be observed in group C, in which intake differences were abolished. The results from the binary choice trials  are shown in F1,4 = 27.35, p = 0.006). This difference was not observed for the group in which intake was controlled .We also analysed the temporal aspects of state changes on choice. Because the long-term rate of gain offered by DA was higher than that offered by DD, in group NC the difference in cumulative intake between the “High Intake” and “Low Intake” treatments must have increased over the trials in a session. Accordingly, the rate of increase in the strength of preference for FA over FD along the trials  was significantly higher in the latter than in the former treatment  versus trinary  trials . We make two types of comparisons, between-treatments and within-treatments. In the between-treatments comparison we compare the binary trials of one treatment with the trinary trials of the other. For example, we compare the binary trials of treatment “Low Intake”  against the trinary trials of treatment “High Intake” , and vice versa. In the within-treatments comparisons, we compare binary versus trinary trials within the same treatment . Notice that within a given treatment (within-treatment comparisons), accumulated intake was the same in binary and trinary trials for both groups of subjects (C and NC), whereas between treatments (between-treatment comparisons), accumulated intake differed between binary and trinary trials for the group of subjects in which intake differences were not controlled (NC). Therefore, if intake, rather than purely cognitive effects, is the cause of changes in preference for the focal options, apparent violations of regularity and of the constant-ratio rule should be observed only in the between-treatments comparisons for group NC. Conversely, if the presence of the decoys has a cognitive effect upon preferences that is independent of state, such violations should be observed both in the between- and within-treatments comparisons. p > 0.1). For group NC, the observed directions of preferences were, in all cases, consistent with all predictions shown in 1,4 = 7.8, p = 0.049; 1,4 = 9.2, p = 0.039; p > 0.1; The presence of apparent violations when the effect of the decoys on intake was allowed and their absence when the effect was abolished was again consistent with the hypothesis that these apparent irrationalities were caused by differences in intake brought about by exposure to the decoys. The hypothesis is further confirmed by the absence (both for the group that received supplementary feeding and the one that did not receive it) of violations in the within-treatment comparisons, when the cognitive effect of the decoys was allowed but state was neutralised.In group NC, all birds reached the stability criteria. In group C, however, one bird did not reach the criteria in treatment “Low Intake”, and another did not reach stability in treatment “High Intake”. We therefore reanalysed the data for this group excluding these two birds. The results were the same, namely, in none of the tests for this group was rationality breached.Our aim in this study was to foster the development of a solid interdisciplinary basis upon which to compare research on economic rationality in humans and nonhumans, and to investigate whether normatively inspired hypotheses of animal behaviour may be systematically misleading, as they implicitly assume rationality. To this end, we examined whether violations of economic rationality in animals that have recently been reported in the literature represent real violations of rationality caused by the use of comparative cognitive mechanisms of choice as proposed for humans or, alternatively, to unwittingly imposed differences in the state of the subjects. To do this requires testing whether one can reproduce the reported breaches of rationality and whether they are abolished when cognitive effects are allowed but state differences are eliminated. A further test requires generating such violations by changes of state alone. We have achieved all of these conditions in our experiments.Why should preferences be modulated by energetic state? To start dealing with this question, it is necessary to start by considering why choices do not go exclusively to the option with maximum value. From an evolutionary perspective, one possibility is that subjects are adapted to some level of ambiguity , and tracking these properties requires some level of response to each available option . If partIn F1,8 = 24.6, p = 0.01) and in the experiment with decoys, but only for the group of subjects for which intake was not controlled . It is thus possible that these potential differences in motivational state led subjects to pay less attention to the alternatives during a choice opportunity in the treatment “High Intake”, resulting in the observed differences in preference levels.From a mechanistic perspective, it is also possible that, under conditions of higher energetic intake, animals are less motivated to search and work for food. Our data support this possibility. The time subjects took to start working once presented with any option in no-choice trials  was significantly longer in the treatment “High Intake” than in “Low Intake” in the experiment without decoys  among three flower types differing in volume and concentration of sucrose  in binary (target and competitor) and trinary  contexts. The birds experienced both contexts consecutively. In each of them, they made repeated choices between the available flower types until a minimum number of 150 choices for the target and competitor had been reached. The strength of preferences for the competitor over the target increased significantly in the presence of the decoy (trinary context), and the authors interpreted these results as being inconsistent with the use of absolute evaluation mechanisms as normally postulated by functional accounts of behaviour. Yet, the resulting differences could have been caused by exposure to energetically different contexts. Net rate of energy intake of the target, competitor, and decoy were, respectively, 81.9, 92.0, and 59.5 J/s. Considering, for instance, an average proportion of choices for the decoy of about 20% (as described in the study), subjects would necessarily experience a higher intake rate in the binary than in the trinary context unless they modified the relative allocation of responses. Therefore, if the interval between consecutive foraging bouts did not differ systematically between contexts, cumulative gain along the 150 choices would have been lower in the trinary context, favouring preference for the option offering the higher net rate of intake, as reported. Further analyses on the extent to which state differed between contexts, testing for differences in inter-bout intervals, variability in choices, and unplanned differences in nutrient balance  independently, not integrated into a single expression of value. In the conditions described by Subjects were 28 naïve starlings captured in Oxford (English Nature licence # 20020068). After capture, the birds were kept outdoors and, during the experiment, transferred to individual indoor cages (120 cm × 60 cm × 50 cm) that served as housing and testing chambers. Lights were on between 0500 and 1900 h, and temperature ranged from 12 °C to 16 °C. Subjects were visually but not acoustically isolated. The experiments took place between June and October 2002.Each cage had a panel with a central food hopper and three response keys. Computers running Arachnid language  served for control and data collection. Rewards were units of Orlux pellets, crushed and sieved to an even size (0.025 ± 0.005 g). Automatic pellet dispensers  delivered rewards at a rate of 1 unit/s. Each option was signalled by a different key colour .Subjects were first trained to peck at keys to obtain rewards until all birds pecked at least 80% of the food opportunities. A discrete trials procedure with two types of trials (no-choice and choice) was then employed. No-choice trials provided the birds information about each alternative, but also contributed to their rate of intake. These trials started with one key blinking. The first peck caused the light to stay steadily on. The first peck after the programmed delay had elapsed triggered the delivery of the programmed amount of food, followed by a fixed ITI of 60 s, during which all keys were off. Since food was delivered following the first peck after the end of the designated delay, experienced delays were slightly longer than those programmed .Choice trials began with two or three keys (depending on whether choice was binary or trinary) simultaneously blinking. The first peck on any of them caused the pecked key to turn steadily on and the others to turn off. After that, the trial continued as in no-choice trials. The order and sides in which the options were presented was randomised. After the sessions, subjects were fed ad libitum with turkey crumbs for at least 2 h, then supplemented with ten mealworms, and then food deprived until the beginning of the experimental sessions on the following morning.In all experiments  we used a within-subjects design with two treatments. The within-subjects design was preferred owing to the high level of variability between individual starlings' energetic requirements, which would have prevented accurate control of energetic state between groups. We therefore focus our analyses on within-subjects comparisons, since variations in the energetic state of subjects between groups