Syzygium aromaticum (L.) Merr. & Perry. (clove) has been used in Unani medicine since ancient times for the treatment of male sexual disorders. The present study is aimed to investigate the effect of 50% ethanolic extract of clove on general mating behaviour, libido, potency along with its likely gastric ulceration and adverse effects on sexually normal male albino rats.The flower bud of The suspension of the extract was administered orally at the dose of 100, 250, and 500 mg / kg, to different groups of male rats (n = 6) once a day for seven days. The female albino rats involved in mating were made receptive by hormonal treatment. The general mating behaviour, libido and potency were determined and compared with the standard reference drug sildenafil citrate. The probable gastric ulceration and adverse effects of the extract were also evaluated.Oral administration of the extract significantly increased the Mounting Frequency, Intromission Frequency; Intromission Latency, Erections; Quick Flips, Long Flips as well as aggregate of penile reflexes and caused significant reduction in the Mounting Latency and Post Ejaculatory Interval. The most appreciable effect of the extract was observed at the dose of 500 mg/kg. The test drug was also found to be devoid of any conspicuous gastric ulceration and adverse effects.The results indicated that the 50% ethanolic extract of clove produced a significant and sustained increase in the sexual activity of normal male rats, without any conspicuous gastric ulceration and adverse effects. Thus, the resultant aphrodisiac effectivity of the extract lends support to the claims for its traditional usage in sexual disorders. Syzygium aromaticum (L.) Merr. & Perry. (Family: Myrtaceae) an evergreen tree 10–20 m in height indigenous to India, Indonesia, Zanzibar, Mauritius and Sri Lanka [Herpes simplex [Clove is the dried flower bud of ri Lanka ,4, carmiri Lanka ,6. It isri Lanka  and usefri Lanka . Clove iri Lanka . It inhi simplex . Phytoch simplex , phenyl  simplex , β caryo simplex , eugenol simplex . Eugenol simplex , and has simplex  and smoo simplex . Phyto c simplex , showed The present study thus, is aimed to investigate the aphrodisiac effect of 50% ethanolic extract of clove in detail, using multiple parameters along with its probable gastric ulceration and adverse effects in sexually normal male albino rats. The doses used in the study were selected according to the Freirich , multiplS. aromaticum (clove) was procured from the market . A voucher (S721) sample was kept for further reference. The clove was crushed to coarse powder and sieved through No. 20 mesh size. The extraction was carried out by mixing the powdered clove with 1:3 w/v in 50% ethanol v/v by Soxhlet apparatus for 6 h. The extract was filtered and the solvent from the filtrate was removed by rotary evaporator under reduced pressure and low temperature. The yield of extract was 10.40% w/w in terms of dried starting material. It was yellowish and of pleasant smell. The extract was preserved in a refrigerator.The authenticated dried flower bud of Sildenafil citrate was purchased from Zydus Cadila, . Other drugs used were ethinyl oestradiol , progesterone  and 5% xylocane ointment ad libitum throughout the study except during the experiment. The study design was approved by the ethical committee of the Department for animal care and use.Twelve weeks old male and female albino rats of wistar strain weighing 350–400 g and 225–275 g respectively, were used for the study. They were housed singly in separate standard cages and maintained under standard laboratory conditions  with free access to solid pellet diet  and water Since clove in Unani medicine is orally administered, therefore, the extract of clove was suspended in distilled water using Tween 80 (1%) for oral administration. Sildenafil citrate and ethinyl oestradiol were also suspended in distilled water using Tween 80 (1%) separately, for oral use. Progesterone was dissolved in olive oil for subcutaneous injection. All the drug solutions were prepared just before administration.The test was carried out by the methods of Dewsbury and Davis Jr  and Szecth day after commencement of the treatment of the male animals. The experiment was conducted at 20:00 h in the same laboratory and under the light of same intensity. The receptive female animals were introduced into the cages of male animals with 1 female to 1 male. The observation for mating behaviour was immediately commenced and continued for first 2 mating series. The test was terminated if the male failed to evince sexual interest. If the female did not show receptivity she was replaced by another artificially warmed female. The occurrence of events and phases of mating were called out to be recorded on audio-cassette as soon as they appeared. Their disappearance was also called out and recorded. Later, the frequencies and phases were determined from cassette transcriptions: number of mounts before ejaculation or Mounting Frequency (MF), number of intromission before ejaculation or Intromission Frequency (IF), time from the introduction of female into the cage of the male upto the first mount or Mounting Latency (ML), time from the introduction of the female up to the first intromission by the male or Intromission Latency (IL), time from the first intromission of a series upto the ejaculation or Ejaculatory Latency (EL), and time from the first ejaculation upto the next intromission by the male or Post Ejaculatory Interval (PEI). In the second mating series only the EL was recorded. The values for the observed parameters of the control, test and standard animals were statistically analysed by using one-way analysis of variance (ANOVA) method.The female animals were artificially brought into oestrus (heat)  by the Sth day at 20:00 h. The penis was exposed by retracting the sheath and 5% xylocaine ointment was applied 30, 15 and 5 min before starting observations. Each animal was placed individually in a cage and the receptive female rat was placed in the same cage. The number of mountings was noted. The animals were also observed for intromission and ejaculation. The MF in control, test and standard animals was statistically analysed by employing one-way analysis of variance (ANOVA) method.The test was carried out by the method of Davidson , modifieth day, the test for penile reflexes was carried out by placing the animal on its back in a glass cylinder partial restraint. The preputial sheath was pushed behind the glans by means of thumb and index finger and held in this manner for a period of 15 min. Such stimulation elicits a cluster of genital reflexes. The following components were recorded: Erections (E), Quick Flips (QF), and Long Flips (LF). The frequency of these parameters observed in control, test and standard groups was statistically analysed by using one-way analysis of variance (ANOVA) method.The effect of the test drug was studied according to the methods described by Hart and Haugen  and Hartth day all the animals were killed and the stomach was then incised along the greater curvature and washed carefully with physiological saline. Any gastric lesions were observed immediately using a magnifying glass. The number of erosions per stomach was assessed for severity, according to the score of Cioli et al [The male animals (350–400 g) were divided into 4 groups of 6 animals each. Group 1 represented the control group, which received 10 ml/kg of distilled water. Groups 2–3 received suspension of the extract orally at the doses of 100, 250 and 500 mg/kg, respectively, daily for 7 days. After the treatment, on 8li et al  : (o) abAll treated rats were observed at least once daily for any overt sign of toxicity , stress  and changes in behaviour . In addition food and water intake were noted.The significance of difference between the means was determined by one-way analysis of variance (ANOVA) with post-hoc't' test. P value <0.05 was considered as significant.1) (P < 0.0001) and decreased Mounting Latency (ML) (P < 0.0001), Intromission Latency (IL) (P < 0.01) in a significant manner. The dose 250 mg / kg of the extract significantly increased the MF (P < 0.001), IF (P < 0.05) and significantly reduced the ML (P < 0.001), EL1 (P < 0.05); Post Ejaculatory Interval (PEI) (P < 0.001) and did not significantly alter the IL (P < 0.61), Ejaculatory Latency in second series (EL2) (P < 0.41). Whereas the dose of the test drug at 100 mg/kg significantly increased the MF (P < 0.05), PEI (P < 0.05) but did not significantly affect the IF (P < 0.61), ML (P < 0.61), IL (P < 0.61); EL1 (P < 0.61); EL2 (P < 0.81). However, the standard drug increased the MF (P < 0.0001), IF (P < 0.0001); EL1 (P < 0.0001), EL2 (P < 0.0001); PEI (P < 0.0001) as well as decreased ML ((P < 0.0001) and IL (P < 0.0001) in a highly significant manner (Table The data obtained with the mating behaviour test indicated that the clove-extract at the dose of 500 mg/kg increased the Mounting Frequency (MF) P < 0.0001), Intromission Frequency (IF) (P < 0.001) ; Ejaculatory latency in first series  in a significant manner (P < 0.001). The extract at the doses of 100 mg/kg and 250 mg/kg did not significantly alter the MF. The standard drug strikingly increased the MF (P < 0.0001). Intromission and Ejaculation were found absent in control, test and standard groups (Table The test for potency exhibited that the higher dose (500 mg/kg) of the test drug significantly increased the frequency of Erections (E) (P < 0.0001), Quick Flips (QF) (P < 0.0001), Long Flips (LF) (P < 0.0001) as well as the aggregate of these penile reflexes (TPR) (P < 0.0001). The extract at the dose of 250 mg/kg significantly increased the E (P < 0.01), LF (P < 0.05) and TPR (P < 0.05) but comparatively less than the higher dose of the extract and standard drug, and did not significantly increase the QF (P < 0.10). whereas, the test drug at the dose of 100 mg/kg did not alter the E (P < 0.78), QF (P < 0.78); LF (P < 0.44) and TPR (P < 0.44) in a significant manner Table .Seven days treatment of low and high doses of the extract caused no significant ulceration in gastric mucosa of albino rats. Moreover, there were neither treatment related defects nor overt clinical signs of toxicity, stress or changes in behaviour and appearance evident. The food and water intake of all test drug treated rats remained similar to those of the control group.S. aromaticum) was tested in animal experimentation for its effect on sexual behaviour, and sildenafil citrate was used as the standard referent.In the present study, clove (1) and in second series (EL2) was studied. The test drug significantly increased the EL1 and EL2 as compared to control animals, whereas a highly significant increase was observed with the standard drug.The study showed that the 50% ethanolic extract of clove possesses significant sexual function enhancing activity as observed in sexual behaviour tests. Mating behaviour test revealed that the test drug significantly increased the Mounting Frequency (MF) and Intromission Frequency (IF) as compared to control but less than that of the standard drug. The (MF) and (IF) are considered as the indices of both libido and potency. So, this is an indication that the test drug possesses a sexual function improving effect. The premature ejaculation is one of the important causes of sexual dysfunction, so the assessment of Ejaculatory Latency in first series (ELThe test drug was found to produce a significant reduction in the Mounting Latency (ML) and Intromission Latency (IL) as compared to control while a highly significant decrease was found in ML of animals treated with sildenafil citrate. This is also an evidence of the sexual function improving effect of the clove.The Post Ejaculatory Interval (PEI) is considered as an index of potency and libido, and also a parameter of the rate of recovery from exhaustion after first series of mating. PEI was found significantly decreased with clove-extract and also with the standard drug. The test drug decreased PEI either by enhancing the potency and libido or by producing lesser exhaustion in the first series of mating or by both.The effect of the test drug on libido was also evaluated by testing the MF after genital anaesthetization which does away with the reinforcing effect of genital sensation thus affording the study of pure libido or intrinsic sexual desire. During the experiment the extract produced a significant increase in the MF of sexually normal male rats whereas the efficacy of the standard drug, as expected, was found to be highly significant. The MF was much reduced in control, test and standard animals in comparison with the MF of corresponding groups in mating behaviour test where the penis had not been anaesthetized. None of the control, test and standard animals were observed