The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis.3H]thymidine, [5-3H]uridine and L-leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D.E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis.The morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization. Antimicrobial proteins and peptides are widely expressed in both plants and animals. A variety of natural antibiotics belonging to different classes such as defensins, cathelicidins, cercopins and protease inhibitors  are founE. coli. Their antimicrobial activities are structure dependent and salt tolerant and their mechanism of action involves interacting with and permeabilizing bacterial membranes. However, structural evidence for the membrane changes in E. coli induced by these peptides is still lacking. Further, it is not still clear whether bacterial killing by HE2 peptides involves only membrane permeabilization or whether the peptides interact with specific targets inside the bacteria to inhibit metabolic processes as reported for other antimicrobial proteins is not yet demonstrated. In this study, using transmission and scanning electron microscopy, we provide further evidence that HE2 peptides induce significant structural changes in E. coli consistent with their membrane dependent mechanism of action as reported earlier. Further, we show that HE2 peptides inhibit E. coli DNA, RNA and protein synthesis suggesting that their antimicrobial action may also involve targets inside the bacteria as well as membrane permeabilization.In the epididymis, a major organ of the male reproductive tract, immature sperm released from the testis undergo sequential maturation to acquire forward motility and fertilizing ability. A wide variety of proteins including antimicrobial proteins released into the lumen of epididymis bind sperm and are thought to play an important role in epididymal immunity in addition to their role in sperm maturation . ExampleE. coli and purified as described previously thymidine, [5-3H]uridine and L-leucine respectively as described [6 mid-log phase E. coli resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with varying concentrations of HE2 peptides and 2.5 μl/ml of either [methyl-3H]thymidine (20 Ci/mmol), [5-3H]uridine (25.5 Ci/mmol) or L-leucine (59.5 Ci/mmol) for different time periods. After incubation, bacterial suspensions were added to 10% ice-cold trichloroacetic acid and allowed to stand in ice for 40 min. Samples were then collected on 2.4 cm GF/C glass microfiber filters  using vacuum filtration and washed thoroughly with 5% TCA and 70% ethanol. The filters were then dried and placed in scintillation vials containing 5 ml of EcoScint scintillation cocktail  and counts were obtained in a LKB 1214 Rackbeta liquid scintillation counter  for 1 min for each filter. Statistical analyses using Student's t-test were performed using Sigma Plot software . Values shown are Mean ± S.D.The effects of HE2 peptides on escribed . 1 × 106E. coli exposed to HE2 peptides. The three images shown for each treatment were documented in different fields of view and are intended to represent the range of responses seen in the bacteria. In contrast to the smooth continuous double membrane structure clearly visible in untreated bacteria ]leucine into DNA, RNA and protein was studied in the presence of 2–25 μg/ml peptides. A dose and time dependent inhibition of DNA synthesis by HE2α peptide was observed  thymidine than [5-3H]uridine and L-leucine, suggesting DNA synthesis is more sensitive to their antimicrobial action. It is possible that in bacteria that are extensively damaged by HE2 peptides, inhibition of macromolecular synthesis may result simply from the total breakdown of the cells. However the electron micrographs show that only some bacteria appear to be exuding cell contents after the 30 minute treatment. Thus during the first 10–20 minutes exposure to HE2 peptides, some peptides may be entering through pores too small for major cytoplasmic release. The early inhibition of DNA and RNA synthesis in bacteria where little loss of cell contents has occurred, may result from specific interaction of the synthetic machinery with HE2 peptides. Further studies are required to identify specific molecular targets within the bacteria and to establish whether HE2 interactions with these targets can be beneficial to the host by slowing bacterial proliferation.The mechanism of action of antimicrobial proteins is primarily thought to be membrane dependent involving membrane permeabilization and disruption. Structural characteristics of antimicrobial peptides tend to play an important role in their mechanism of action. For example, β-defensins are cationic in nature and with β-sheet rich amphipathic structures stabilized by the three disulfide motif . The catenectins , human nenectins , pleurocenectins  and the enectins . In thisIncreasing recognition of the ability of a number of proteins on the sperm surface to kill bacteria has led to the proposal that they may defend against microbial attack in both the male and female reproductive tracts. The cathelicidin hCAP18 on sperm is processed by the prostate-derived protease, gastricsin to release the active peptide ALL-38 and is found in the female reproductive tract after intercourse . A membeE. coli consistent with their membrane dependent mechanism of action [E. coli DNA, RNA and protein synthesis.In conclusion, we report that the epididymal antimicrobial peptides HE2α, HE2β1 and HE2β2 induce striking morphological changes in f action . In addiSY performed the electron microscopy studies, radioactive incorporation assays and wrote majority of the manuscript. KGH prepared the recombinant peptides. SHH and FSF supervised and coordinated the work and the preparation of the manuscript. All authors read, commented upon and approved the final manuscript.