of different subjects would hinder the comparison of their preference levels. The pairing of options with colours was balanced across subjects and changed between treatments. Treatment order was balanced across birds. Subjects were given one resting day with ad libitum food between treatments. Each treatment lasted for 20 sessions. Data from the last five sessions were used for analyses.In the discrimination experiment, we used a between-subjects design with six male starlings, split into two groups of three birds each. Group 1 chose between 1 and 2 units of food, and group 2 chose between 2 and 5 units. Rewards were delivered after the first peck on the corresponding key. Subjects experienced two sessions per day, at 0800 and 1300 h. Each session consisted of 84 trials, divided into 21 blocks of four trials each: two no-choice (each option presented once) followed by two choice trials.To investigate the potential effect of different intake rates on preference between the target options, we used ten birds  in a within-subjects design with two treatments, which differed with respect to the amount of supplementary food delivered to the starlings. One of the treatments simulated the intake effect  of experience with decoy DA (treatment “High Intake”) on 25% of the foraging opportunities . The other treatment simulated the intake effect of experience with decoy DD (treatment “Low Intake”) on 25% of all foraging opportunities. To achieve this we delivered unconditionally the reward corresponding to the appropriate decoy once per experimental block (details below), after the ITI that followed the last trial of that block. Unlike the trials with the pair of focal options, no action was needed on the part of the subjects to receive the unconditional reward, nor was any specific discriminative stimulus associated with it.There were three daily sessions, at 0600, 1000, and 1400 h. Each session consisted of 36 trials, grouped into 12 blocks of three trials each. Each block started with two no-choice trials , followed by an ITI and an unconditional food delivery in which the amount of the simulated decoy was delivered after the delay corresponding to that decoy had elapsed. The third and last trial of each block was a choice between the two focal options.Twelve birds were randomly assigned to two groups  of six birds each . All subjects experienced two treatments, one with decoy DA (treatment “High Intake”) and another with DD (treatment “Low Intake”). In group C, the differences in intake rate between treatments caused by the exposure to the energetically different decoys were eliminated with supplementary feeding.Three daily sessions started at 0500, 0900, and 1400 h. Each session consisted of 63 trials, grouped into seven blocks of nine trials each: three no-choice trials followed by six choice trials in a random order . To equalise intake between treatments in group C, we adopted the following procedure. We calculated the maximum obtainable amount of food and delay per block for treatment “High Intake” (the treatment offering the higher cumulative delay and amount), and in both treatments delivered supplementary rewards up to this amount and delay twice per block. Thus, in every block of trials we equalised intake and total time to the same value in both treatments. The supplement was delivered after the fourth and the ninth trial of each block, after adding the appropriate delay to the ITI. In both treatments, supplements in the middle of the block were followed by a 5-min no-food interval to prevent satiation. Blocks were separated by 10-min intervals.According to the principle of IIA, the strength of preference between two options should be independent of the presence of other (less preferred) options. These other options may either form part of the general situational background and be absent at the time of the choice, or form part of an enriched set of options at choice time. To test whether differences in background led to breaches of IIA, we compared choice proportions in binary choices  between the two treatments by conducting separate tests for each group of subjects. To test the temporal effects of potential state changes over the trials in the experimental sessions , we calculated the slope of the regression of trial number against  proportion of choices for FA over FD and tested whether the group of slopes was different between treatments for both groups of subjects.We also tested for differences in preference between the focal options across contexts, comparing binary with trinary choice trials. We performed two analyses. First, we tested whether the relative strength of preference between the focal options differed between the binary and trinary contexts. Relative preferences were calculated asp is the relative preference for FA over FD when the alternatives indicated inside the curly brackets were present, and n is the number of choices for FA within the same set of alternatives. D stands for either of the decoys (DA or DD). The second term in the denominator follows the same notation. According to a strong probabilistic version of IIA known as the constant-ratio rule  in five consecutive sessions (against session number) was not significant and the standard deviation of these proportions did not exceed 0.20.


Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling.Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting.2-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K+ flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells.Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased betaOur data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer. Breast cancer is the leading cancer in women  and estrRecent studies in human cancer cell lines or in animal models have shown that the growth of adenocarcinomas of the lungs, pancreas and colon are under β-adrenergic control -15. Stud2-adrenergic receptors in all six breast cancer cell lines tested , whereas β1 receptors were not found in two estrogen non-responsive cell lines  -dCTP . Reactions were run with the following conditions: 1 cycle of 2 min. at 94°C, then 31 cycles of 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec. A 10 μl sample of each PCR reaction was heated at 95°C for 3 min., then loaded into a 5% TBE-urea Ready Gel . This underwent electrophoresis at 200 V in TBE buffer until the xylene cyanol dye front reached the bottom of the gel. The gel was transferred to filter paper, dried and exposed to film or imaged on a Molecular Dynamics 445 SI phosphoimager . A 100 bp DNA ladder (Invitrogen-Life Technologies) was exchange labeled with T4 polynucleotide kinase and 30 μCi [γ-32P] ATP .Preliminary experiments were done with MDA-MB-453 cells to determine a cycle number of PCR amplification that is within the linear range, which is critical for meaningful results to compare expression levels between samples and to determine the mixture of 18S primers/18S competimers (Ambion-Classic II). The 18S ribosomal RNA primers/competimers are used as an invariant internal control, which allows correction for sample variation. Results indicated this was 31 cycles of PCR and a 1:9 18S primer/competimers ratio. For experimental treatments, as described before , cDNA waThe GIRK-1 primers for real time PCR are forward 5'-ctctcggacctcttcaccac-3' and reverse 5'-gccacggtgtaggtgagaat-3' . and the internal TaqMan probe is 6-FAM-tcaagtggcgctggaacctc-TAMRA , annealing temperature 62°. GIRK2 primers-forward 5'-gacctgccaagacacatcag-3' and reverse 5'-cggtcaggtagcgataggtc-3'  and the internal TaqMan probe is 6-FAM-gtgcaatgttcatcacggcaac-TAMRA (bases 837–859), annealing temperature 56°. GIRK4 primers-forward 5'-agcgctacatggagaagagc-3' and reverse 5'-aagttgaagcgccacttgag-3'  and the internal TaqMan probe is 6-FAM-accggtacctgagtgacctcttca-TAMRA (bases 301–324), annealing temperature 62°. Reactions were run on a Cepheid SmartCycler . Reaction conditions are 200 μM dNTPs, 0.3 μM gene specific primers, 0.2 μM TaqMan probe, 4 mM (GIRK1) or 6 mM (GIRK2or4) magnesium acetate, 2 μl cDNA and 1.5 U MasterTaq  and MasterTaq buffer in a final volume of 25 μl. TaqMan beta-actin detection reagents (Applied Biosystems) were used with the same reaction conditions as above except a 5 mM magnesium concentration was used and this was run at 95° for 120 seconds, followed by 45 cycles of 95°, 15 seconds; 68°, 30 seconds.4(3))  was added to MDA-MB-453 breast cancer cell line suspensions of 1 × 106 cells at a final concentration of 150 × 10-9 M. Fluorescence intensity measurement after treatment of the cells was obtained from a FACS Vantage/SE Cell Sorter .We determined inward potassium flux in these cells by flow cytometry via the method of Krjukova et al. . The negFollowing incubation with agents as detailed in the Figure legends, cells were washed twice with phosphate buffered saline and lysed with cold RIPA lysis buffer containing protease inhibitors . Cell lysates were collected from culture plates using a rubber policeman, and protein collected by centrifugation. Protein concentrations were determined by BCA protein assay . Aliquots of 20 μg protein were boiled in 2x loading buffer  for 4 minutes, then loaded onto 10% Tris-HCl-Polyacrylamide gels , and transferred electrophoretically to nictrocellulose membranes. Membranes were incubated with primary antibodies  and appropriate secondary antibodies . In all western blots, membranes were additionally probed with an antibody for actin (Sigma) to ensure equal loading of protein between samples. The antibody-protein complexes