to show Intromission or Ejaculation because their occurrence depends upon local genital sensation which was obstructed due to anaesthetization.The effect on potency was also evaluated by testing the effect of the drug on the frequency of penile reflexes namely Erections (E), Quick Flips (QF), and Long Flips (LF). The test drug significantly increased the frequency of all the components of penile reflexes  in the test animals as compared to control group but comparatively lesser than the standard drug. The aggregate of penile reflexes (TPR) was also found increased in the animals treated with the extract and sildenafil citrate. Therefore, the experiment revealed that the test drug produced a marked increase in potency.The spices are reported to produce an increase in gastric acid secretion by a cholinergic mechanism , and so,The results of the present study clearly proved that the clove is endowed with sexual function improving activity. This is in consonance with our earlier study showing sexual function improving effect of the test drug in male mice. However, the established drug i.e. sildenafil citrate exhibited, as expected, tremendous activity. With regard to the mechanism of the test drug, it is difficult to interprete the mechanism involved in potentiation of sexual function. The drugs induced changes in neurotransmitter levels or their action at cellular levels could change sexual behaviour . Hence, Moreover, research should be aimed at isolating the active principle(s) responsible for aphrodisiac activity and the mechanism by which the drug enhances sexual function. In addition, to discover the applied effective concentration or dosages of the extract, more studies are also required.The present results indicated that the 50% ethanolic extract of clove possesses potent aphrodisiac activity in normal male albino rats without any gastric ulceration and adverse effects and provided scientific evidence in favour of the claims made in Unani medicine that the clove is clinically useful as sexual invigorator in males.The author(s) declare that they have no competing interests.T-Supervised the design and coordinatrion of the study.SA-Practically conducted the design of the study.AL-Participated and performed the statistical analysisIA-Participated in the drafting of manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We show that a 2′-O-methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans, injection of the 2′-O-methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2′-O-methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7-containing protein–RNA complex. Thus, we demonstrate that 2′-O-methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2′ O-methyl RNA oligonucleotides can block small RNA function in vivo - providing a tool to determine the biological functions of siRNA and microRNA. These oligonucleotides can also be used to identify small RNA-associated proteins that mediate silencing pathways2′- The endoribonuclease Dicer produces two types of small regulatory RNAs that regulate gene expression: small interfering RNAs (siRNAs) and microRNAs (miRNAs) . In animDrosophila S2 cells . RNAi is a powerful method for the study of gene function in animals and plants and has even been proposed as a therapy for treating genetic disorders and viral infections. Biochemical studies in S2 cells  and affiS2 cells  or immunS2 cells  from culfor RNAi a, 2002b.for RNAi .C. elegans, the miRNAs lin-4  luciferase (Pp-luc) mRNA target  strand of the siRNA duplex, thereby blocking incorporation of the guide (antisense) strand into RISC  to the target RNA than a 2′-O-methyl oligonucleotide. These data suggest that specific proteins in the RISC facilitate either target finding, target binding, or both. Consistent with this idea, inhibition of RISC function was incomplete using 21-nt 2′-O-methyl oligonucleotides (data not shown). Thus, target sequence flanking the site of complementarity to the siRNA guide strand may play a role in target RISC binding. Perhaps an active mechanism that involves target sequences flanking the siRNA facilitates the search for the target sequence; future studies will clearly be needed to test this idea.Thus, the interaction of 2′--O-methyl oligonucleotides are stoichiometric, irreversible, sequence-specific inhibitors of siRNA function in RNAi reactions using Drosophila embryo lysate. Can 2′-O-methyl oligonucleotides block siRNA function in vivo? To address this question, we carried out sequential transfection experiments using 1 nM, 5 nM, 10 nM, or 25 nM siRNA duplex. siRNA was transfected on the first day; then, reporter and control plasmids were cotransfected together with various amounts of 2′-O-methyl oligonucleotide on the second day. Silencing of Pp-luc, relative to the sea pansy (Renilla reniformis) luciferase (Rr-luc) control, was measured on the third day. For each siRNA concentration, we determined the concentration of 2′-O-methyl required for half-maximal inhibition of RNAi . The data fit well to a sigmoidal curve, consistent with the idea that, at these concentrations, increasing amounts of siRNA do not produce a corresponding increase in RISC activity. Higher concentrations of siRNA were not examined because they produce sequence-independent changes in gene expression  As a control, we also tested whether the oligonucleotide could block the activity of a let-7-containing RISC assembled in vitro in Drosophila embryo lysate. Addition of this 2′-O-methyl oligonucleotide efficiently blocked target RNA cleavage directed by the endogenous let-7-programmed RISC in the HeLa S100 extract and by the RISC programmed with exogenous let-7 siRNA duplex in Drosophila embryo lysate . This finding suggests that single-stranded 2′-O-methyl oligonucleotides are not efficiently transmitted to the progeny of injected animals. To circumvent this problem, we next injected 2′-O-methyl oligonucleotides directly into larvae and examined the phenotypes of the injected animals. The lin-4 miRNA functions in L1/L2 larvae, and we have found that, in our hands, L1 larvae do not survive microinjection (data not shown); thus, it was not possible to assay for inhibition of lin-4 function by direct injection. In contrast, let-7 functions during the L4 stage, and we found that L2 and L3 larvae survive the microinjection procedure  strain and compared the penetrance of the phenotypes with those observed for injection into wild-type. Consistent with the idea that the injected oligonucleotide specifically inactivates let-7, the absence of alae- and vulval-bursting phenotypes were both suppressed in the lin-41(ma104) mutant strain (lin-41(ma104) strain). The observed suppression (64%) was nearly identical to that reported for a let-7, lin-41 genetic double mutant  of the miRNAs lin-4 and let-7  is less than the amount of siRNA transfected. These data suggest that only a fraction of the transfected siRNA forms RISC. Furthermore, the data are consistent with stoichiometric and irreversible binding of the 2′-O-methyl oligonucleotide to RISC in vivo.Our in vitro and in vivo studies using 2′-O-methyl oligonucleotide by RISC and, by inference, recognition of target RNA by RISC are qualitatively different from the simple binding of two complementary nucleic acids by basepairing. We observed that RISC function was far more readily inhibited by binding a 2′-O-methyl oligonucleotide to RISC than by binding the same 2′-O-methyl oligonucleotide to the site of RISC recognition on a target RNA. A clear implication of this finding is that RISC does not acquire its RNA target by a passive basepairing mechanism that zippers together 21 nt of complementary RNA. Thus, RNAi is not merely a form of antisense inhibition in which the antisense strand is stabilized in a duplex. Rather, an active mechanism—perhaps involving target sequences flanking the region of complementarity—underlies the specificity and efficiency of RISC targeting.Our data hint that recognition of the 2′--O-methyl oligonucleotides to probe miRNA function in vivo. Injection of a 2′-O-methyl oligonucleotide complementary to the let-7 miRNA into C. elegans larvae phenocopied a let-7 loss-of-function mutation, demonstrating that 2′-O-methyl oligonucleotides can disrupt the function of a single miRNA in vivo. These data, combined with our studies in vitro and in cultured cells, show the promise of 2′-O-methyl oligonucleotides as a tool for dissecting the function of the numerous miRNAs found in a wide range of organisms. In this regard, 2′-O-methyl oligonucleotides provide a tool similar in practice, but mechanistically distinct from, RNAi itself and thus may facilitate the study of small RNA function in cases in which classical genetic mutations in miRNA genes are unavailable.Finally, we have shown the utility of 2′Drosophila embryo lysate preparation, in vitro RNAi reactions, and cap-labeling of target RNAs were as described elsewhere  were 5′-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUC C-3′ and 5′-Bio-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUC C-3′ (complementary to the Pp-luc siRNA sense strand); 5′-GGA CAU UUC GAA GUA UUC CGC GUA CGU GAU G-3′ and 5′-Bio-A CAU UUC GAA GUA UUC CGC GUA CGU GAU GUU-3′ (complementary to the Pp-luc antisense strand); and 5′-Bio-UCU UCA CUA UAC AAC CUA CUA CCU CAA CCU U-3′ (complementary to let-7); 5′ biotin was attached via a six-carbon spacer arm.Synthetic siRNA  was deprotected according to the manufacturer, annealed b, 2001c,O-methyl oligonucleotide (10 pmol) was incubated for 1 h on ice in lysis buffer containing 2 mM DTT with 50 μl of Dynabeads M280  to immobilize the oligonucleotide on the beads. To ensure that the tethered oligonucleotide remained in excess when more than 50 nM siRNA was used, 20 pmol of biotinylated 2′-O-methyl oligonucleotide was immobilized. For RISC capture assays, siRNA was preincubated in a standard 50 μl in vitro RNAi reaction for 15 min at 25°C. Then, the immobilized 2′-O-methyl oligonucleotide was added to the reaction and incubation continued for 1 h at 25°C. After incubation, beads were collected using a magnetic stand . The unbound supernatant was recovered and an aliquot assayed for RISC activity as previously described  using Lipofectamine 2000  according to the manufacturer's protocol, first with various concentrations of siRNA targeting Pp-luc mRNA. After 6 h, the cells were washed with PBS and the media replaced. On the next day, the cells were cotransfected with Rr-luc-expressing (0.1 μg/well) and Pp-luc-expressing (0.25 μg/well) plasmids and 2′-O-methyl oligonucleotides using Lipofectamine 2000 (GIBCO) according to the manufacturer's protocol. The luciferase activity was measured 24 h later with the Dual Luciferase assay kit  using a Mediators Diagnostika  PhL luminometer.HeLa S3 cells were transfected in a 24-well plate  was injected into either wild-type (N2) or lin-41(ma104) L2 larvae. Injection of L2 larvae was essentially as described elsewhere  and Mini Complete Protease Inhibitor cocktail (1 tablet/10 ml solution)  using a stainless-steel Dounce homogenizer . The homogenized extract was clarified by a centrifugation at 13,817 × let-7 miRNA, the beads were boiled for 10 min in 20 μl of SDS loading buffer . Proteins were resolved by SDS-PAGE on an 8% gel and transferred to Hybond-C membrane . To detect GFP-tagged ALG-1, ALG-2, and RDE-4 proteins, the membrane was incubated overnight at 4°C with either monoclonal anti-GFP (Roche) or an affinity-purified polyclonal anti-RDE-4 antibody (To recover the proteins associated with the antibody  diluted Immunoprecipitation of GFP-tagged ALG-1/ALG-2 complexes was performed by preclearing worm extract with 50 μl of protein G–agarose beads (Roche) per 5 mg of total protein for 1 h at 4°C. The cleared extract was then incubated with 10 μg of monoclonal antibody anti-AFP 3E6  for 1 h at 4°C followed by 50 μl of protein G–agarose. The agarose beads were then washed three times with ice-cold homogenization buffer.let-7 miRNA was monitored by Northern blotting. RNA was eluted from the immobilized 2′-O-methyl oligonucleotide by digestion with 1 mg/ml proteinase K in 200 mM Tris–HCl (pH 7.5), 25 mM EDTA, 300 mM NaCl, 2% (w/v) SDS at 50°C for 30 min, followed by extraction with phenol–chloroform, and recovered by precipitation with ethanol. Recovered RNA was resuspended in 10 μl of formamide-loading buffer , heated to 100°C for 2 min. RNA was resolved on a 15% denaturing polyacrylamide gel, transferred to Hybond-N membrane (Amersham Biosciences), and detected by Northern blot analysis using a 5′-32P-radiolabeled antisense let-7 RNA probe (UAU ACA ACC UAC UAC CUC AUU) as described elsewhere (Depletion of lsewhere .http://www.ncbi.nlm.nih.gov/Genbank/) accession number for Photinus pyralis is X65324 and for Renilla reniformis is AF025846.The GenBank (http://www.sanger.ac.uk/Software/Rfam/index.shtml) accession numbers for the let-7 family members are MI0000060–MI0000068, MI0000433, and MI0000434.Rfam (http://www.ncbi.nlm.nih.gov/LocusLink/) ID numbers for the genes discussed in this paper are alg-1 (181504), alg-2 (173468), bantam (117376), let-7 (266954), lin-4 (266860), lin-41 (172760), miR-14 (170868), and rde-4 (176438).The LocusLink (