Thus, evidence indicates that altered expression of MMPs may play a role in the aggressive phenotype seen in African American women.To date there are 26 human matrix metalloproteinases (MMPs) which are classified according to their substrate specificity and structural similarities. The four major subgroups of MMPs are gelatinases, interstitial collagenases, stromelysins, and membrane-type matrix metalloproteinases (MT-MMPs). This study investigates the expression of 26 MMPs, which have been shown to play a role in cancer metastasis. Breast tissues and cell lines derived from African American patients and Caucasian patients were assayed to demonstrate alterations in the transcription of genes primarily responsible for degrading the extracellular matrix (ECM). The expression levels of the extracellular matrix and adhesion molecules were analyzed using the gene array technology. Steady state levels of mRNAs were validated by RT-PCR analysis. Total RNA was isolated from tissue and cell lines and used in the RT-PCR assays. From this data, differential expression of MMPs between 6 breast cancer cell lines and 2 non-cancer breast cell lines was demonstrated. We have performed an  In 2003, it was estimated that approximately 1.3 million Americans would be diagnosed with invasive cancer. Of this group, racial/ethnic minorities account for a disproportionate number of these cancers . Thus a The extracellular matrix (ECM) is a complex structural entity surrounding and supporting organs and tissues of the body. The ECM plays a key role in cell-cell signaling, wound repair, cell adhesion and tissue function. Recent studies suggest that cell adhesion proteins located on breast cancer cells interact with the ECM . This inThe matrix metalloproteinases (MMPs) are a family of structurally and functionally related endoproteinases that are involved in the degradation of the ECM. Currently, there are 26 identified human matrix metalloproteinases, which are classified according to their substrate specificity and structural similarities . AbnormaMMP expression has bee shown to be elevated during development, pregnancy, and involution and has been shown to be related to tumor cell invasiveness . This stAll cell lines were purchased from American Type Culture Collection . Cells were propagated in the recommended media and given new media every 2 to 3 days until 90% confluent . Cells were collected by low speed centrifugation and lysed by adding 200 μl of Lysis/Binding Solution. An equal volume of 64% ethanol was added to the lysate. The lysate/ethanol mixture was transferred to the RNAqueous Filter Cartridge and centrifuged for 1 minute at 13,400 rpm. The flow through was discarded and 700 μl of Wash Solution 1 was added to the RNAqueous Filter Cartridge and centrifuged for 1 minute. The column was washed twice with 500 μl of Wash Solution 2/3 and eluted with 110 μl Elution Solution. Isolated RNA was quantitated using the UltraSpec 2000 (Pharmacia Biotech). All RNA samples were stored at -70°C in RNA elution solution until further use.The Extracellular Matrix and Adhesion Molecule gene arrays were obtained from SuperArray . The array membranes were pre-hybridized with GEA hybridization solution and denatured salmon sperm DNA at 60°C for two hours. For each RNA sample, a labeling mix consisting of 4 μl 5X GEA labeling buffer, 2 μl biotin-16-dUTP, 1 μl RNase inhibitor, 1 μl reverse transcriptase, and 2 μl RNase-free water was prepared and an aliquot of 3 μg of RNA was added to each respective thin-walled PCR tube. The cDNA labels were created using a cycle of 3 minutes at 70°C, 2 minutes at 42°C, and an additional 90 minutes at 42°C. Two microliters of stop buffer was added and the mix denatured at 94°C for 5 minutes. The labeled cDNA was added to the membrane and allowed to hybridize overnight. The membranes were washed with 2X SSC/0.1% SDS and 0.1X SSC/0.5% SDS, blocked with blocking solution, and the probes were detected using AP-Strepavidin, specific buffers, CDP-Star and subsequent exposure to X-ray film for 30 seconds to 5 minutes. The autoradiograms were analyzed using ScanAlyzer