were detected as previous described  or by thThe estrogen-responsive  and estrogen non-responsive  human breast cancer cell lines were screened for the presence of the GIRK1 potassium channel by RT-PCR analysis. The ER(+) cell lines MCF-7, MDA-MB-361 and ZR-75-1 and the ER(-) cell line MDA-MB-453 expressed mRNA for the GIRK1 channel Figure , and GIR2 adrenergic receptor but not the β1 receptor [2-adrenergic receptor mRNA (p < 0.0079 by t-test) (data not shown).To determine if GIRK channels are functionally linked with β-adrenergic receptors in breast cancer cells expressing this ion channel, we decided to investigate the ER(-) cell line MDA-MB-453. This ER (-) cell line, which was the only ER(-) cell line tested that expressed GIRK1, was used for further experiments due to the fact that ER(-) breast cancers have a poorer prognosis than ER(+) cancers ,3. In adreceptor . MDA-MB-receptor . Using rT) were calculated for each sample, which will be lower for samples with more mRNA expression. CT values for GIRK 1 expression were significantly (p < 0.001 by t-test) lower for propranolol treated cells (27.808 ± 0.107) (SD) as compared to control MDA-MB-453 cells (28.964 ± 0.338) (SD). Actin CT values were unchanged between control (13.666 ± 0.286) (SD) and propranolol treated cells (13.404 ± 0.427) (SD). The exposure to propranolol caused a slight decrease in GIRK2 mRNA expression (p < 0.04 by t-test) in the treated cells, opposite the result we found for GIRK1. Control  and propranolol . GIRK4 expression levels were unchanged between control and propranolol treated cells, indicated by real time PCR. Control  and propranolol treated . By contrast, MDA-MB-453 cells treated for 30 minutes daily for 7 days with 1 μM propranolol did not show changes in GIRK1 mRNA expression levels ) was added to MDA-MB-453 breast cancer cell line suspensions of 1 × 106 cells at a final concentration of 150 × 10-9 M. Fluorescence intensity measurement after treatment of the cells was obtained from a FACS Vantage/SE Cell Sorter. An increase of dye fluorescence corresponds to membrane potential depolarization and K+ flux. The β2 selective agonist, formoterol hemifumarate (1 μM), added to MDA-MB-453 cell suspensions at the same time as the fluorescent dye lead to a 2X increase of fluorescence inside the cells, indicating inward potassium movement trimethine oxonol  after exposure to a dose of tritiated NNK equivalent to the amount of NNK found in two packs of cigarettes. Stimulation of Erk 1/2 was also seen using 1 μM of the beta-adrenergic agonist formoterol, but only at 150 minutes (data not shown).We also determined signaling events in MDA-MB-453 cells that are affected by beta-adrenergic agents. Increased activation of Erk 1/2 was seen in MDA-MB-453 cells after treatment with 100 pM NNK at times ranging from 15–150 minutes Figure . The conOur data demonstrate expression of the G-protein inwardly rectifying potassium channel 1 (GIRK1) in 67% of the breast cancer cell lines tested, with higher levels in ER(+) cell lines. Approximately 40% of primary human breast cancers were found to express GIRK1 and expression of GIRK1 was not found to be correlated with ER status . These d2-adrenergic mRNA was decreased by the same treatment (data not shown). Addition of propranolol for 7 days for only 30 minutes daily had no effect on GIRK1 gene expression. Treatment for a shorter period of time (24 hours) also had no effect on GIRK1gene expression in our studies. The 6 day continuous exposure to propranolol caused a barely detectable decrease in GIRK2 mRNA expression and no change in GIRK4 mRNA expression levels. Longer treatment times may be necessary for gene expression changes in GIRK2 or GIRK4 similar to gene expression changes that are seen in GIRK1.The majority of experiments in the present study were done with the ER(-) cell line MDA-MB-453 since it was the only ER(-) cell line tested that expressed GIRK1, and because ER(-) breast cancers have a poorer prognosis than ER(+) cancers ,3. We sa1or β2 receptors [2 and β3 adrenergic receptors were found to be correlated with a decreased risk for breast cancer [2 adrenergic receptor in the six breast cancer cell lines used in this study, but expression of β1 in all the estrogen responsive cell lines but not in two ER(-) cell lines (MDA-MB-435S and MDA-MB-453) [Although there were no short-term effects of beta-adrenergic agents on GIRK gene expression, we detected other cellular effects. The beta-adrenergic agonist formoterol hemifumarate stimulated potassium influx in MDA-MB-453 cells, and this influx was prevented by the GIRK channel inhibitor clozapine. NNK, a high affinity agonist for beta-adrenergic receptors  increaseeceptors . Heteroleceptors . Two polt cancer , suggest-MB-453) . Further-MB-453) .All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK potassium channels are possible in these breast cancer cell lines. This is the first report that implicates β-adrenergic receptors and G-protein inwardly rectifying potassium channels 1 (GIRK1) in the regulation of human breast cancer cells and suggests a potential role of the tobacco nitrosamine NNK in breast cancers expressing these regulatory pathways. Beta-adrenergic antagonists have both long term effects on gene expression and beta-adrenergic agonists have short term effect on potassium flux and cellular signaling pathways.The author(s) declare they have no competing interests.HP carried out the majority of experiments and participated in the design of the study, and helped draft the manuscript. QY carried out the western blots. YC was involved in relative competitive RT-PCR studies. HS conceived of the study and helped draft the manuscript.The pre-publication history for this paper can be accessed here:


Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-Å resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.The  The Drosophila protein PGRP-SA is found to have an intrinsic activity to cleave L,D-configured peptide bonds, which exist only in prokaryotes. This work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria Drosophila, two distinct signal transduction pathways are involved in the pathogen-specific innate immune response by inducing the expression of a panel of specific antimicrobial peptides  in response to bacterial infections . PGRPs cosophila . Remarkalt flies . Similarlt flies .N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) plus a stem peptide chain consisting of D- and L- (or meso-) amino acids, with the third amino acid being most frequently lysine in Gram-positive bacteria and diaminopimelic acid (DAP) in Gram-negative bacteria. Recently, Several PGRPs have been shown to bind peptidoglycan (PG) , an esseDrosophila. We tested the activity of rPGRP-SA in vivo by injecting the protein into wild-type (wt) and PGRP-SA–deficient seml). These results demonstrate that rPGRP-SA expressed in insect cell culture medium is active in vivo.To facilitate molecular characterization of PG recognition and signal transduction mediated by PGRP-SA, we overexpressed and purified recombinant PGRP-SA (rPGRP-SA) in a baculovirus-insect cell expression system. PGRP-SA is a secreted protein circulating in the hemolymph (the insect blood) of reatment A and 1B.. luteus C. As litml flies D. InjectM. luteus or Enterococcus faecalis and to DAP-type PGs from Escherichia coli or Pseudomonas aeruginosa, but not to amidated DAP-type PGs from Bacillus  at Argonne National Laboratory. The crystal structure was determined by molecular replacement, using the structure of PGRP-LB as a search model. PGRP-LB is a zinc amidase similar in structure to T7 lysozyme  and is 2tyrosine . The Cystyrosine A. The otan PGRPs C.The most prominent feature of PGRP-SA is a long surface groove demarcated by residues of the H2 helix from one side and of the L1–L4 loops from the other side, with the residues from B3, B4, and B7 of the central β sheet forming the base of the groove A. These M. luteus by in vitro PG-binding assays . As a result, the culture medium from the C48A mutant failed to restore the semlPGRP-SA phenotype after injection in Drosophila -L-Ala-γ-D-Glu-meso-DAP-D-Ala, and analyzed the reaction mixtures afterwards by high performance liquid chromatography (HPLC) . The Km value of rPGRP-SA for its substrate GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-meso-DAP-D-Ala was 21.4 ± 1.8 μM and the catalytic constant kcat was 0.48 ± 0.02 h−1. The small turnover number of PGRP-SA estimated for hydrolyzing DAP-PG substrates is comparable to the kcat of small G proteins such as Ras GTPases. We also tested different other PG-related tetrapeptide compounds as substrates and found that rPGRP-SA hydrolyzed the same peptide bond in DAP-containing muropeptides but had no detectable activity on all the lysine-type compounds tested (unpublished data). This enzymatic activity of rPGRP-SA was not inhibited by ethylenediaminetetraacetic acid (EDTA) or by phenylmethylsulfonyl fluoride (PMSF) (unpublished data). We observed that the two Ser158 mutants, S158C and S158A, did not exhibit any detectable activity  . To our al D-Ala B to 5E. -SA does A. In the-SA does B. The fa-SA does . To test-SA does G and 6A. culture .L,D-carboxypeptidase, from E. coli, has been identified and characterized  with the docking groove residue(s) to help orient the peptide bond between DAP and D-Ala. However, understanding the structural basis of the selectivity to DAP-PG over lysine-PG would require a cocrystal structure of PGRP-SA and a lysine-type PG ligand. As the PG docking groove