Batrachochytrium dendrobatidis has been implicated in the decline and extinction of numerous frog species worldwide. In Queensland, Australia, it has been proposed as the cause of the decline or apparent extinction of at least 14 high-elevation rainforest frog species. One of these, Taudactylus eungellensis, disappeared from rainforest streams in Eungella National Park in 1985–1986, but a few remnant populations were subsequently discovered. Here, we report the analysis of B. dendrobatidis infections in toe tips of T. eungellensis and sympatric species collected in a mark-recapture study between 1994 and 1998. This longitudinal study of the fungus in individually marked frogs sheds new light on the effect of this threatening infectious process in field, as distinct from laboratory, conditions. We found a seasonal peak of infection in the cooler months, with no evidence of interannual variation. The overall prevalence of infection was 18% in T. eungellensis and 28% in Litoria wilcoxii/jungguy, a sympatric frog that appeared not to decline in 1985–1986. No infection was found in any of the other sympatric species. Most importantly, we found no consistent evidence of lower survival in T. eungellensis that were infected at the time of first capture, compared with uninfected individuals. These results refute the hypothesis that remnant populations of T. eungellensis recovered after a B. dendrobatidis epidemic because the pathogen had disappeared. They show that populations of T. eungellensis now persist with stable, endemic infections of B. dendrobatidis.The chytrid fungus  Recapture experiments provide evidence that some amphibian species can now persist with infections of the pathogenic chytrid fungus and suggests, for example, that frogs and fungus might be coevolving Lauranctil 1998  and was til 1998 . B. denddeclines , althougdeclines  had direB. dendrobatidis infection on toe tips collected between 1994 and 1998 from six species of frogs at Eungella National Park in east-central Queensland, Australia ((Rheobatrachus vitellinus) disappeared suddenly from relatively undisturbed rainforest streams, and it is now considered to be extinct ((Taudactylus eungellensis) also disappeared, but it was later found to have persisted in a few small populations  of the 474 toes assessed. Four species showed no sign of infection, while we detected infections on 58 (18.4%) of 316 toes of T. eungellensis and 13 (34%) of 47 toes of frogs identified at the time as Litoria lesueuri . The most parsimonious model  included each of these predictors but no interaction terms.Using a logistic model with season , species, and site as predictor variables, we found significant effects of each variable on infection, corrected for the effects of the other variables . A significantly smaller proportion was infected at Rawson Creek (10.5%) than at Dooloomai Falls (26.7%) or Tree Fern Creek (25.0%). There was a marginally significant difference overall in the infection levels among males, females, and subadults . However, when site was included in the model, any suggestion of a difference in infection level between males, females, or subadults disappeared. When we separated the main T. eungellensis populations, it was apparent that the difference in prevalence between those categories was influenced by the population at Tree Fern Creek, where 46% of males (n = 13) and no females or subadults were infected  differeinfected . When weT. eungellensis varied significantly among seasons , but not among years . Further, there was no evidence that the pattern of seasonal variation in infection changed among years . Infection was most prevalent (37.8%) during the winter months (1 June to 31 August) and least prevalent (11.3%) during the summer months (1 December to 28/29 February). Comparing the two sites with the largest sample sizes, Dooloomai Falls and Rawson Creek, there was no evidence that they had differing seasonal patterns of infection , although the level of infection overall was much higher at Dooloomai Falls . Infection levels were much higher in winter and spring combined than in summer and autumn , and there was no evidence of infection levels differing between winter and spring or between summer and autumn . The seasonal changes in infection at the two sites are shown in At these three sites, levels of infection among B. dendrobatidis was detected in 13 of the 47 L. wilcoxii/jungguy examined, giving an estimated prevalence of 27.7%, with a 95% confidence interval ranging from 15.6% to 42.6%. There was no evidence that prevalence of infection differed among males, females, and subadults ; among sites ; or among seasons  (each of the above Δdev terms is corrected for the other terms in the model).L. wilcoxii/jungguy and T. eungellensis was hampered by the fact that prevalence of infection on T. eungellensis differed between sites, and that the sampled populations of the two frog species had largely disjunct distributions. When the data were pooled over all sites, there was insufficient evidence to reject a null hypothesis of equal prevalence in the two species, whether the effects of season were allowed for  or not .Comparing the prevalence of infection between L. wilcoxii/jungguy and T. eungellensis recaptured at any stage later in the study, grouped by their infection status at first capture. A logistic model with recapture as the response and species and infection status as predictors produced no evidence that the effect of B. dendrobatidis infection on recapture probability differed between the species . In both species, the probability of recapture was significantly lower for infected frogs than for uninfected frogs , correcting for the substantially higher overall recapture rate of T. eungellensis relative to L. wilcoxii/jungguy.B. dendrobatidis may affect survival, but confounds the probability of recapturing a frog that is present at a site with the probability that a frog is still present at the site. The more sophisticated analysis that follows separates these two components, although it cannot distinguish between death and permanent emigration from the site. For brevity, we refer to continued presence at the site as “survival.”This simple analysis suggests that Investigating mark–recapture data for both Rawson Creek and Dooloomai Falls, we found that the best model (on the basis of minimizing the AIC) for which there were sufficient data to estimate almost all parameters had the probability of recapture varying with time but not group , and the survival probability differing between the groups but constant through time. However, further analysis in both cases showed that there was no evidence that survival differed between infected and uninfected frogs: At both sites, the group effect was due to survival being highest in females, intermediate in males, and lowest in subadults ; Table 4T. eungellensis, a rainforest frog that almost disappeared as a result of major die-offs in the Eungella area in the mid 1980s, now persist with stable infections of B. dendrobatidis. This does not imply that this pathogen cannot have been responsible for the decline. One hypothesis that can be discarded, however, is that B. dendrobatidis drove frog populations to low levels, consequently became locally extinct itself, and that frog populations subsequently recovered or stabilised in the absence of the pathogen. Two other hypotheses are consistent with our observed results.Our results show unequivocally that remnant populations of B. dendrobatidis was not responsible for the initial decline of T. eungellensis populations. These populations declined more than a decade before this chytrid was formally identified, and the declines occurred over a very short period , our results show that infection with B. dendrobatidis did not inevitably lead to rapid death in T. eungellensis.Our mark–recapture analysis did not find consistent evidence that captured . TogetheL. wilcoxii/jungguy appears not to have declined at the same time as T. eungellensis and R. vitellinus and, in our data, it has a high prevalence of infection. It therefore is a candidate reservoir host. Whether the prevalence of B. dendrobatidis differs between the sibling species L. wilcoxii and L. jungguy at sites where they are sympatric would be an interesting question, but cannot be answered from our study. The L. lesueuri complex has a widespread distribution throughout streams on the east coast of Australia  were histologically prepared in transverse sections stained with Ehrlich's haematoxylin for skeletochronological assessment . For both mark–recapture and analysis of prevalence, we grouped the data into 3-mo austral seasons . Mark–recapture modelling of survival requires that several assumptions be satisfied. Most critically, every marked individual present in the population at a given time has the same probability of recapture as all other members of its group, and the same probability of surviving to the next time interval. We used a parametric bootstrap (With the software MARK , we usedootstrap  to test ootstrap . Since t


To determine whether healthcare professionals perceive the pain of infants differently due to their understanding of that infant's level of risk for neurological impairment.Neonatal Intensive Care Units (NICU's) at two tertiary pediatric centers. Ninety-five healthcare professionals who practice in the NICU  participated. They rated the pain , distress (0–10), effectiveness of cuddling to relieve pain (0–10) and time to calm without intervention (seconds) for nine video clips of neonates receiving a heel stick. Prior to each rating, they were provided with descriptions that suggested the infant had mild, moderate or severe risk for neurological impairment. Ratings were examined as a function of the level of risk described.F  = 4.4, p = .02]. No differences in ratings were found due to participants' age, gender or site of study. Physicians' ratings were significantly lower than nurses' across ratings.Professionals' ratings of pain, distress, and time to calm did not vary significantly with level of risk, but ratings of the effectiveness of cuddling were significantly lower as risk increased [Professionals provided with visual information regarding an infants' pain during a procedure did not display the belief that infants' level of risk for neurological impairment affected their pain experience. Professionals' estimates of the effectiveness of a nonpharmacological intervention did differ due to level of risk. Research on pain in infants has progressed considerably over the past twenty years. The nature and frequency of procedural pain in the neonatal intensive care unit (NICU) is now understood , many meHowever, much less is known about the pain experienced by neonates who are at risk for neurological impairment (NI), as most studies of neonatal pain have either excluded this group or have not examined data specific to them within larger data sets. We do know that this group represents approximately 10% of infants admitted to the Neonatal Intensive Care Unit  and thatThe crucial first step of pain management is pain assessment. Without a valid and reliable approach to assessing pain, and the demonstrated efficacy of interventions for pain, decisions about pain management may not improve care. However, even with valid, reliable pain assessment tools, the characteristics of healthcare providers may affect ratings provided by them. These characteristics can include the healthcare providers' views of pain interventions , lack ofThe present study was designed to move beyond self-report of beliefs to examine whether healthcare professionals' judgments of pain in neonates are affected by their perception that a neonate has mild, moderate or severe risk for neurological impairment.Research in this area is only emerging, but has important implications for how healthcare professionals deliver care to this vulnerable population. In a previous study using questionnaires, we found that caregivers of children with severe cognitive impairment view the pain of children with more severe impairment as reduced . A seconThese studies suggest that those who manage the pain of infants at risk for, or children with, intellectual deficits believe that pain is less for those at greater risk or who have greater impairment. These results may explain why we also found infants at risk for neurological impairment receive less pain treatment in the NICU . HoweverOne hundred and one healthcare