and GEArray Analyzer .4, 1.0 μl AMV reverse transcriptase, Tfl DNA polymerase and 1 μg of total RNA in a 0.5 ml thin walled Eppendorf tube on ice. The reaction mixes were then vortexed for 5 seconds and centrifuged. The PCR cycling profile was as follows: 48°C for 1 minute for reverse transcription of the RNA into cDNA, 94°C for 4 minutes to deactivate the reverse transcriptase, and 30 cycling sequences of denaturing at 94°C for 45 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute with a final extension at 72°C for 10 minutes. An aliquot of 20 μl of each RT-PCR reaction were run on 1.2% agarose gels, stained with ethidium bromide, photographed and subjected to densitometic measurements using the Chemi-Imager Tm 4000 .The RT-PCR reactions were performed in a P/E GeneAmp 9700 thermocycler , using the Access RT-PCR system . The reaction mixes were prepared by combining 27. 5 μl of nuclease free water, 10 μl of AMV, 1 μl Tfl 5X reaction buffer, 2 μl dNTP mix, 50 pM of upstream primer, 50 pM of downstream primer in 1.5 μl volume each, 3 μl 25 mM MgSOGene arrays were utilized to explore and compare the expression levels of extracellular matrix adhesion molecules in AA and CAU breast cancer cells. The array revealed elevated expression in 36% of the genes in AA samples when compared to their CAU counterparts. Of those elevated genes, 31% were from the cell membrane adhesion molecules group, 17% from the extracellular matrix proteins functional gene group, 37% from the proteases category, and 14% were protease inhibitors. Initial results of the gene array indicated a significant elevation of the proteases (data not shown). To further evaluate this, we proceeded with direct analysis of all known MMPs.Comparison of the individual relative densities between AA and CAU women revealed elevated expression in 12 of the 26 MMPs  .Gene Array analysis of AA and CAU breast cancer cells indicates that there is altered expression of the genes in the Extracellular matrix and adhesion molecules, particularly the proteases. This group included 17 MMPs of which ten displayed elevated expression in AA women.RT-PCR was performed to confirm the results of the gene array. We observed elevated expression of 12 MMPs in AA cell lines when compared to their CAU counterparts. These include one gelatinase (MMP-9), two interstitial collagenases , 3 stromelysins , two MT-MMPs (MMP-14 and -15) and 4 uncategorized MMPs . There was no MMP-17 expression detected in any of the cell lines (Figure Studies have shown that normal mammary gland expression of MMPs is low except during times of development, pregnancy, and involution ,15,16. HOur results suggest that there is altered expression of MMPs in cell lines derived from AA and CAU women. It also demonstrates that there is greater expression of MMPs in AA women than in CAU women. This investigation indicates that altered expression of MMPs may play a role in the aggressive phenotype seen in AA women. This evidence suggests that the elevated expression levels of 12 MMPs may be a contributing factor in the higher mortality rates of AA breast cancer patients. This study is significant because it may reveal biomarkers of metastasis in AA women. To date, this is the first study to extremely investigate MMP expression in cell lines derived from African American patients.MMP-Matrix metalloproteinases, MT-MMP-Membrane-type matrix metalloproteinases, AA-African American women, CAU-Caucasian, RT-PCR-Reverse transcriptase Polymerase Chain Reaction, ECM-Extracellular matrixJAM performed the microarrays and RT-PCR experiments, was involved in tissue culture and prepared the first draft of the manuscript. HFY was responsible for primer design, and performed data analysis and densitometric readings of the gene arrays and RT-PCR. KL maintained all cells and tissues and assisted in the editing of this manuscript. MJ provided cell lines, training in microarray performance and editing. AAD conceived the study and participated in its design, coordination and funding, as well as preparation of the manuscript. All authors read and approved the final manuscript.


Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information.Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions.et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the Bayesian methods decreased classification performance.In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO) functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen  A number of techniques have been developed that exploit combinations of protein features in training data and can predict protein-protein interactions when applied to novel proteins. Our study is motivated by a study by Jansen et al. [ provides more details about the quantification of these variables. Their Bayesian method predicts protein-protein interactions genome-wide by probabilistic integration of genomic features that are weakly associated with interactions . The model was used for two separate predictions of probabilistic interactomes (PI), one of which (PIE) is built on four high-throughput experimental interaction data sets, and the other (PIP) on the mRNA expression, Gene Ontology, MIPS functional and coessentiality data. Within the PIP sub-network, different genomic features are assumed to be independent in prior. In addition, this method involved discretizing the raw data into groups and representing the two mRNA expression profiles (cell cycle and Rosetta compendium data) by their first principal component for computational convenience.Proteins transmit regulatory signals throughout the cell, catalyze large numbers of chemical reactions, and are crucial for the stability of numerous cellular structures. Interactions among proteins are key for cell functioning and identifying such interactions is crucial for deciphering the fundamental molecular mechanisms of the cell. As relevant genomic information is exponentially increasing both in quantity and complexity, n et al. , who pron et al.  complexen et al.  as gold n et al.  and the n et al. , biologin et al.  and the n et al. , and hign et al. -10. The Our current study focuses on assessing the contributions of different types of genomic data towards predicting protein-protein interactions. This may help us to understand which genomic features have the closest biological relationship with protein-protein interactions and hence to construct a better prediction model. As prediction rules involving less relevant information may have lower prediction accuracy, our analysis can give us insights into how to construct more parsimonious models with comparable or better prediction accuracy. A potential disadvantage of the Bayesian network approach may be that the data discretization can obscure information contained in the raw genomic data. Thus, in addition to assessing the information content of the data sources, we also propose alternative non-Bayesian models that fully utilize the data without discretization. These methods, such as logistic regression and random forests, do not require prior knowledge, and we can evaluate the importance of the different genomic features in the context of these methods.. In doing so, we find that some of the features have stronger influence on the overall prediction. While this might be true for the larger problem as well, there are a number of caveats that one has to keep in mind, such as that the features that are present in this subset might not be the strongest in the whole set of 18 million protein pairs.To accurately and quantitatively assess the information contributions of different genomic features, we construct in essence a simplified problem that has some but not all of the elements of the original study. Here, we only look at a subset of the data from  comprisiHere, we construct models for predicting protein-protein interactions that, given the gold standards, are basically dichotomous classifiers. Multiple logistic regression  is one cThe logistic model has the advantage that it provides an estimated probability that a pair of proteins interact, and is readily available in standard statistical packages. In this paper, the logistic regression analysis was generated using PROC LOGISTIC in SAS/STAT software, Version 9 of the SAS System. Moreover, we can evaluate the importance of different genomic features by variable selections. Among many available schemes, we chose stepwise variable selection that is widely used in standard packages. Stepwise selection is a greedy search algorithm that selects variables with the best marginal prediction power given the current model. To quantify the importance of the predictor variables to the model fitting, we can use the deviance measure1 - log L0),-2 on all the features for random forest, logistic regression and the Bayesian network method. Figure Information assessment of different genomic data may help us understand their relationship with protein-protein interactions, and form a guideline for future model development.We saw that the MIPS and Gene Ontology functional similarities were the two most important information sources under both the logistic regression and random forests methods. Histograms of the MIPS and GO functional similarity data Figures  and 4 shErr) defined as follows.In the following paragraphs, we show quantitatively that the MIPS and Gene Ontology functional similarities are the dominating information contributors for predicting protein-protein interactions, while other genomic features have negligible benefit and can not provide credible predictions by themselves. We examine the performance of random forests using three different genomic feature sets: (i) all genomic features included, (ii) MIPS and Gene Ontology functional similarities only, and (iii) genomic features other than the MIPS and Gene Ontology functional similarities. The random forest performance is evaluated with the classification error  and f2(X) for the gold standard positives and negatives, respectively. Denote a classifier by C(X). Then the classification error can be written asI(A) is an indicator function equal to 1 when A is true and 0 otherwise. A minimal classification error Errmin can be computed by minimizing (1) across the space of X. It is easy to see thatwhere C(X) = I(f1(X) >f2(X)). With this formula, we can estimate the optimal (minimum) classification error based on any estimates of f1(X) and f2(X). In our study, f1(X) and f2(X) are estimated by their empirical density functions.is achieved at Table We also estimated the classification errors using the other genomic features within the random forest framework. Table Moreover, we compared the ROC curves Figure  of the rIn the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed in . Random We considered alternative non-Bayesian methods such as logistic regression and random forest for predicting protein-protein interactions. These existing methods do not require prior information needed for the Bayesian approach, and can fully utilize the raw data without discretization. The logistic model performs similarly as the Bayesian method in terms of classifications and, like the Bayesian method, produces estimated probabilities that two proteins interact. As a dichotomous classifier, the random forest method outperforms the other methods considered and efficiently uses the information, although it is computationally more expensive. In particular, its importance measure provides a more objective assessment of different genomic features on predicting protein-protein interactions than simply considering contributions to model fitting. These findings are motivation to look for other, more sensible data resources and superior models.We found that the data discretizations used in the Bayesian methods decreased classification performance. We note here that the genomic features datasets investigated here themselves are highly processed versions of the datasets they were derived from and that there may be better ways to take the original data into account.Another caveat is that the predictions might be just defining groups of proteins that have the same genomic properties as the protein complexes in the MIPS data. This does not necessarily mean that they really represent protein complexes. Rather, they may represent groups of proteins that have the same properties as protein complexes.In this analysis we have looked at the relative weights of various features in predicting protein-protein interactions based on the previous study in . We lookX1, X2, ..., Xn. Let Y = 1 when two proteins interact, i.e., they are in the same complex, and Y = 0 when not. The logistic model is of the formDenote the gold standards by random variable Y and the other genomic features by X consists of X1, X2, ..., Xn and their interaction terms.where the random vector The stepwise selection procedure starts from a null model. At each step, it adds a variable with the most significant score statistics among those not in the model, then sequentially removes the variable with the least score statistic among those in the model whose score statistics are not significant. The process terminates if no further variable can be added to the model or if the variable just entered into the model is the only variable removed in the subsequent elimination. Here, the score statistic measures the significance of the effect of a variable.sensitivity and specificity. Sensitivity is calculated by dividing the number of true positives (TP) through the number of all positives, which equals the sum of the true positives and the false negatives (FN); specificity is calculated by dividing the number of true negatives (TN) through the number of all negatives, which equals the sum of the true negatives and the false positives (FP).Receiving operator characteristic (ROC) curve  is a graSensitivity = TP/(TP + FN), Specificity = TN/(TN + FP).specificity on the X axis and sensitivity on the Y axis. A good classifier has its ROC curve climbing rapidly towards upper left hand corner of the graph. This can also be quantified by measuring the area under the curve. The closer the area is to 1.0, the better the classifier is; and the closer the area is to 0.5, the worse the classifier is.The ROC curve plot shows 1 - NL and BW conducted the major part of the data analysis, and created all the tables and figures, under the supervision of HZ. RJ and MG provided the data sets for the analysis and contributed to the discussion on the comparisons of different methods. All authors read and approved the final manuscript.The complete-information subset in ZIP file.Click here for file