is lined with residues that are highly diverse among different PGRPs, indicating that each PGRP protein may bind to a specific set of PG fragments, determining the structure of PG ligand-bound PGRP-SA will also provide important insights into PG recognition specificity of this family of proteins. However, so far, no cocrystal of a PGRP protein with a PG compound has been obtained.In the present study, we characterized the PG docking groove of PGRP-SA through a combined structural and functional analysis, and we showed that this surface groove mediates both PG sensing and Toll signaling and that Ser158 in the groove is involved in PG interaction, Toll activation, and the newly discovered eraction A. TherefDrosophila possesses a high number of genes encoding serine proteases and serine protease inhibitors (serpins) (serpins) . Serine serpins) . A hemolserpins) . As PGRPserpins) . Based oserpins) . AlthougDrosophila PGRP-SA will be presented elsewhere. Briefly, full-length PGRP-SA  with a 6×His tag at the C terminus was overexpressed in insect Hi-5 cells using the Bac-to-Bac baculovirus expression system  and purified with Talon metal affinity resins  followed by size exclusion on a Superdex 75 column  pre-equilibrated in 20 mM Tris-HCl (pH 7.8) and 300 mM NaCl. The purified protein was analyzed by N-terminal sequencing and mass spectrometry to ensure its identity and purity. Crystallization was carried out at 21 °C by the hanging-drop vapor diffusion technique. The protein formed plate-like clusters over a reservoir containing 2.0 M NaKPO4 (pH 6.2). Single crystals were produced by two successive rounds of streak- and macroseeding and were cryoprotected in reservoir solution supplemented with 30% glycerol before data collection.Details on the cloning, expression, purification, and crystallization of recombinant We collected X-ray diffraction data using synchrotron radiation at the 19-ID beamline at APS. The diffraction images were processed and scaled with the HKL2000 package . The posPoint mutations were generated by a PCR-based strategy using the QuikChange Kit , and the identities of the mutagenized products were verified by sequencing.+, Diptericin-lacZry), P flies were used as wild-type strains   . DrosophsemlPGRP-SA female adults (3–4 d old) using a Nanoject apparatus . One hour later, flies were infected with a thin needle previously dipped into a concentrated culture of M. luteus or given an injection of 9.2 nl of M. luteus PG (5 mg/ml). Flies were then incubated for 24 h at 25 °C. A highly purified solution of M. luteus PG was produced and injected in flies as described by A quantity of 9.2 nl of water or rPGRP-SA protein was injected into the thorax of wild-type or Drosomycin quantification from whole animals, RNA was extracted using RNA TRIzol (Invitrogen). cDNAs were synthesized using SuperScript II (Invitrogen) and PCR was performed using dsDNA dye SYBR Green I  on a Lightcycler (Roche). All samples were analyzed in duplicate and the amount of mRNA detected was normalized to control Rp49 mRNA values. We used normalized data to quantify the relative levels of a given mRNA according to cycling threshold analysis (ΔCt).For g for 5 min, was washed with 1 ml of binding buffer followed by a 5-min spin and finally dissolved in 10 μl of SDS buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The PG-bound rPGRP-SA was visualized by Coomassie Blue staining.The assay was performed at 4 °C by incubating 20 μg of purified wild-type or mutant rPGRP-SA with 300 μg of insoluble PGs, prepared as described previously , in 300 18 5μ reverse-phase HPLC column  and elution was performed at 0.6 ml/min with buffer A (50 mM sodium phosphate [pH 4.45]) for 10 min and then with a gradient of methanol in buffer A (from 0% to 25% in 50 min). Peaks were detected at 215 nm. In all cases, substrates and products were purified and desalted by HPLC, and their identity was confirmed by amino acid and mass spectrometry analyses.The activity was tested in 20 mM HEPES (pH 7.4) containing 2.5 mM EDTA, 50 μM substrate, and enzyme (25 μg of wild-type or mutant rPGRP-SA) in a total volume of 50 μl. After incubation for the indicated period at 37 °C, the mixture was injected on a Nucleosil 100 Chttp://www.rcsb.org/pdb/) under accession number 1S2J.The atomic coordinates and structure factors have been deposited in the Protein Data Bank (


Topographic reorganization of central maps following peripheral nerve injury has been well characterized. Despite extensive documentation of these physiological changes, the underlying anatomical correlates have yet to be fully explored. In this study, we used Golgi impregnation and light microscopy to assess dendritic morphology following denervation of the glabrous hand surface in adult primates.After survival durations that permit complete physiologically-defined reorganization, we find a systematic change in the dendritic arborization pattern of both layer II/III pyramidal and layer IV spiny stellate cells in the contralateral hand region of area 3b, compared to unaffected cortical areas. In general, our analyses indicate a progressive expansion of distal regions of the dendritic arbor with no appreciable changes proximally. This pattern of distal dendritic elaboration occurs for both basilar and apical dendrites.These observations are consistent with the notion that latent inputs gain expression in reorganized cortex after nerve injury via their influence through contacts with more distally located termination sites. The ability of the nervous system to modify its output in accordance with experiential demands is a central tenet of neuronal plasticity. For many years, the view of critical periods permeated our beliefs; almost dictating that plasticity beyond such epochs was, at best, minimal. The seminal experiments of Merzenich, Kaas and colleagues ,2 have pWhile physiological techniques are frequently used to characterize topographic (re)organization of central maps, the underlying anatomical correlates have not been thoroughly investigated. Using intracellular injection techniques, thalamic axons have been reported to innervate a much broader sector of cortex than necessary to represent typical receptive field size, suggesting the existence of "latent" inputs ,15. DisiIn this study, we report our examination of neurons in two cortical layers; spiny stellate cells in layer IV, as this is the primary input target of thalamocortical axons; and pyramidal neurons in layer II/III, as supragranular changes have been shown to precede somatotopic modification in the granular cell layer . We predFigure We found a greater number of intersections in the distal halves of dendritic arbors of pyramidal cells in deprived relative to control cortex. For basilar dendrites, there is a 92.0% increase in the number of intersections in the distal arbors of deprived cells relative to control cells  were socially housed with food and water available ad libitum. In six animals, the median and ulnar nerves to one hand were transected following the principles of animal care detailed in NIH publication no. 86–23. The local institutional animal care and use committee approved all procedures prior to initiation of any experiments. Briefly, monkeys were anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (25–30 mg/kg) and xylazine (0.5–1.0 mg/kg). Their forearms were shaved and prepared for surgery with alternate scrubbings of povidone-iodine and alcohol. Under sterile conditions an incision was made along the midline of the ventral forearm, the median and ulnar nerves were located by blunt dissection and cut about midway between the elbow and wrist. The epineural sheath of the proximal stump was retracted 0.5–1.0 cm, and the exposed nerve avulsed. The empty epineural sheath was re-extended, folded back upon itself and ligated. The nerve stumps were repositioned and the incision closed with sutures. Post-surgically, all subjects received penicillin, dopram hydrochloride, and dexamethasone injections. Subjects were permitted to recover for a period of time previously shown sufficient to permit complete reorganization of the hand representation in cortical area 3b . Two additional subjects served as naïve controls.Adult squirrel monkeys (Following electrophysiological mapping of the affected cortical areas, animals were overdosed and perfused transcardially with 0.9% saline. Brains were extracted and immersed in a modified Golgi–Cox solution for 11 days, thereafter dehydrated and embedded in celloidin. Tissue blocks were sectioned coronally at 150 μm in thickness for morphological assessment and every fourth section in the series was cut at a thickness of 90 μm for Nissl staining to facilitate cytoarchitectonic identification of area 3b borders. Free-floating sections were processed and mounted on glass, according to previously reported procedures .Analysis of dendritic morphology was conducted blind to experimental condition on thoroughly impregnated cells using methods described by Sholl . For eacWe have shown previously that dominant and latent inputs from the three nerves innervating the hand have overlapping territories in area 3b ,32, withJDC participated in design of study, conducted the histological processing, drafted the manuscript: JAT conducted some of the histological processing: CLW participated in design of study: DRS participated in design of study: PEG participated in design of study, conducted statistical analyses, conceived and coordinated the study. All authors read and approved the final manuscript.