professionals, with at least one year of experience working with infants with neurological impairment in the NICU, were recruited from two tertiary level university affiliated NICU settings in central and eastern Canada. They were recruited through information provided by the research nurses and notices posted within their centers. Each participant was paid a small honorarium for their participation, and all provided informed consent. The study was approved by each health centre's Research Ethics Board.Participants completed a questionnaire that requested information regarding their age, gender, education and work experience.Nine video clips were viewed by each participant. The 30 second video clips depicted term and preterm neonates of Caucasian descent experiencing a heel stick and squeezing for blood collection. Video clips were of the infants' faces only, with most lying on their sides and all bundled. Audio was not included. Prior to each videoclip, a verbal description of the neonate suggesting he/she was at mild, moderate or severe risk for neurological impairment was provided to the participant. These descriptions had been previously recorded on audio tape by a researcher to ensure each participant was read the description in an identical fashion. Descriptions were counterbalanced such that each videoclip was described for some participants as having either mild risk, moderate risk, or severe risk within each of two orders of presentation. Thus, each participant viewed three infants that were described as having mild, moderate or severe risk for neurological impairment, but the level of risk, and the order in which the clips were presented varied. Examples of the descriptions provided to participants are shown in Table Participants rated the pain of infants shown on videotape from 0 (no pain) to 10 (extreme pain). They also rated each infant's pain using a Faces Pain Scale . These measures were chosen because they are easy to use and were feasible for this experimental task. Although many validated measures of neonatal pain exist, these are multidimensional in nature. As such, they require the person using them to have access to information regarding the infant's physiological status, something we were unable to provide in the context of this task.r = .74), moderate (.r = .56) and severe risk for neurological impairment (r = .60).The 7 face scale , was incParticipants provided a rating of how effective they believed a behavioural intervention (i.e. cuddling) would be for minimizing the procedural pain for each infant  and of how long (seconds) they believed it would take each infant to calm without intervention.To ensure that the descriptions provided were valid depictions of infants at each level of risk and were understood and accepted by participants, participants were asked to rate the level of risk they believed each infant had for future neurological impairment .Participants took part in small groups of 5 to 6 professionals that were randomly assigned to one of the two orders of presentation. They completed the demographic questionnaire and the rating tasks were explained to them. They were then shown the nine video clips. After each videoclip was viewed, the participants were provided with time to make their independent ratings for that videoclip before the next was shown. Participants were not permitted to interact with each other until all tapes had been viewed and rated. After ratings were complete, they were debriefed regarding the purpose of the study and a discussion of their experience was facilitated by the research assistant.n = 88) to 7% (n = 1) of responses .Preliminary analyses indicated that two participants were missing more than 10% of ratings. As per an a priori decision as to how to handle missing data, their data were excluded from further analyses. The remaining 99 participants were missing 0%  = 4.4, p = .04). A more detailed look at the data using stem and leaf plots revealed 4 participants in one group had extreme scores for ratings of Time to Calm  relative to the other participants in that group . The data of these four participants were removed. A second RM ANOVA revealed no significant effect due order of presentation of the video clips , a significant effect of Rating  and a significant interaction between the two [F  = 3.4 p = .002]. Thus, there was no overall effect due to the level of risk described, but level of risk described did affect some ratings.The mean ratings provided for video clips of infants described as having mild, moderate or severe risk for neurological impairment are depicted in Table F  = 2.9, p = .06] and a significant effect on ratings of the perceived effectiveness of cuddling , but nonsignificant effects on Faces Pain Scale ratings , distress  and time to calm . As Table t(94) = 2.5, p = .01] or moderate risk . The difference in ratings between those described as at mild or moderate risk were nonsignificant . Thus, participants believed that beyond a moderate level of risk, the effectiveness of cuddling dropped significantly.One-way RM ANOVA's revealed Level of Risk had a marginal effect on participants' ratings of pain on the 0–10 scale , the second revealed a nonsignificant main effect of Gender , and the third indicated Age also did not significantly effect ratings on the five measures . Thus, participants' ratings did not vary due to their institution, gender or age.Three Mixed Measures ANOVA's were used to examine the effect of participants' characteristics on the five ratings ratings. The first result indicates a nonsignificant main effect of Site . However, the main effect of Level of Risk was nonsignificant and the main effect of Profession only approached significance . Participants' ratings were not affected by their professional background. The interaction between Rating and Level of Risk was significant , but all other interactions were nonsignificant. Games-Howell post-hoc comparisons revealed a significant difference in the ratings provided by staff nurses and physicians (p = .004) and a difference between respiratory therapists and physicians that approached significance (p = .06). As shown in Table To examine the effect of participants' profession on their ratings, three groups were included in a Mixed Measures ANOVA: staff nurses n = 34), physicians (n = 11), and respiratory therapists (n = 17). Residents and Fellows, Nurse Managers and Educators and Specialists, and other professionals were not included due to small numbers in those groups. The analysis revealed a significant main effect of Rating Scale [, physiciEighty-nine participants provided information regarding their amount of professional experience. Correlations indicated that years of experience were not correlated significantly with any of the five ratings provided after corrections for multiple tests. Thus, the importance of an infants' level of risk for neurological impairment was neither greater nor less as experience in this setting increased.Overall, the professionals in this study did not rate the pain of neonates differently when provided with information indicating those infants had mild, moderate or severe risk for neurological impairment. The professionals' perception of the infants' level of risk also did not affect their ratings of the infants' distress, or their belief in how long the infant would take to calm after pain without intervention. Professionals did perceive that cuddling would be significantly less effective for infants at high risk for neurological impairment than for infants with mild or moderate impairment. However, this effect was not large, and, although it was statistically significant, it may be spurious. Further research should examine whether beliefs regarding pain experience in this group and beliefs regarding the effectiveness of cuddling and other nonpharmacological interventions are truly independent. These results are inconsistent with the results of our previous questionnaire study indicating professionals, with similar levels of experience in neonatal intensive care settings, perceive the pain experience of infants as reduced as their level of risk for neurological impairment increases . There aThe professionals who participated in this study were asked to rate the risk for neurological impairment of each infant they viewed on videotape. Asking them to do this may have alerted them to the purpose of the study and elicited efforts on their behalf to provide ratings that were unbiased. However, their ratings of the perceived effectiveness of cuddling did vary by level of risk for impairment, suggesting attempts to appear unbiased do not fully explain the results found.relative to the pain experience of those without risk [In our previous studies, questionnaires elicited beliefs about the pain experience of infants and children with varying levels of risk out risk -12. In cIt is also possible that the beliefs expressed by professionals in our previous study  do not mThe finding that pain ratings did not vary due to level of risk for neurological impairment raises questions about our previous study that revealed infants at risk for neurological impairment receive less pain treatment in the NICU . When a Further research is needed to reconcile the current results with beliefs that risk for neurological impairment does affect pain experience expressed by a similar group of professionals in our previous survey  and the Another finding in this study warrants discussion. Professionals' judgments of the effectiveness of cuddling decreased with increasing risk for neurological impairment, despite their having judged pain as similar in intensity. This result is similar to a finding by Fanurik et al. . They foThe current results, along with those of Fanurik's group , raise tIf professionals and caregivers do believe pain behaviour is more reflexive, and that pain experience is more physiologically based when a child has neurological impairment, it could explain the current results regarding the effectiveness of cuddling. Professionals viewing the video clips may have perceived the behavioural responses of the infants with different levels of risk for impairment as being similar in intensity. Nonetheless, they may have interpreted the behaviour of those with more risk as more of a reflexive response to a physiological insult, while they saw the behaviour of those with lesser risk as reflecting a more multidimensional pain experience incorporating both physical and psychological suffering. Thus, we could again speculate that they may have felt cuddling, an intervention that would address physical and psychological aspects of pain, would be more effective for the less impaired groups. This phenomenon would not be novel or unique. For most of recorded history, there has been a belief that cognitive interpretation of pain was necessary for pain to result in long-term negative consequences. This belief was often the justification for poorer pain management for both children and infants . AlthougThe current study has several limitations. Professionals were asked to rate the pain experience of infants receiving heel sticks from videotape. Although this may approximate the real situation in a NICU setting, it is not identical. In a NICU setting, professionals would have rich information from the environment, previous contact with an infant, physiological data, and medical records that guide their assessment of pain. They would also view this infant within the context of all other infants in the unit. Professionals here were also asked only to provide ratings of pain intensity. As the results suggest, this is only one dimension of pain and may not be the dimension that plays the largest role in their judgments regarding pain in a clinical setting. The professionals here were experienced in the types of pain experienced in the NICU and may have held a priori beliefs about the painfulness of this procedure that moderated their judgments. Research suggests professionals' beliefs regarding the painfulness of a procedure play a large role in their assessments of children's pain ,9,25.The current study indicates professionals' perception of the pain intensity of infants does not differ due to their understanding of the infants' level of risk for neurological impairment. Professionals also view cuddling as less effective for infants at greater risk for neurological impairment. Further research is needed to examine the reasoning behind the judgments made by healthcare professionals and to clarify why they might view an intervention as less effective for infants with greater risk of neurological impairment, despite having rated their pain intensity as similar to that of infants at lesser risk.The authors declare that they have no competing interests.The study was conceived and designed by BS, PM & LB with