The plastic response of fine roots to a changing environment is suggested to affect the growth and form of a plant. Here we show that the plasticity of fine root growth may increase plant productivity based on an experiment using young seedlings (14-week old) of loblolly pine. We use two contrasting pine ecotypes, "mesic" and "xeric", to investigate the adaptive significance of such a plastic response.The partitioning of biomass to fine roots is observed to reduce with increased nutrient availability. For the "mesic" ecotype, increased stem biomass as a consequence of more nutrients may be primarily due to reduced fine-root biomass partitioning. For the "xeric" ecotype, the favorable influence of the plasticity of fine root growth on stem growth results from increased allocation of biomass to foliage and decreased allocation to fine roots. An evolutionary genetic analysis indicates that the plasticity of fine root growth is inducible, whereas the plasticity of foliage is constitutive.Results promise to enhance a fundamental understanding of evolutionary changes of tree architecture under domestication and to design sound silvicultural and breeding measures for improving plant productivity. In anPhenotypic plasticity is the potential of an organism to alter its phenotype in changing environments -19. PhenLoblolly pine is the most important tree species for fiber production in the southern US . BecauseP < 0.001). These values increased to 102% and 199% for these two ecotypes, respectively, when the trees were treated for 14 weeks (P < 0.001).After 4 weeks of treatment, trees receiving the high nutrient treatment displayed 22% ("xeric") and 47% ("mesic") greater stem biomass than those under the low fertilizer treatment (P < 0.01), as compared to the significance level when the proportion was not held constant (P < 0.0001). These dependent relationships suggest that increased stem biomass due to better nutrient supplies was attributable to both increased foliage investment and decreased energetic costs of fine root construction.Allometric analysis was used to evaluate the influences of foliage and fine-root biomass partitioning on stem growth which arise from differences in nutrient supply. On both harvesting dates, the proportion of foliage biomass to total plant biomass increased markedly, whereas the proportion of fine root biomass decreased significantly, with better nutrient supplies. As an illustration, we use Figure py←1 = -0.09), whereas the plasticity of fine-root biomass partitioning, i.e., decreased partitioning of biomass to fine roots under higher fertilization, had a significant impact on the corresponding increase of stem biomass . Plasticity between different growth stages indicates the dependence of plastic responses on the timing and sequences of developmental events. Ecotypic variation in developmental plasticity represents different genetic bases involved in relevant developmental events [Foliage and fine roots have complementary roles in uptake of resources; the former in energy and carbon uptake and the latter in water and nutrient uptake ,22. Mechl events .Ecotypic differentiation of loblolly pine could be explained by limits of plasticity. Quantitative evolutionary genetic models predict that the phenotypic plasticity of a trait is costly or physiologically limiting when the trait is forced to respond to environmental variation ("passive" response). DeWitt et al.  delineatOur analysis suggests that the plasticity of fine-root biomass proportion is physiologically limiting, whereas the plasticity of foliage biomass proportion is not. The relationship of the shoot biomass residuals was positive with the degree of plasticity of foliage biomass proportion Fig. , but negForest tree form (biomass partitioning) is highly plastic in response to changes in nutrient levels. The carbon budgets for forest trees show a surprisingly large role of roots. Under low nutrient conditions that predominate in natural forests, 60–80% of photosynthate is allocated below ground, compared with 30% for high nutrient levels [Progress towards domestication in trees will be slowed by long generation times, but is likely to be based on the exploitation of interactions between genotypes and yield associated with various types of agronomic methods , as have been shown for herbaceous crop plants. However, the environmental uncertainties during the long life span of trees have caused some breeders to consider the value of plasticity as a trait itself. And plasticity could obscure the relationship between phenotype and genotype, making selection less efficient. Efforts to domesticate forest trees will be enhanced by a deeper knowledge of phenotypic plasticity .Our study of biomass partitioning in relation to varying nutritional levels in loblolly pine supports the previous hypothesis, proposed by Linder and Axelson , that thPinus taeda L.). We used two contrasting loblolly pine ecotypes from regions that differ in soil resource availability. One of the ecotypes, known as the "Lost Pines" of Texas, is adapted to droughty conditions and low soil fertility and is denoted by "xeric", whereas the other, Atlantic Coastal Plain, is adapted to more moderate conditions and is denoted by "mesic". Adaptive differentiation between the contrasting "xeric" and "mesic" ecotypes has been previously characterized [Phenotypic plasticity was evaluated for fine roots and biomass partitioning of a commercially important forest tree species, loblolly pine  and fine root biomass (py2←) to stem biomass through the change of nutritional levels were estimated by solving the following regular equations:Path analysis was used to identify the cause-effect relationships in a complex system . Path anpy 1←+ r12py 2←= ry1r12py 1←+ py 2←= ry2ry 1and ry 2are the family correlation coefficients of the plasticity of foliage biomass and fine-root biomass with the plasticity of stem biomass, respectively, and r12 is the family correlation coefficient between the plasticity of foliage biomass and fine-root biomass. Residuals were estimated to evaluate the degree of determination for the path analysis [where analysis . All datRW designed the study, carried out the experiment, analyzed the data and drafted the manuscript. JEG participated in the experiment. SEM participated in the design of the study. DMO participated in the design and coordination. All authors read and approved the final manuscript.