Candida albicans is a polymorphic fungus causing serious infections in immunocompromised patients. It is capable of shifting from yeast to germinating forms such as hypha and pseudohypha in response to a variety of signals, including mammalian serum. We have previously shown that some of the large 25S components of ribosomal RNA in Candida albicans get polyadenylated, and this process is transiently intensified shortly after serum exposure just prior to the appearance of germination changes.We now present data that this process also involves the small 18S subunit of ribosomal RNA in this organism. Unlike the large 25S subunit, polyadenylation sites near the 3' end are more variable and no polyadenylation was found at the reported maturation site of 18S. Similar to 25S, one or more polyadenylated mature sized 18S molecules get intensified transiently by serum just prior to the appearance of hypha.The transient increase in polyadenylation of both the large and the small subunits of ribosomal RNA just prior to the appearance of hypha, raises the possibility of a role in this process. Candida species are now among the most important pathogens especially for the immunocompromised host. They are the fourth most common organisms recovered from blood cultures in hospitalized patients [Candida albicans the most frequently isolated of the species, is a polymorphic organism. It can switch from a yeast form (blastospore) to a filamentous phase (hypha and pseudohypha) in response to a variety of external stimuli, including mammalian serum. Mutants defective in this serum response, also show a reduced capacity to cause disease in a murine model, [patients . Candidae model,  suggestiCandida albicans polyadenylates some of its 25S ribosomal RNA (rRNA) and the polyadenylation site corresponded to the large subsegment 3'-end maturation [It is widely accepted that the production of the RNA components of ribosomes in eukaryotes proceeds through the transcription of large pre-RNA molecules by RNA polymerase I (Pol I), that get processed into the final large and small subsegments . We haveturation . We alsoWe have found polyadenylation to occur both in yeast grown in YPD and in those exposed to serum for 5 minutes. In all cloned PCR products the number of adenines in the chain exceeded those in the poly-T primer (DT12) used to generate them. Furthermore, the anchor sequence assures us that we did not extend an inappropriately annealed primer. Unlike our data with the 25S subunit, where the attachment was to one of two thymidines one base apart at the reported maturation site , the polUBI4 was also up-regulated by serum but its intensity remained the same at 15 minutes, while the 18S band returned to baseline assuring us that the 18S enhancement was not as a result from an error in RNA loading. We previously found ACT1 [Saccharomyces cerevisiae has recently been described in similar amounts [Similar to 25S, polyadenylation of 18S subunit was enhanced by serum exposure and this is shown in Figure  amounts .To further confirm that the 18S increase from time zero to 5 minutes is real, we performed real-time PCR reactions and the results are shown in Figure The 18S band in Figure These data indicate that 18S subunit mirrors the large 25S molecule as regards to polyadenylation and its response to serum, suggesting that this is not an incidental phenomenon. They do differ from the 25S subunit in that polyadenylation occurs both upstream and downstream to but not at the reported 3'-end maturation site, while for the 25S subunit, the polyadenylation was found to be exclusively at the maturation site. Perhaps the 3'-end of the 18S subunit plays an important role in the recognition of start sites on mRNA, and is vigorously protected from modifications. This would also suggest that polyadenylation of 18S has a role outside of the ribosome.Saccharomyces cerevisiae that was found to be increased in mutants lacking the degradative function Rrp6p [Saccharomyces cerevisiae [S. cerevisiae has the inherent capacity to utilize Pol II for rRNA transcription but that this capacity is suppressed by a mechanism that includes UAF. These mutants though, switched to Pol II transcription exclusively. Our data with C. albicans differs in that both polyadenylated and non-polyadenylated forms are produced simultaneously. Conrad-Webb and Butow [S. cerevisiae. The template utilized by this strain was an episomal copy of ribosomal DNA that contained a Pol II promoter sequence overlapping with the Pol I promoter. Recently, circular and linear rDNA plasmids have been reported in C. albicans [Escherichia coli has been reported [One of the basic questions raised by our original data was whether these polyadenylated transcripts are products of Pol I and get polyadenylated following maturation cleavage or are newly transcribed by Pol II. These data do not resolve this question. While finding of multiple polyadenylation sites in 18S with most of them clearly not corresponding to a reported maturation site, might result from transcription by an enzyme other than Pol I, it is just as likely that they may represent inappropriate cleavage by the ribosomal RNA processing apparatus and these products are being readied for degradation by polyadenylation. The recent report of polyadenylation in revisiae  in cellsalbicans  for the reported ,11 incluC. albicans is unknown. Open reading frame analysis of the 18S subunit indicates that translation into protein is unlikely, as it would result in peptides shorter than 40 amino acids. Multiple polyadenylation sites upstream and downstream from the reported maturation site suggest, that these may be inappropriately processed molecules that are being readied for degradation, though one of the downstream sites may be an A2 processing site. The mere up-regulation of polyadenylation by serum prior to germination also does not indicate a role in hypha formation as other genes such as ubiquitin and actin also respond similarly. There are aspects to our new 18S and our previous 25S data that leave the possibility for a role in germination open. These include the transient nature of this up-regulation for both subunits just prior to germination and the possible selective nature of this process involving 18S.The role of polyadenylation of rRNA in Candida albicans' polymorphic behavior.The ribosome is central to cellular function and the RNA component of this organelle assumes critical structural and catalytic roles. Our initial unexpected finding of polyadenylation of a portion of the large rRNA subunit is now extended to the small subunit. That this modification also involves the other major component of the ribosome points to a biological role for this process. The fact that the transient up-regulation of RNA polyadenylation from both subunits just precedes the phenotypic expression of germination, suggests a possible role in regulating Candida albicans SC5314 (obtained from W. Fonzi) [2O) was utilized to induce germination. Yeast cells were grown overnight in YPD at 30°C, harvested by centrifugation, washed once in H2O and transferred to FBS pre-heated to 37°C at 1–5 × 106 cells ml-1. Fonzi)  was grow-1 lyticase (ICN), in a volume 1/5 that of the volume of the of the original cell culture. The digestion proceeded for 10–20 minutes at 30°C. Adequacy of the digestion was monitored by testing a small drop of cell suspension in SDS for viscosity [RNA from cells at various growth conditions was obtained as follows. Incubating mixtures were rapidly cooled in an ice-water bath and were thereafter centrifuged at 4°C and washed with ice cold water once. Cell walls were digested by suspending the pellet in a buffer containing 1 M sorbitol, 0.1 M EDTA, 0.1%, v/v, β-mercaptoethanol and 100 U mliscosity . MixtureEcoRI digestion and gel purification, and was labeled with P32 by using random priming. As a control a 499 bp UB14 probe was also generated with the primers 5'-GAAGTCGAATCTTCTGACACCATCG-3' (P3) and 5'-TGGTGGAATACCTTCTTTGTCTTGG-3' (P4). The primer design was based on the UB14 sequence (Accession No. Z54197) reported by the Stanford Candida Genome project's World Wide Web site [Candida albicans rRNA [Samples of 50 ng of poly(A) RNA were electrophoresed on 1.2% formaldehyde agarose gel blotted to nylon membranes. A 262 bp long fragment of 18S was generated from reverse transcribed total RNA with the primers 5'-TCGATGGAAGTTTGAGGCAA-3' (P1) and 5'-ATTCAATCGGTAGTAGCGACGGGC-3' (P2) based on the previously published sequence . After cloning into pCR2.1 (Invitrogen), and sequencing  to confirm that it was 18S specific, the insert was released by ans rRNA . PhosphoTo confirm that the polyadenylated form of 18S is increased by five minutes, total RNA was predigested with RNase-free DNase I (New England Biolabs), then reverse transcribed with Superscript II RT (Invitrogen) utilizing an anchored polyT primer 5'-AATTCGGCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3' (DT12) to generate cDNAs from polyadenylated RNA molecules. The same reaction also contained the primer P6, specific for 5S rRNA subunit that does not get polyadenylated . Using tTo identify polyadenylation positions involving 18S, total RNA was heated at 70°C for 5 minutes to minimize secondary structures. Reverse transcription was done with the anchored polyT primer DT12, utilizing Superscript II RT (Invitrogen). PCR products were amplified with primers DT12 and P1 which is situated 398 nucleotides from the reported 3' end of the molecule using HotStarTaq (Qiagen). To show the presence of a mature sized polyadenylated molecule, amplification was also carried out with DT12 and 5'-TATCTGGTTGATCCTGCCAGTAGTC-3' (P8) situated at the 5' end of 18S using FailSafe PCR System (Epicentre). Amplified products were subcloned into pCR2.1 (Invitrogen), a number of clones were picked and sequenced . For the full-length clone, only parts of the 5' and 3' ends were sequenced.HL and CPW carried out and analyzed experiments. JF conceived of the study, designed experiments, analyzed data and wrote manuscript. All authors have read and agree with final manuscript.