assistance from all remaining authors. The study was conducted under the supervision of PM, BS, KO, AO. Statistical analyses were conducted by LB, with assistance from PM, BS and JB. Interpretation of results were conducted by LB, PM, BS, JB, CC, LF, KB and AO. The manuscript was prepared by LB, and edited by PM and BS, with review and assistance from all remaining authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Diabetes mellitus (DM) is an important public health concern, the impact of which is increased by the high prevalence of co-existing chronic medical conditions among subjects with DM. The aims of this study were therefore to (1) evaluate the impact of DM and co-existing chronic medical conditions on health-related quality of life (HRQoL) ; (2) to determine the extent to which the SF-6D (a single-index preference measure) captures the multidimensional information provided by the SF-36 (a profile measure).Using data from a cross-sectional, population-based survey of Chinese, Malay and Indians in Singapore, we developed 9 separate multiple linear regression models, with each SF-36 scale or SF-6D index score being the dependent variable for one model. The influence of DM and a second chronic medical condition (hypertension (HTN), heart disease (HD), musculoskeletal illnesses (MS)) and their interactions were studied after adjusting for the influence of potential confounding variables.Among 5,224 subjects, the prevalence of DM, HTN, HD and MS were 5.9%, 10.7%, 2.4% and 26.6% respectively. DM lowered SF-36 scores by more than 2 points on 3 SF-36 scales and lowered SF-6D scores by 0.03 points. Subjects with DM and HTN, DM and HD or DM and MS experienced further lowering of SF-36 scores exceeding 2 points on at least 6 scales and further lowering of SF-6D scores by 0.05, 0.08 and 0.10 points respectively. Generally, DM and co-existing medical conditions exerted additive effects on HRQoL, with the exception of DM and heart disease, where a subtractive effect was noted. SF-6D index scores generally reflected the patterns of influence of DM and chronic medical conditions on SF-36 scores.DM and chronic medical conditions generally reduced HRQoL in this multiethnic general population in an additive, rather than synergistic or subtractive fashion. In this study, the SF-6D was a reasonably good summary measure for the SF-36. Diabetes mellitus, the prevalence of which is reaching epidemic proportions in many parts of the world, is an increasingly important public health concern. In the United States, diabetes is present in 8% of the adult population, and is associated with a two-fold increase in age-adjusted mortality ,2. In adDiabetes has detrimental effects on a range of health outcomes including health-related quality of life (HRQoL) -12. For Subjects with diabetes and multiple co-existing chronic medical conditions have poorer HRQoL than those without these conditions ,17. For In combination, the influence of multiple chronic medical conditions on HRQoL may exhibit an additive, synergistic or possibly subtractive relationship. Assuming that each chronic medical condition results in the lowering of HRQoL, in an additive relationship, the combined effect of two or more chronic medical conditions on HRQoL approximates the sum of the independent effect of each of these conditions, while in a synergistic relationship, the combined effect is greater than the sum of the independent effect of each of these conditions and in a subtractive relationship the combined effect is smaller than the sum of the independent effect of each of these conditions Figure . For exaTwo published studies have evaluated the relationship between diabetes and other chronic medical conditions on HRQoL in the general population ,24. In tThe primary purpose of this study was therefore to evaluate the impact of diabetes and co-existing chronic medical conditions on the physical, mental and social domains of HRQoL in the general population. We also sought to determine if the impact of diabetes and co-existing chronic medical conditions on HRQoL would be additive, synergistic or subtractive. For these purposes, we measured HRQoL using both the SF-36 and the SF-6D. The SF-36 is a comprehensive, generic HRQoL measure that has been extensively validated and allows comparison of HRQoL in subjects with various chronic medical conditions. The SF-6D is a single index utility score derived from the SF-36 which measures health preference, and which is therefore suitable for pharmacoeconomic analyses to assist healthcare resource allocation. We sought to determine the extent to which the SF-6D would reflect the multidimensional information provided by the SF-36.st January 1998 and ability to read a newspaper in English or Chinese; Chinese, Malay or Indian ethnicity as reflected in the National registration identification card, which reflects parental ethnicity; and status as free-living adults in the community. Persons older than 65 years of age were excluded because of low literacy rates in this age group in Singapore [We analyzed data from a cross-sectional, population-based disproportionately stratified sample of ethnic Chinese, Malays and Indians listed in the 1996 electoral register for the Bishan-Toa Payoh district  in Singapore from April 1998 to January 1999, details of which have been reported previously  and are ingapore . FieldwoThe SF-36 measures perceived health in the areas of physical functioning (PF), role-physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role-emotional (RE), and mental health (MH), with higher scores (range 0–100) reflecting better perceived health. The UK English  and ChinThe SF-6D is a six-dimensional health classification system assessing physical functioning (PF), role limitations (RL), social functioning (SF), pain (PN), mental health (MH), and vitality (VT), with 4 to 6 levels per dimension ,30. A SFThe Family Functioning Measure is a 3-item Likert scale assessing the quality of interactions among family members , with hiData from participants completing English and Chinese SF-36 versions were pooled to increase the power and representativeness of our study. This approach was supported by previous work demonstrating equivalence of these SF-36  and SF-6To study the influence of diabetes and other chronic medical conditions on HRQoL, we developed 9 multiple linear regression models, with each SF-36 scale and the SF-6D index score being the dependent variable for a separate model. Each model was built in 3 stages. The first stage assessed the effect of diabetes on HRQoL while adjusting for the influence of sociodemographic factors. The second stage assessed the independent effects of diabetes and a second chronic medical condition on HRQoL while adjusting for the influence of sociodemographic factors. The third stage additionally assessed any interactions between diabetes and this second chronic medical condition on HRQoL. Thus, each model studied the effect of diabetes and one other chronic medical condition. The absence of a significant interaction term would suggest that the influence of two chronic medical conditions on HRQoL scores was additive. The presence of a significant interaction term would suggest that a synergistic or subtractive relationship existed, the former if the coefficient for the interaction term was negative, the latter if the coefficient was positive . Ethnicity was coded using dummy variables. Educational level was used as proxy for socioeconomic status. In anticipation that if the number of subjects with a given chronic medical condition was less than 50, the numbers of subjects with this medical condition and diabetes would be very small and not amenable to meaningful interpretation, two chronic medical conditions, stroke and cancer, were thus excluded from analysis because of small numbers of subjects reporting these conditions (n<50). Two other chronic medical conditions were subsequently excluded from analysis because of small numbers of subjects with diabetes and these conditions .Complete data were available from 5,224 of 5,420 subjects in the study, with 196 (3.6%) subjects excluded from analysis (110 because of missing demographic information and 86 because of missing responses to the SF-36). Table The influence of chronic medical conditions on individual SF-36 scales is presented in Table The influence of chronic medical conditions on SF-6D scores is also presented in Table Characteristics of subjects with diabetes, with and without other co-existing chronic medical conditions, are shown in Table The impact of co-existing chronic medical conditions on adjusted SF-36 scores of subjects with diabetes is presented in Table The influence of diabetes and co-existing chronic medical conditions on SF-6D scores is presented in Table In this multiethnic, population-based study, we found that subjects with diabetes experienced lowering of HRQoL as compared to subjects without diabetes. The presence of other chronic medical conditions in subjects with diabetes led to further lowering of HRQoL, the effect of which was generally additive. Our findings further underscore the importance of preventing and treating complications of diabetes to prevent further deterioration in HRQoL among subjects with diabetes, and also highlight the need to identify factors that may be modulated to improve HRQoL in these subjects.Our findings are important for several reasons. First, this is the first study showing that the combination of diabetes and a second chronic medical condition may adversely affect the mental domains of HRQoL as measured by the vitality, social functioning and mental health scales of the SF-36  ,24. SecoWe found that the effect of diabetes on HRQoL was generally mild, with greater impact on the SF-36 scales measuring physical ) relative to mental health components . This was not surprising, given that our subjects were recruited from the general population and were therefore likely to have less severe illness than subjects in hospital or clinic-based studies. Further, other studies have shown that impact of diabetes on HRQoL is intermediate, relative to other chronic medical conditions . For exaWe also found that with the exception of subjects with diabetes and heart disease, the presence of co-existing chronic medical conditions in subjects with diabetes generally resulted in further significant lowering of HRQoL. Our results are important because they demonstrate that the impact of these co-existing chronic medical conditions in diabetes is not only in increasing healthcare costs ,37 and mth or 6th decade of life [In our study, the relationships between diabetes and other chronic medical conditions on HRQoL were largely additive. This is in contrast with the study by Gaynes et al. , where s of life , often m of life  (althoug of life ,43. In c of life . Hence, A secondary objective of this study was to understand the extent to which the single index SF-6D captured information from the multidimensional SF-36. We found that in general, the impact of co-existing chronic medical conditions on the SF-36 was well-reflected in the SF-6D. However, the subtractive effects of diabetes and co-existing heart disease on SF-36 scores were not reflected in SF-6D scores. This suggests that there is some inevitable loss of information associated with a reduction from a multi-dimensional scale to a unidimensional scale. Hence, more studies are needed to evaluate the adequacy of the SF-6D as a summary measure of the SF-36.We recognize several limitations of this study. First, identification of chronic medical conditions was based on self-report which may not be as accurate as physician diagnoses. However, reliability of self-report has been found to be acceptable for conditions that required medical or laboratory diagnostic procedures, including diabetes -47. SecoIn conclusion, in this large, multiethnic, population-based study, we found that subjects with diabetes experienced lowering of HRQoL as compared to subjects without diabetes. The co-existence of other chronic medical conditions in subjects with diabetes led to further lowering of HRQoL, the effect of which was generally additive. Finally, we found that the SF-6D is a reasonably good summary measure of the SF-36 although more studies are needed to confirm this observation.ADL – Activities of Daily Living, DM – Diabetes mellitus, HD – Heart disease, HRQoL – Health-related quality of life, HTN – Hypertension, MS – Musculoskeletal illnesses, OR – Odds ratio, SF-36 – Medical Outcomes Short-Form 36.JT conceived of the study, and participated in its design and coordination. HL Wee and YB Cheung participated in the design of the study and performed the statistical analysis. SC Li and KY Fong participated in the design of the study and its coordination. All authors read and approved the final manuscript.