S. haematobium infection in a cohort of schoolchildren attending grade 3 in a rural part of the province.Schistosomiasis is one of the major health problems in tropical and sub-tropical countries, with school age children usually being the most affected group. In 1998 the Department of Health of the province of KwaZulu-Natal established a pilot programme for helminth control that aimed at regularly treating primary school children for schistosome and intestinal helminth infections. This article describes the baseline situation and the impact of treatment on Schistosoma haematobium infection, treated with praziquantel and re-examined four times over one year after treatment in order to assess the impact of treatment and patterns of infection and re-infection.Primary schoolchildren from Maputaland in northern KwaZulu-Natal were examined for S. haematobium was low and appeared to be limited to the hot and rainy summer. Analysis of only one urine specimen per child considerably underestimated prevalence when compared to the analysis of two specimens, but both approaches provided similar estimates of the proportion of heavy infections and of average infection intensity in the population.Praziquantel treatment was highly efficacious at three weeks after treatment when judged by egg reduction rate (95.3%) and cure rate of heavy infections (94.1%). The apparent overall cure rate three weeks after treatment (57.9%) was much lower but improved to 80.7% at 41 weeks after treatment. Re-infection with S. haematobium infection necessitate regular treatment of schoolchildren in the area. The seasonal transmission pattern together with the slow pace of re-infection suggest that one treatment per year, applied after the end of summer, is sufficient to keep S. haematobium infection in the area at low levels.According to WHO guidelines the high prevalence and intensity of  Schistosoma haematobium being the most common species . Schonfection . Howevernfection . Some stnfection ,11.In 1998 the Department of Health of the province of KwaZulu-Natal (KZN) in co-operation with the Department of Education established a pilot programme for helminth control that aimed at regularly treating primary school children for schistosome infections and intestinal helminth infections . All chiOur objectives were to describe the pattern of schistosome infection at baseline, to monitor the impact of treatment in our study population and to assess re-infection after treatment in order to develop recommendations for future control activities.The study was conducted in central Ingwavuma district in northern KwaZulu-Natal Figure . The stuThe study population Table  was recrAll pupils who provided a urine specimen during the pre-treatment survey were included in the analysis of infection patterns at baseline but only children who had been treated with praziquantel were included in the analysis of the post-treatment surveys. Of these, another 4.6% who reported having received additional treatment for schistosome infection while our study was ongoing were excluded from analysis. Otherwise only children who refused to participate or who were absent or unable to produce a specimen during each of our repeated visits were not included in the analysis of the respective surveys. They were included, however, for those surveys where they participated in order not to increase bias due to the likely difference in disease status between absentees and pupils who attended school .®, Bayer) without regard to infection status. In order to facilitate administration of the drug, the nurses had been provided with a dosing sheet that showed the correct dosage for different bodyweights. The weight of the children was determined using an ordinary bathroom scale. Because one Biltricide® tablet containing 600 mg of praziquantel can be subdivided into four segments of 150 mg the required dose can be administered relatively accurately. The drug was administered with a glass of water after the children had eaten a peanut butter sandwich which was provided by the treatment team. Children were asked to swallow the tablets with some water in front of one of the team in order to monitor adherence. Because praziquantel is considered a safe drug and has been used extensively since its introduction in the early 80ies possible side effects of treatment were not monitored systematically [Treatment in all primary schools in the entire district was carried out in April and May 1998 by school nursing teams from the two local hospitals as part of the first treatment campaign of a provincial helminth control programme. The study team assisted the nurses with treatment and also recorded those of the study population who were treated and those who were not. All consenting children from all grades were treated for schistosome infection with a single dose of 40 mg/kg praziquantel  and albendazole treatment was repeated in October 1998 [Children were also treated for intestinal helminth infection with 400 mg albendazole  had egg counts of 50 or more EPC, the WHO [S. haematobium infection were moderate.In the pre-treatment survey 68% of the study population were found infected with  the WHO  thresholS. haematobium prevalence of boys slowly increased from 60% in the youngest to 79% in the oldest age group whereas the youngest girls had a much lower prevalence (37%) but the increase with age was steeper.Figure Of the 1109 children who participated in the baseline survey only 852 (76.8%) were treated with praziquantel. 228 children (20.6%) did not receive treatment either because they refused to be treated (7 children) or because they were absent on treatment day (221) and for 29 children (2.6%) it is unclear whether or not they were treated. Differences between the treated and untreated groups (excluding children of unclear treatment status) regarding sex, age, infection status and infection intensity were small and not statistically significant.S. haematobium infection status, CR and ERR over 53 weeks after treatment are summarised in Table In Table p < 0.001 for both prevalence and intensity using the sign test for equality of paired observations [No discernible re-infection took place between 3 and 41 weeks after treatment (Table rvations ).S. haematobium. The geometric mean EPC including uninfected children of 0.44 was still far below the corresponding figure before treatment (16.08) and this was also true for the geometric mean EPC when excluding uninfected children (58.2 before treatment and 6.6 at 53 weeks after treatment).The group at risk of re-infection Figure  was defiFigure S. haematobium infection in girls and boys might hint at different water contact patterns.The results of our baseline survey are in agreement with a survey conducted in the area about 20 years earlier. Schutte et al.  found prThe proportion of children treated in our study can not be regarded as representative for the control programme in general, because study participants were more informed about it than their schoolmates. However, the fact that only about three quarters of the children were treated is very disappointing. Although only 3 children did not consent to participate in our study, and only 7 of those who consented openly refused to be treated absenteeism was unusually high during the first round of treatment. This improved greatly in the second treatment half a year later, when – according to the opinion of school staff – pupils and their parents had realised that treatment was beneficial and had only mild and transient side effects. Unfortunately this second round of treatment did only include albendazole but not praziquantel because the schedule of the provincial treatment programme only provided one treatment for schistosome infection per year.Praziquantel treatment resulted in drastic reductions of infection intensity and prevalence of heavy infections as soon as three weeks after treatment, which is in accordance with the literature . The redS. haematobium egg counts [The high variability of repeated g counts  renders All this indicates that the reporting of measures of infection intensity is not only important because they are a better indicator of population morbidity than prevalence ,15, but Because of the slow decrease in total prevalence after treatment it did not seem appropriate to restrict analysis of re-infection to those children who were egg-negative three weeks after treatment. According to our above reasoning this would have excluded a number of children who had been treated successfully but were still excreting old eggs. On the other hand we did not want to restrict the analysis to those children who were found egg-negative at 41 weeks after treatment. Even though this was the survey where we found the lowest prevalence, we might have excluded children who had been treated successfully, but had become re-infected again before this survey. Therefore we included all children who were found egg-negative at either 3, 16 or 41 weeks after treatment into the group at risk of re-infection. We are, however, aware that this definition is likely to include some uncured children who were still harbouring low level infections.S. haematobium transmission occurred mainly during the hot and humid summer. According to the literature, S. haematobium has a pre-patent period (infection to egg-excretion by the host) of about eight to ten weeks [S. haematobium transmission and the development of their Bulinus globosus snail hosts [Our data indicate that en weeks ,30. Thusil hosts ,32. MoreS. haematobium transmission is well documented for the highveld region of Zimbabwe[Seasonality of  Zimbabwe with pat Zimbabwe. A study Zimbabwe.S. haematobium infection in the area indeed necessitate regular treatment of schoolchildren, that praziquantel treatment is highly efficacious in reducing the proportion of moderate and heavy infections and that one treatment per year after the end of summer is sufficient to keep infection intensities at low levels. Because levels of S. haematobium infection in the study area seem to be among the highest in the country [Our study shows that according to WHO guidelines  the high country  the lattBecause we would not want to discourage future attempts to control schistosomiasis in the region and elsewhere we would finally like to stress that the above described money and time consuming intensive examinations are only required when doing research but that for a control programme a minimum of surveillance is sufficient .The authors declare that they have no competing interests.ES conceived of the study and designed it together with AO, PM, JDK and CCA. ES conducted the field work with contributions from AO, JDK, CCA and WB, did the statistical analysis and drafted the manuscript. All authors contributed to the final version of the manuscript and read and approved it.The pre-publication history for this paper can be accessed here:


As there are a number of ways to divide up the anatomical structure of an organism, each may be represented by more than one valid partonomic (part-of) hierarchy. This raises the issue of how to represent and integrate multiple such hierarchies.Anatomy ontologies play an increasingly important role in developing integrated bioinformatics applications. One of the primary relationships between anatomical tissues represented in such ontologies is In this paper we describe a solution that is based on our work on an anatomy ontology for mouse embryo development, which is part of the Edinburgh Mouse Atlas Project (EMAP). The paper describes the basic conceptual aspects of our approach and discusses strengths and limitations of the proposed solution. A prototype was implemented in Prolog for evaluation purposes.With the proposed name set approach, rather than having to standardise hierarchies, it is sufficient to agree on a suitable set of basic tissue terms and their meaning in order to facilitate the integration of multiple partonomic hierarchies. Drosophila, zebrafish and C elegans, can be found on the Open Biological Ontologies web site ; hasPart.TID1 TID2;• TID2 is an immediate part of TID1, i.e. For the evaluation of the name set representation, we use an extended version of the tissue predicate (view handling is ommitted from the protoype description to keep our examples simple) :ext_tissue.• S, T, FN: as above;• NSp: positive name set of tissue represented by a list ;• NSpL: length of NSp;• NSn: negative name set of tissue represented by a list ;• NSnL: length of NSn;For example, the embryo mesenchyme tissue of Figure ext_tissue.Tp Tc is true:The following Prolog clause is used to determine whether subPart :-ext_tissue,ext_tissue,NSpLc > NSpLp,ord_subset,ord_disjoint.proper subset relationships.Predicates ord_subset and ord_disjoint from the Prolog library were used to implement the set theoretic aspects of the representation. Although these predicates support ordered sets, this is not required for our representation (but there were no unordered set predicates in the library). NSpLc > NSpLp is required to enforce Tp Tc is true:The following two Prolog clauses are used to determine whether not_immediate_subPart :-subPart,subPart.immediate_subPart :-subPart,not not_immediate_subPart.The Prolog implementation given is not particularly efficient and there are a number of optimisations that could be put in place. However, as the purpose of the prototype was not to deliver a robust application for end-users, but a reference implementation of the proposed approach for evaluation purposes, it proved entirely sufficient.The paper makes no claims over the relative merits of different implementation strategies for the proposed approach. Alternatives to Prolog include using a relational database system or an ontology language, such as OWL (more details of OWL available from W3C ). The laFor evaluation purposes, a number of tests were carried out on the name set representation of the Mouse Atlas anatomy. These are discussed here, together with some general observations about the proposed approach.The first assumption that must hold is that no two tissues (at any given stage) have the same name set representation. This was tested usingtest1 :-ext_tissue,ext_tissue,T1 not = T2.test1 returns no, i.e. no two different tissues with the same name sets were found, as required.To test whether all part-of relationships can be reconstructed from the name set representation, we usedtest2 :- immediate_subpart, not hasPart.test3 :- hasPart, not immediate_subPart.Both, test2 and test3 return no, i.e. the name set representation does not lead to any part-of relationships that are not intended (test2), and all existing part-of relationships are found through the name sets (test3), as required.group in EMAP. A recently identified need for a group has been for all  tissues at Theiler stage 17. Using predicateThe smallest form of part-of hierarchy integration is the addition of a new tissue node, which is equivalent to adding a immediate_subPart_ns.• S: stage ID;• NSp: positive name set of new tissue node (group);• NSn: negative name set of new tissue node (group);• T: tissue ID of immediate sub-part of tissue identified by name sets and stage;we can write a "query" in the form:immediate_subPart_ns, tissue,writeName(FN), nl, fail.and obtain the following result:  ...Similarly, using predicate immediate_superPart_ns, we obtain:immediate_superPart_ns is analogous, and its Prolog implementation very similar, to immediate_subPart_ns. Details are, therefore, omitted.The correctness of these results was confirmed by one of the biologists who created EMAP. Other, similar tests, worked equally well. A constraint put on all of these cases, however, is that the name set of the new group tissue must only contain names that are already used in the existing hierarchy.This raises the question of how to deal with the introduction of new component names. For example, the addition of a group  cannot automatically be carried out, since the existing hierarchy does not use head in its name sets. For the integration to work, it is first necessary to add head to the appropriate name sets in the existing hierarchy. This can be done at the highest appropriate levels, since sub-parts inherit all name set elements from their super-parts, and may therefore not require as much effort as one initially expects.For the head example, however, we did identify two additional problems which are likely to be typical in this context. Firstly, some agreement needs to be reached as to what in fact is considered to be part of the newly introduced tissue. In our example: how much of the neck is anatomically considered to be part of the head? The second problem deals with the fact that an existing tissue may need to be divided further in order to obtain the appropriate subparts for the newly introduced tissue. For example, the carotid artery runs from the head into the body of the mouse embryo, i.e. only a part of carotid artery is actually part of the head. Hence, the carotid artery needed to be divided into two subparts, one for the head section of it, one for the rest. In our name set approach, the former contains head in its positive name set, while the latter contains head in its negative name set. Of course, only the head section part becomes part of the head. Neither of these two problems presents any direct consequences for our approach.O1 includes midbrain as one of the parts of the brain, but no further detail, and O2 is a brain anatomy ontology that divides the midbrain into cerebral aqueduct, floor plate, lateral wall, etc., then we would find {brain, central nervous system, embryo, midbrain, mouse, nervous system, organ system} as the positive name set for midbrain in O1, and {brain, cerebral aqueduct, midbrain} as the positive name set in O2, resulting in {brain, central nervous system, cerebral aqueduct, embryo, midbrain, mouse, nervous system, organ system} – the union of these previous two name sets – as the representation of midbrain in the merged ontology. The meaning of the component names in the intersection of the two original names sets, {brain, midbrain} must have been used in a consistent manner for the merger to work, though many of the component names will differ across the ontologies, because of the different levels of granularity, e.g. the terms nervous system and organ system are unlikely to be found in the brain specific ontology. When merging ontologies of different granularity, the same principle as before applies: shared component names must be used in a consistent manner. Assuming ontology component names thus far, shows that some of them have additional structural complexity and if one wishes to take advantage of the semantics of these complexities, the proposed name set representation would need to be extended. For example, at Theiler stage 18 the tissue  has two subparts, called  and . The naming, hence, reflects lineage relationships between tissues, and the identity of a tissue is partially established by that relationship. Although extensions to the name set representation could be developed to allow the inclusion and subsequent reasoning over such information, it would lead to a semantic overloading of the name sets and for simplicity are, therefore, not considered further – the (component) name is treated as an atomic string describing a tissue, while the lineage relationship is modelled externally to the name sets.Taking a closer look at these "basic tissue terms", called Theoretically, merging two part-of hierarchies can be accomplished by systematically (top-down) adding each tissue from one hierarchy into another, i.e. conceptually the problem can be reduced to iteratively adding "group nodes" as discussed above.part-of relationships specified. Instead, a much more flexible solution is offered without having to sacrifice the interoperability across multiple data sets annotated with these anatomical concepts.The approach discussed in this paper will not work where there has been no agreement on the basic component terms, and as such is different from already existing work on merging autonomous ontologies. This raises two questions: what is the basis on which these terms should be agreed and what benefits are to be obtained from the proposed solution if such agreement has to be reached before these partonomic hierarchies can be merged. With respect to the first question, if a basic term, for example skin, exists, then it must be possible to dissect the mouse to a level that separates all the corresponding tissue from the rest of the mouse tissues, e.g. separate all skin tissue from the rest of the mouse. Other examples of basic terms are, therefore, head, skeleton, limb and forelimb. At this point scientists are then free to use combinations of these terms (for the positive and negative name sets) to describe the anatomical concepts they are interested in, e.g. {head, skin} to refer to the skin of the head. The different anatomy hierarchies created by different scientists can then be automatically merged using the approach proposed in this paper. Hence, to answer the second question from above, the benefit of our solution lies in the removal of the need for multiple scientists to agree on a single anatomy partonomy where all tissue concepts are defined and their Essentially, the solution is based on the transitivity property of the structural part-of relationship. As such, one could imagine implementations other than the one based on name sets to achieve the same result. The basic idea, however, would be the same. Using the name set concept makes the solution more directly accessible to biologists, who are more familiar with naming anatomical concepts than using computer generated IDs. We believe that the same approach may be applicable in other ontology areas, which have similarly transitive relationships, but since we have not tested this idea, we shall not elaborate on it in this paper.part-of relationships. Some preliminary studies suggest that where there are different types and these types are organised in an is-a hierarchy, the proposed integration mechanism will still work at the level of the common part-of type. For example, let H1 be a part-of hierarchy based on part-of-type-1, and let H2 be a part-of hierarchy based on part-of-type-2. If both, part-of-type-1 and part-of-type-2, are specialised versions of the more general part-of-type-0, i.e. part-of-type-1 is-a part-of-type-0 and part-of-type-2 is-a part-of-type-0, then we can use the proposed approach to integrate H1 and H2. The integrated hierarchy, however, would only support part-of-type-0 semantics. Our work in this area is still in its early phase and beyond the scope of this paper. Further details will be reported elsewhere.Also, the work described here only deals with the integration of hierarchies that are based on the same type of The work presented in this paper has focused on the issue of integrating different partonomic hierarchies in one species, mouse. We note that a similar approach may be useful when trying to integrate partonomic hierarchies across different organisms. This is subject of current research work, however, and will be reported on separately.part-of. For any given organism, however, there is more than one way to divide it into parts and subparts, thus leading to more than one valid partonomic hierarchy. To be able to interoperate between bioinformatics resources that make use of these anatomy ontologies, the corresponding hierarchies must be reconciled in some way. The paper addresses the problem that unique identifying names for tissues often reflect the partonomic hierarchies in which they are used. Although these names are in fact ordered sets (the order implying a particular hierarchy) of "component names", the order in these sets is not necessary to uniquely identify any tissue. Also, the sets of components in names can be used to derive all part-of relationships in the hierarchy. Based on these observations, we have developed a name set representation which facilitates integration of different partonomic hierarchies. Although this does not eliminate the requirement to agree on a set of suitable basic tissue terms and their meaning, it does remove the need to standardise the partonomic hierarchies. The proposed approach has been tested for the anatomy ontology of the Edinburgh Mouse Atlas. A Prolog prototype was implemented for evaluation purposes.Anatomy ontologies play an important role in bio-medical informatics. One of the key relationships modelled in such ontologies is that of 1Tj is a direct subpart of Ti, if Tj is part of Ti and there is no other tissue Tk such that Tj is part of Tk and Tk is part of Ti. If such a tissue Tk exists, Tj is an indirect subpart of Ti.AB developed the name set representation, implemented the prototype and carried out parts of the evaluation. YY provided input with respect to the current implementation of EMAP and EMAGE in relation to the proposed name set representation. DD carried out part of the evaluation process. RB contributed to the development of the name set representation. DD and RB are overall project leaders of EMAP and EMAGE. All authors have contributed to the writing and/or revision of the paper.