Metastasis is the leading cause of cancer-related death. Bone marrow (BM) is a frequent site for the settlement of disseminated tumor cells which occurs years before overt metastases signal incurability.Here we describe a new method to assess the initial stage of metastasis development in cancer patients. By immunomagnetic selection with HER2/neu and EpCAM as catcher antigens single disseminated tumor cells can be enriched from BM samples. To examine whether the immunomagnetic enrichment technique may change gene expression in the selected tumor cells, we performed differential expression profiling with the breast cancer cell lines MCF-7 and BT474 as models. The profiles were performed using 1.2 Cancer Arrays (Clontech) containing 1176 cDNAs that can be grouped into different functional categories, such as signal transduction, cell cycle, adhesion, cytoskeleton plasticity, growth factors and others.The reproducibility of the gene expression profiling between duplicate cDNA-array experiments was assessed by two independent experiments with MCF-7 breast cancer cells. Scatter blot analysis revealed a good reproducibility of the cDNA array analysis (i.e. less than 10% difference in the gene expression between the arrays). Subsequent comparative cDNA-array analyses of immunobead-selected and unselected MCF-7 and BT474 cancer cells indicated that the antibody incubation during the immunomagnetic selection procedure did not considerably alter the gene expression profile.The described method offers an excellent tool for the enrichment of micrometastatic tumor cells in BM without largely changing the gene expression pattern of these cells. To predict and monitor therapeutic responses the assessment of the gene expression profile of disseminated tumor cells seems to be of utmost importance. However, it is uncertain to which extent incubation with antibodies used for immunomagnetic isolation of these cells might affect their expression profile. We have addressed this aspect, using monoclonal antibodies against two prominent antigens, EpCAM and HER2/neu that are frequently and independently expressed on micrometastatic tumor cells  dATP  labeled cDNA synthesis as previously described [Total RNA was isolated using the peqGold TriFast™  according to the manufacturer's instruction. In order to remove genomic DNA contamination, a DNase step was included using the DNA-free™ kit  according to manufacturer's instructions. RNA was dissolved in RNase-free Hescribed . The cDNThe membranes were exposed to phosphoimager plates  for 3 days, and plates were scanned with the phosphoimager Fuji Bas (Raytest) at a 100 μm-resolution. The images were analyzed using the Imagene 5.5 software . The data of the arrays were normalized on the basis of the genes ubiquitin, HLAC and beta actin. The ratio between antibody-treated and non-treated cells was calculated for each gene. Ratios lower than 0.5 or higher than 2 were considered as differentially expressed if at least one sample showed an expression above 0.5. We performed duplicates of each experiment and created scatter blots with the SPSS software for windows.In a recent work  we demonTo test whether the antibody incubation during the immunomagnetic enrichment approach affects gene expression in the selected cells, we applied cDNA-array analysis. We subsequently evaluated whether the immunomagnetic enrichment method affected gene expression in the selected cells. This aspect is of utmost importance for further molecular description of disseminated tumor cells and has not been addressed before. The profiles were performed using 1.2 Cancer Arrays (Clontech) containing 1176 cDNAs that can be grouped into different functional categories, such as signal transduction, cell cycle, adhesion, cytoskeleton plasticity, growth factors and others [As models, we used the breast cancer cell lines MCF-7 and BT474 (ATCC HTB-22 and HTB-20), because they express heterogeneous levels of the target antigens HER2/neu and EpCAM comparable to micrometastatic breast cancer cells in vivo . The repR) can specifically block cell proliferation and affect gene expression in HER2/neu-positive breast cancer cells [Here, we investigated whether an immunomagnetic enrichment procedure for micrometastatic cancer cells present in BM aspirates leads to significant changes in the gene expression pattern of the enriched tumor cells. In order to mimic the biological conditions of a tumor type with frequent BM involvement, we used two breast cancer cell lines (MCF-7 and BT474). Both cell lines expressed the target antigens, EpCAM and HER2/neu, for immunomagnetic separation at different levels  and theyer cells ,14. Furter cells . Howeverer cells . Taken tThus, we conclude that the immunomagnetic selection protocol described here might be useful for experimental approaches aimed to determine the gene expression profile and genome of disseminated CK-positive cells ,18. As w


By incorporating this concept in left ventricular (LV) models to simulate filling and systolic phases, we have obtained the time-varying expression for Ea and the LV-volume dependent expression for Ep.Description of the heart as a pump has been dominated by models based on elastance and compliance. Here, we are presenting a somewhat new concept of time-varying passive and active elastance. The mathematical basis of time-varying elastance of the ventricle is presented. We have defined elastance in terms of the relationship between ventricular pressure and volume, as: a is expressed as: ; Epis represented as: . Ea is deemed to represent a measure of LV contractility. Hence, Peak dP/dt and ejection fraction (EF) are computed from the monitored data and used as the traditional measures of LV contractility. When our computed peak active elastance  is compared against these traditional indices by linear regression, a high degree of correlation is obtained. As regards Ep, it constitutes a volume-dependent stiffness property of the LV, and is deemed to represent resistance-to-filling.Using the patient's catheterization-ventriculogram data, the values of passive and active elastance are computed. Ea) can be used to characterize the ventricle's contractile state, while passive elastance (Ep) can represent a measure of resistance-to-filling.Passive and active ventricular elastance formulae can be evaluated from a single-beat P-V data by means of a simple-to-apply LV model. The active elastance (E The heart may be conceived as a pump that receives blood from a low-pressure system and raises it to a high-pressure system. Although the mechanism responsible for generation of wall stress (and hence left ventricular (LV) pressure) is contraction of the myocardial fibers, an analytical formulation linking the myocardial and LV dynamics is still lacking. In the absence of this formulation, a popular way of linking LV pressure and volume dynamics is by means of LV compliance (or elastance) ,2. AlthoThe concept of compliance or elastance was first employed for blood vessels , by relaDefares  avoided Classically, ventricular compliance is defined, at any point in time, as the change in ventricular volume concomitant with the change in ventricular pressure, such thatC = dV / dP, E = dP / dV     (1)If C is assumed constant, this equation becomes a linear relation, whose integration givesV = CP + Vc     (2)c is an integration constant. If the compliance varies with time, then all terms in equation (2) may vary with time, as:where VV (t) - Vc (t) = C(t)P(t)     (3)In this context, Suga  opted fod represents the unstressed LV volume corresponding to zero LV-pressure, obtained by drawing a tangent to P-V curves at end-ejection, as illustrated in Figure to represent the elastance of the contracting LV, where Vmax and of the volume-axis intercept (Vd) of the tangent to the P-V curve at end-ejection (as a measure contractility of the cardiac muscle) is not only unreliable  = ρh / 4πR2, for a spherical LV model (in mmHg/(ml/s2));E represents LV elastance (mmHg/ml).LV) is caused by both Ea variation as well as Ep decrease. The instantaneous time-varying ventricular elastance (E) is the sum of (i) volume-dependent passive elastance (Ep) and (ii) active elastance (Ea) due to the activation of the LV sarcomere. Hence,Likewise during ejection, the LV pressure variation (dPE = Ea + Ep     (6)p and Ea, and then their formulations. The passive (unactivated) myocardium exhibits properties of an elastic material, developing an increasing stress as strain increases, as occurs during ventricular filling. The passive stress-strain relation of myocardial muscle strip is nonlinear, and therefore cannot be described by Hooke's law. As an approximation, it follows an exponential relationship  = Vi = dPLV,i     (9)i and PLV,i are the monitored LV volume and pressure at this instant, and Eped is the passive elastance at the end-diastolic phase.where i is a time instant during the isovolumic contraction and relaxation, V and Ep is constant), the governing equation (5) becomes VdE = dPLV, which can be represented as:Also, during isovolumic relaxation  = Vi  = dPLV,i     (10)pes is the passive elastance at the end-systolic phasewhere ENow, applying equations (9 & 10) to the case shown in the Figure 1. For isovolumic contractionEa,1 = 0     (11-a)Ea,2 = (P2 - P1)/V2 + Ea,1 = 0.029477 mmHg/ml     (11-b)Ea,3 = (P3 - P2)/V3 + Ea,2 = 0.103771 mmHg/ml     (11-c)Ea,4 = (P4 - P3)/V4 + Ea,3 = 0.253584 mmHg/ml     (11-d)Ea,5 = (P5 - P4)/V5 + Ea,4 = 0.463599 mmHg/ml     (11-e)2. For isovolumic relaxationEa,18 = (P18 - P17)/V18 + Ea,17 = Ea,17 - 0.058954 mmHg/ml     (11-f)Ea,19 = (P19 - P18)/V19 + Ea,18 = Ea,17 - 0.177824 mmHg/ml     (11-g)Ea,20 = (P20 - P19)/V20 + Ea,19 = Ea,17 - 0.312656 mmHg/ml     (11-h)Ea,21 = (P21 - P20)/V21 + Ea,20 = Ea,17 - 0.463599 mmHg/ml     (11-i)a,17 at end-ejection is unknown. For different representative values of Ea,17, we can get different Ea curves. We need to determine the optimal value of Ea,17, such that Ea can be described by a smooth curve to fit both isovolumic contraction and ejection phases. In Figure a,17 = 1.1 mmHg/ml, and the polynomial expression for Ea(t) to fit its above calculated values during isovolumic contraction and relaxation. However, in order to (i) more suitably represent Ea to correspond with its role during the cardiac phases , and (ii) because of the sigmoidal shape of Ea curve and its variation resembling the LV pressure variation during systole  as:Now in the above expressions 11(f-i), ESo that its constituent parameters have physiological significance as indicated following equation (8).a(s) during isovolumic contraction, when u(t-d) = 0, we employ the expressionHence, to compute Ea0, τC and ZC to fit the monitored pressure-volume data. Then, to compute Ea(s) during isovolumic relaxation, we employ the total expressionand determine its parameters EτR and ZR to fit the measured pressure-volume data.and determine its remaining parameters d, a is depicted in Figure a can be employed as an index of contractility.For the sample data of Figure During the diastolic filling phase, equation (5) becomesp is constant at a particular volume Vi, equation (13) becomesNow because Ei and PLV,i are the monitored LV volume and pressure at this time, and M = ρh / 4πR2. For the patient data . Therefore, from equation (15), we can calculate the values of Ep at various instants during filling phase. We then plot Ep vs V, in Figure p, we obtain the values of the parameters Ep0 and zp, as:where i is a time-instant during diastolic filling, Vzp = 0.0395 ml-1, Ep0 = 4.375 × 10-3 mmHg / ml     (16)p function (corresponding to its expression given by equation 7) as follows:and the EEp = 4.375 × 10-3 eV 0.0395    (17)p as a measure of LV resistance-to-filling.We now propose to adopt Ea and Ep are varying. During ejection and filling phases, Ep can be calculated at any time using equation (17). Likewise, Ea can be calculated during ejection and filling phases using equation (8), once its parameters have been determined by employing equation (12-b & 12-b) during isovolumic contraction and relaxation phases. Their values during the cardiac cycle are listed in Table Hence during ejection, both Eequation 7. LikewiThe analyses, presented herewith, are now applied to clinically obtained data consisting of the subject's left ventricular (instant-to-instant) dimensions (obtained by cineangiocardiograph) and chamber pressure (obtained by cardiac catheterization). For each subject, passive and active elastances are determined from the left ventricular data. Table a vs incremental pressure (P-Ped) for this patient HEL. Note that the elastance is much higher at late-ejection than early ejection. This is because of a continuing sarcomere stress development and shortening. The active elastance reaches its maximum value at late-ejection (point 15), and thereafter decreases. However as shown in Figure The variations of model-derived nonlinear passive and active elastances for the subject HEL are shown in Figure p vs LV volume plots, in Figure p increases exponentially with increase LV volume; the parameters (EP0 and zp) characterize this relationship. The passive elastance curve is steeper for a stiffer myocardium, with a corresponding bigger value of the exponential coefficient Zp (subject ML). The Ea vs incremental pressure (P-Ped) plots, in Figure a during systole, which in turn governs the generation of LV pressure.Figures a variation is by means of the Ea vs normalized time (t/ts) plot, shown in Figure a,max could be regarded as an index of LV contractility. Hence, we decided to plot Ea,max vs the traditional contractility indices of EF and (dP/dt)max. These plots are displayed in Figures a,max has a high degree of correlation with both EF and (dP/dt)max. It is interesting to compare our correlation-coefficient value (0.8972) with the value of 0.89 obtained by Mehmel et al [d)]es.Yet another way to study Eel et al , althougThe active elastance curve can explain some critical LV physiological phenomena, namely LV pressure generation during isovolumic contraction and LV suction during early filling. The rapid decrease in elastance during isovolumic relaxation extends into the filling phase, and can explain the decrease in LV pressure (Vi - Vi-1) + Vi      (18)P term, based on the calculation of its value being of the order of 10-2 compared to (i) (Vi - Vi-1), which is of the order of 100 and (ii) Vi which is the order of 101.by neglecting the i can be less than Pi-1 (or that Pi - Pi-1 < 0) only if  is negative, i.e., active elastance is decreasing. Now for subject H.E.L  are:In equation 18), it is seen that P8, it is Ep,22 = 0.2768 mmHg/ml, Ea,22 = 0.0393 mmHg/ml, Ea,21 = 0.1140 mmHg/ml, V22 = 105 ml, V21 = 90.6 ml     (19)P22 - P21) = -3.22 mmHg, confirming decrease of pressure during early filling.Substituting these values into equation (18) gives .Hence, our novel concept of "decreasing E10 & t9), as follows:Similarly, both the active and passive elastances can explain LV pressure variation during ejection, using equation (18). Let us show how this happens by just taking two instants (V10 - V9) + V10     (20)PP10 - P9 = 1.92 mmHg, which is approximately equal to the actual value of 2 mmHg.Substituting these computed values (listed in Table P / (V - Vd) [max (given by ESPVR) as illustrated on Figure Previous works have described elastance as a derived parameter from LV pressure-volume data, based on the definition of (V - Vd) ,10,12-14p and Ea enable us to explain the phenomena of (i) LV suction during early filing, (ii) LV pressure rise during isovolumic contraction (iii) LV pressure variation during the ejection phase, and (iv) LV pressure drop during the relaxation phase. Both the concepts of Ea and Ep are made possible by our redefining elastance and compliance asOur definitions of EdP = d(EV) = d(V/C) = VdE + EdV     (21)a enables us to explain (i) the generation of pressure during isovolumic contraction and decrease of pressure during isovolumic relaxation, when the volume is constant, and (ii) the decrease of pressure during the rapid filling phase.Our concept of Ea along with increasing Ep during filling, which enables us to explain the LV suction effect during early filling, is indirectly supported by the work of Shoucri [Our concept of decreasing E Shoucri . He has a, as provided by us.However, the determination of this active fiber stress in his paper is empirical and not based on a well-defined concept and expression for Ea and Ep are invoked to explain the variations of pressure during the ejection and filling phases. From the viewpoint of intrinsic indices of LV assessment, Ep can represent LV myocardial stiffness property and resistance to LV filling. On the other hand, Ea has been shown to correspond to LV contractility, by means of Figures a,max and (dP/dt)max as well as EF.Finally, both Ep and Ea) with an arterial lumped-parameter model  in order to simulate and explain the mechanisms of chronic hypertensive states [a itself as a measure of LV adaptation to chronic hypertension induced in the circulation system.In future, we can also couple our LV model  = ρh, ρ is the myocardial density, and u is the radial displacement, dPel and dPLV are the incremental elastic-recoil and left ventricular pressures .where the myocardial element mass, Now since,r1 = r2 = RBy assuming we can write:ρs is the surface density, M = ρsh, and V is the LV volume. Now, referring to Figure A-1,where andRe is the resistance to LV filling , ρf is the blood density and v is the blood velocity at site 2.where LVdP) as:Let us now define incremental elastic recoil pressure , we haveIntroducing the term elastance (= 1/C), we can put downLiang Zhong carried out the elastance studies, participated in data acquisition, performed statistical analysis and drafted the manuscript. Dhanjoo N. Ghista conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Eddie Y-K Ng participated in coordination and helped to draft the manuscript. Soo T Lim participated in its design and coordination. All authors read and approved the final manuscript.