For decades, scientists accepted that the nucleic acids, DNA and RNA, packed with thousands of protein-coding genes, were the sole purveyors of genetic information; all inherited traits, from eye color to shoe size, must be stored and expressed through nucleic acid mechanisms. But prions are an exception. These misshapen proteins are capable of growing, replicating, and infecting other cells—that is, they are heritable. And all without a scrap of DNA. Most famous as the culprit behind bovine spongiform encephalopathy, or mad cow disease, prions also occur naturally in some organisms and may play important roles in their growth and development.Prion-forming proteins normally exist as benign cellular components, such as enzymes or receptors. But they possess the innate ability to alter their three-dimensional structure, or fold, which changes their function and makes them almost impossible to destroy. Like other misfolded proteins, such as those responsible for Alzheimer's and Huntington's diseases, prions pack together and form aggregates. But what distinguishes prions from simple protein aggregates is their exponential growth and amplification, which allows them to infect new host cells. Prions grow by inducing normal proteins to alter their shape and adhere to an initial aggregate “seed.” These growing masses are then thought to divide with the help of “chaperones,” cellular proteins that aid in protein folding and transport, resulting in smaller prion particles called propagons. The propagons are then distributed to both mother and daughter cells during division, thereby infecting the next generation of cells. Though this theory of the prion life cycle was proposed a few years ago, scientists are still working out the underlying molecular mechanismsAs they report in this issue, Lev Osherovich and colleagues dissected yeast prions and found that growth and heritability are controlled by two independent and “portable” sequences. Furthermore, the heritability element seems to be the only thing that keeps slow growing protein aggregates from becoming infectious prions. Previous research showed that one end of the yeast protein, Sup35p, is critical for turning this normal housekeeping enzyme into a prion. The “prion-forming domain” of Sup35p consists of two segments: one stretch rich in the amino acids glutamine and asparagine and another made up of several, small series of amino acids, called oligopeptides. Osherovich and colleagues had earlier found another yeast protein, New1p, which had similar segments, though in reverse order.To study the function of these sequences, the team constructed several strains of yeast, each with a small part of the prion-forming domain missing. By watching the behavior of these modified proteins, each fused to a green fluorescent protein for easy observation, the authors could infer the roles of the deleted segment.For both Sup35p and New1p, the authors found that the area rich in glutamine and asparagine was responsible for the aggregation and growth of prions—acting like a patch of Velcro that locks the misshapen proteins together. While this had been suggested by previous research, the authors also found that this sticky sequence only adheres to proteins that mirror its own pattern of amino acids, thereby explaining why prions from different species don't often interact, a phenomenon called the species barrier. The stretch of oligopeptide repeats in Sup35p and New1p, however, was required for the inheritance of prions—the proper division of prion masses and subsequent distribution of propagons during cell division. The authors suggest that oligopeptide repeats function as a secure binding location for the chaperone proteins, which are necessary for heritability, and thus infectiousness, of prions. Their results also help to explain why stable inheritance of prions is rare; while many proteins have stretches of amino acids similar to the described aggregation sequence, few also contain sequences like oligopeptide repeats that permit inheritance.Though both the aggregation sequence and the oligopeptide repeats are required for prion growth and infection, the segments seemed to function completely separately, allowing the authors to create a synthetic prion-forming domain by combining the aggregation element of New1p with the Sup35p replication/heritability element. This artificial prion acted like New1p, again showing that it is the sticky, aggregation element that specifies which proteins will be added to the growing prion mass. Osherovich and colleagues then went on to create another artificial prion by fusing the oligopeptide repeats to an expanded polyglutamine tract, the type of aggregation sequence responsible for the toxic buildup of brain proteins in Huntington's disease. With this simple addition, the slow growing aggregate was transformed into a heritable, infectious prion.By creating artificial hybrid prions, Osherovich and colleagues showed that the two discrete elements of prion-forming domains are portable and work together regardless of their origins. The authors suggest that other artificial prions could be used as a model system to study different types of aggregation sequences, such as those found in the human prion protein responsible for Creutzfeldt-Jakob's disease or the misshapen plaques of proteins that contribute to Alzheimer's disease.


Although various hematologic abnormalities are seen in tuberculosis, immune thrombocytopenic purpura is a rare event.We report a case of a 29 year-old male who was presented with immune thrombocytopenia-induced hemoptysis, macroscopic hematuria and generalized petechiae. The patient was found to have clinical, microbiological and radiological evidence of active pulmonary tuberculosis. The immune thrombocytopenic purpura was successfully treated with anti-tuberculous drugs combined with corticosteroids and high dose immune globulin therapy.Immune thrombocytopenic purpura can be one of the hematological manifestations of tuberculosis which has a global prevalence with increasing incidence secondary to HIV infection. During the past 2 decades, tuberculosis -both pulmonary and extrapulmonary- has re-emerged as a major health problem worldwide. Hematologic abnormalities have been described in association with mycobacterial infections for almost 100 years. Patients with both pulmonary and extrapulmonary tuberculosis (TB) may demonstrate peripheral blood abnormalities and findings may be minimal or profound  were normal. A bone marrow aspiration demonstrated hypercellularity of all cell lines with normal maturation of myeloid and erythroid precursors. Megakaryocytes were increased in number with normal morphology. On bone marrow aspiration hemophagocytosis was not observed. A chest X-ray  units throughout hospitalization. During his hospitalization findings of hemolysis or gastrointestinal bleeding and massive bleeding in another site except hematuria and hemoptysis were not established. A complete blood count at discharge demonstrated a WBC 17 × 109/l, Hb 9.6 g/dl, and platelet count 310 × 109/l -hemolytic uremic syndrome (HUS), hemophagocytic syndrome and disseminated intravascular coagulation (DIC) associated with TB. Idiopathic thrombocytopenic purpura  is an acquired disease of children and adults defined as isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia. Adult ITP typically has an insidious onset with long-lasting histories of purpura (thrombocytopenia for >6 months) and spontaneous remission is uncommon and is likely to be incomplete -13. Ster9/l and he had no side effect thought to be secondary to anti-tuberculous drugs [Several factors are known to cause bleeding in association with infections, of which thrombocytopenia is the most common. The etiology of thrombocytopenia in most cases appears to be increased destruction of platelets such as due to DIC or septicemia without evidence of DIC or platelet adherence to damaged vascular surfaces or direct platelet toxicity caused by the microorganism or involvement of bone marrow. Adult acute immune thrombocytopenic purpura is defined as a bleeding disorder in otherwise healthy person caused by transient destruction of platelets. Although the most important therapy for infection-related thrombocytopenia is that directed at the underlying infection, treatment decisions for immune thrombocytopenic purpura remain controversial and may include single or combination therapy with corticosteroids, intravenous immunoglobulin (IVIg) according to degree of thrombocytopenia or hemorrhage ,14. The us drugs ,16. The In conclusion, since the incidence of tuberculosis is currently increasing in worldwide countries and it may present with different hematologic manifestations, in case of immune thrombocytopenic purpura tuberculosis should also be recalled. Finally, further studies are needed in order to fully characterize the pathophysiology and immunological abnormalities in tuberculosis-related immune thrombocytopenic purpura.