Cellular coactivators interacting with Bel1/Tas are unknown to date.Foamy virus Bel1/Tas trans-activator Bel1/Tas in vivo. Here we show that the Bel1/Tas-mediated trans-activation was enhanced by the coactivator p300, histone acetyltransferases PCAF and SRC-1 based on the crucial internal promoter BRE. The Bel1/Tas-interacting region was mapped to the C/H1 domain of p300 by co-immunoprecipitation and pull-down assays. In contrast, coactivator SRC-1 previously reported to bind to the C-terminal domain of p300 did not directly interact with the Bel1 protein but nevertheless enhanced Bel1/Tas-mediated trans-activation. Cotransfection of Bel1/Tas and p300C with an expression plasmid containing the C/H1domain partially inhibited the p300C-driven trans-activation.Transient expression assays, co-immunoprecipitation experiments, pull-down assays, and Western blot analysis were used to demonstrate that the coactivator p300 and histone acetyltransferase PCAF specifically interact with the retroviral trans-activator capable of partially blocking the strong Bel1/Tas-mediated activation of the C-terminal region of coactivator p300. The potential mechanisms and functional roles of the three histone and factor acetyltransferases p300, PCAF, and SRC-1 in Bel1/Tas-mediated trans-activation are discussed.Our data identify p300 and PCAF as functional partner molecules that directly interact with Bel1/Tas. Since the acetylation activities of the three coactivators reside in or bind to the C-terminal regions of p300, a C/H1 expression plasmid was used as inhibitor. This is the first report of a C/H1 domain-interacting retroviral  Both proteins function by mediating positive or negative cross talk between different signaling pathways and participate in fundamental cellular processes that include embryonic development, cell growth, differentiation, and apoptosis. In addition, they can act as tumor suppressors and, last but not least, directly interact with diverse viral trans-activators to facilitate virus replication or viral activator-mediated transformation [In the sequential model of transcriptional regulation including viral template -3. The ftemplate ,4,5. It ormation ,7.trans-activators, for instance p53 [Several models have been proposed to explain transcriptional activation. Coordinated recruitment of coactivators by diverse transcriptional activators to specific promoter target sites has been shown by a collective effort of many groups . Accordiance p53 .trans-activator proteins were found to interact with distinct domains of p300/CBP and PCAF. Prominent among them are the early adenoviral E1A antigen [Diverse viral  antigen ,11, Epst antigen , and hum antigen ,14. Seve antigen ,16.trans-activator of the acidic class and is known to directly interact with its responsive elements (BRE) [trans-activation domain (TAD) was mapped to the C-terminus of Bel1 with little if any protein homology to other FV Bel1/Tas proteins from different species. Previously, we identified the nuclear factor 1 (NF1) as a repressor of Bel1/Tas-mediated trans-activation [trans-activation. This is the first time that these cellular coactivators have been shown to interact with the Bel/Tas1 trans-activator protein. Furthermore, Bel1/Tas binding to the C/H1 domain of p300 and coactivator-driven trans-activation seem to follow a unique pathway.The apparently nonpathogenic primate foamy viruses (PFV) show a wide host range and tissue tropism and have been developed into vectors that efficiently transduce SCID-repopulating cells . The PFVts (BRE) ,19. Bel1ts (BRE) . Additiotivation . This retivation , we invetivation . Here wetrans-activate the Bel1 internal promoter (IP.BRE), transient reporter gene assays were performed. The IP.BRE that extends from -1 to -192 of the second cap site of the PFV genome was cloned into the pGL3-pro-luc reporter plasmid [trans-activator under the control of the CMV-IE promoter. The results of immunoblotting shown in the lower left panel of Figure To examine whether p300 . In contrast, when the HAT PCAF expression plasmid was used for co-expression, an enhancement of Bel1/Tas-induced activation was detectable at a concentration of 0.02 μg PCAF DNA and 0.5 μg pbel1s . It is intriguing that the distribution of the closely spaced Lysines of Bel1/Tas apparently mimics the correspondingly spaced Lysines in histones. This observation is further complicated by our observation that cotransfection with higher levels of PCAF led to a reduced stability of Bel1/Tas  into pCI-p300N digested with SphI/NotI for expression of residues 1 to 424 of p300. Construct no. 7  fusion proteins pGST-C/H1 (328–424) and pGST-KIX (436–661)  for 6 hr. Cells were harvested and used for immunoprecipitation as described above. The precipitates were analyzed by SDS-PAGE on 12% gels, dried, and exposed on KODAK Biomax MR1 films.Plasmid p300FL was transfected into 293T cells and metabolically labeled with L-[Plasmid pCMV-βgal directing β-galactosidase expression from the CMV-IE promoter was used for normalization of transfection efficiency. Luc reporter gene assays were performed and quantified as described  using a Immunoprecipitation was performed as described previously with minor modifications . LysatesE. coli BL21/DE3 cells after transformation with the corresponding plasmids. Expressed proteins were purified by binding to glutathione Sepharose 4B resin. Each of the GST-fusion proteins bound to glutathione Sepharose 4B were mixed with lysates obtained from pbel1s-transfected 293T cells. After incubation at 4°C overnight in binding buffer [The recombinant GST-C/H1 and GST-KIX proteins were expressed in g buffer  and exteg buffer  or detecCells were harvested two days after transfection by lysis in 1% SDS and the protein concentration was determined using the DC protein assay (BioRad). Identical amounts of proteins were separated by SDS-PAGE on 12% gels, blotted, reacted with monoclonal serum directed against the FLAG epitope of the four different FLAG-tagged p300 (Sigma), or polyclonal serum direct against Bel1 , and dettrans-activation domain; NF1, nuclear factor 1; CMV, cytomegalovirus; GST, glutathione-S-transferase.CBP, CREB-binding protein; C/H1, 2, and 3, Cys/His-rich domains of p300; FL, full-length; HAT, histone acetyltransferase; LTR, long terminal repeat; luc, luciferase; PCAF, p300/CBP-associated factor; Q, Glutamine-rich domain of p300; SRC, steroid receptor coactivator; p300N, p300M, and p300C, amino-, middle and C-terminal regions of p300; PFV, primate foamy virus; IP.BRE, internal promoter Bel1 response element; TAD, HB and WM contributed equally to the manuscript. VO carried out the molecular cloning of p300 derivatives. YN participated in the design of the study. All authors read and approved the final manuscript.


Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early β-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator β-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula β-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that  New insights into the early events of neural induction which render certain cells competent to respond to signals emanating from Spemann's organizer Lens induction is an example of a secondary embryonic induction. Most experimental embryologists concentrated their research on the induction of the neural plate, which is considered the primary embryonic induction  mRNA, a secreted antagonist specific for Nodal-related mesoderm inducers , Noggin (Nog), and Goosecoid in dorsal endomesoderm at the gastrula stage. Second, a technical revolution has taken place with the availability of antisense morpholino oligos (MO) that permit loss-of-function studies in Xenopus  region that contains both prospective neuroectoderm cells and Spemann organizer precursors. The BCNE region also expresses Xenopus Nodal-related 3(Xnr3), a secreted factor with neural-inducing properties that is expressed at high levels in early Xenopus embryos , a gene expressed in anterior endoderm at gastrula . Neural differentiation could also be obtained in the absence of CerS mRNA when additional care was taken to avoid mesodermal contamination. As shown in In embryology, the test of whether cells are specified to form a particular tissue is to culture them in isolation from the rest of the embryo. Dorsal animal cap explants from embryos injected with  did not A and 3B.Xenopus is one of the best-studied vertebrate embryos, it was surprising that this requirement of a region of the blastula for CNS formation had not been reported previously. To investigate this further, we replaced the deleted fragment with various ectodermal grafts. The brain defects could be rescued by transplantation of dorsal, but not ventral, animal cap grafts  or pan-neural markers  were still expressed, indicating that only anterior neural differentiation was eliminated by Chd-MO and that posterior neural induction continues to take place. We also generated a Noggin antisense morpholino oligo (Nog-MO) reagent, which, like Chd-MO, inhibited brain markers  mRNA, or LiCl. These treatments induced multiple neural markers in animal caps , but not of posterior or pan-neural ones  .Two recent studies have described morpholino antisense oligos targeting  factors . Xenopusvolution . Examinaectively A  blastula dorsal animal cells are the sole source of Chd, and anterior CNS differentiation can be completely inhibited by Chd-MO  for 5 min and incubated overnight with reaction buffer containing 10% BM purple solution (Roche) in a Coplin jar in the dark at 4°C. Staining was stopped by incubation in Stop solution , and sections dehydrated in 100% methanol and completely air-dried before mounting in Vectashield medium . In some experiments nuclear lacZ mRNA (kind gift of R. Harland), fluorescein dextran amine or Texas red dextran amine were used as lineage tracers.To fate map BCNE descendants, an improved lineage tracing method was developed. Embryos were injected with 1–4 nl of 1% BDA  in Hin MEMFA . SubsequCerS, pCS2-Chd, pCS2-β-catenin, and pCS2-dnGSK3 were linearized with NotI and transcribed with SP6 RNA polymerase as described previously  for Chd, 800 pg  for β-catenin, and 600 pg  for dnGSK3 mRNA.To generate synthetic mRNAs, the plasmids pCS2-eviously . The folChd-MO1 (5′-ACG TTC TGT CTC GTA TAG TGA GCG T-3′) and Chd-MO2 (5′-ACA GCA TTT TTG TGG TTG TCC CGA A-3′) (Nog-MO (5′-TCA CAA GGC ACT GGG AAT GAT CCA T-3′) (this work); β-cat-MO (5′-TTT CAA CCG TTT CCA AAG AAC CAG G-3′) (Cer-MO (5′-ACT TGC TGT TCC TGC ACT GTG C-3′) (this work); and a control-MO (5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′) (Chd-MO solution (Chd-MO1-SS:Chd-MO2-SS:H2O = 1:1:6), β-cat-MO solution (β-cat-MO-SS:H2O = 1:4), Nog-MO solution (Nog-MO-SS:H2O = 1:1), Cer-MO solution (Cer-MO-SS:H2O =1:1), control-MO solution (control-MO-SS:H2O = 1:1), and Chd-MO/Nog-MO solution (Chd-MO1-SS:Chd-MO2-SS:Nog-MO-SS:H2O = 1:1:4:10). A total of 8 nl (two times 4 nl or four times 2 nl) morpholino solutions were injected at the 2-cell stage or 4 nl (two times 2 nl) of morpholino solution injected at the 8-cell stage.Morpholino oligos were as follows: GA A-3′) ; Nog-MO AG G-3′) ; Cer-MO AT A-3′) . The morFigure S1(A–H) Microinjection of individual 32-cell blastomeres does not faithfully recapitulate the lineage of BCNE grafts at gastrula (compare with (I–L) Diagram indicating the injection of the lower daughter of B1 and the upper daughter of C1 at the 64-cell stage (I), which reliably identify BCNE descendants at stage 9 (J), stage 11 (K), and stage 36 (L). Arrowheads indicate the blastopore. Abbreviations: fp, floor plate; no, notochord.(4.39 MB TIF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers discussed in this paper are for β-catenin (M77013), Cer (U64831), Cer pseudoallele (BG160114), Chd (L35764), Goosecoid (M81481), and Nog (M98807).The GenBank (