We present a biological data warehouse called Atlas that locally stores and integrates biological sequences, molecular interactions, homology information, functional annotations of genes, and biological ontologies. The goal of the system is to provide data, as well as a software infrastructure for bioinformatics research and development.The Atlas system is based on relational data models that we developed for each of the source data types. Data stored within these relational models are managed through Structured Query Language (SQL) calls that are implemented in a set of Application Programming Interfaces (APIs). The APIs include three languages: C++, Java, and Perl. The methods in these API libraries are used to construct a set of loader applications, which parse and load the source datasets into the Atlas database, and a set of toolbox applications which facilitate data retrieval. Atlas stores and integrates local instances of GenBank, RefSeq, UniProt, Human Protein Reference Database (HPRD), Biomolecular Interaction Network Database (BIND), Database of Interacting Proteins (DIP), Molecular Interactions Database (MINT), IntAct, NCBI Taxonomy, Gene Ontology (GO), Online Mendelian Inheritance in Man (OMIM), LocusLink, Entrez Gene and HomoloGene. The retrieval APIs and toolbox applications are critical components that offer end-users flexible, easy, integrated access to this data. We present use cases that use Atlas to integrate these sources for genome annotation, inference of molecular interactions across species, and gene-disease associations.The Atlas biological data warehouse serves as data infrastructure for bioinformatics research and development. It forms the backbone of the research activities in our laboratory and facilitates the integration of disparate, heterogeneous biological sources of data enabling new scientific inferences. Atlas achieves integration of diverse data sets at two levels. First, Atlas stores data of similar types using common data models, enforcing the relationships between data types. Second, integration is achieved through a combination of APIs, ontology, and tools. The Atlas software is freely available under the GNU General Public License at:  One important goal in bioinformatics is to integrate data from disparate sources of heterogeneous biological information. Data integration allows us to assemble targeted data reagents for bioinformatics analyses, and to discover scientific relationships between data. Most public repositories of biological data focus on deriving and providing one particular type of data, be it biological sequences  the source data, 2) the ontology system, 3) the relational data models, 4) the APIs, and 5) the applications  controlled vocabulary . The Atl.The Atlas internal ontology exists largely to help describe Sequence Features as they exists in the GenBank Sequence Feature model, as this is the primary data source for features. Neither the Open Biological Ontologies (OBO)  relationsequence, which stores the sequence string and associated meta-data such as sequencing technique and molecule type; sequence identifiers, for which all identifiers that appear in the records are stored  ), The Atlas system is also being used to determine yeast orthologs of genes that are implicated in human disease (Hieter P: in preparation). The inference being that human genes for which there are yeast orthologs represent essential genes which are candidates for human disease agents. Compiling the reagents for this custom database was straightforward using the Atlas tools. It takes advantage of the linkage between sequence identifiers, Taxonomy, HomoloGene, and OMIM.We have built a data warehouse of biological information with the goal of providing high-throughput, flexible access to the data by means of SQL queries, API-level queries, and end-user application-level queries. Our goal was to create a system that serves as a platform through which information from many sources of data can be interrogated, enabling biologists and computer scientists to easily carry out queries necessary for their research. The data warehouse facilitates complex queries on local instances of GenBank, RefSeq, UniProt, HPRD, BIND, NCBI Taxonomy, HomoloGene, Gene Ontology, OMIM, Entrez Gene, and LocusLink. With previously disparate data now unified in a relational model, SQL can be used to retrieve this consolidated information at once. Though Atlas can act to serve data publicly over the internet, its simple setup enables anyone or any institution to easily serve their own customized data warehouses to their own local users. Installing Atlas in-house to serve local users gives the data provider full control over the data they serve. Giving users access to the system on a high-bandwidth internal network offers convenience and high-performance for large queries, such as retrieving all human ESTs. Such data is then more readily retrieved with lower latency and higher bandwidth than attempting to retrieve the same data over the internet.One of the important strengths of the Atlas architecture is that it allows data integration at two levels. The first level uses a common data model to integrate similar types of data from different sources . The second level uses the APIs, ontologies, and tools to cross-reference disparate types of data.vertebrata), from the RefSeq database. With a single call to the taxonName2Sequences method, the user can accomplish this task. Within these API methods are SQL statements which first retrieve the taxonid from the Taxonomy database. Then using a recursive method, the taxon identifiers for all organisms beneath that given taxon node, are returned. All amino acid sequences for each of these taxon identifiers are then retrieved using taxonId2Sequences , thus leaving only one instance of a factoid in our database. However, as it would not necessarily be our goal to judge the correctness of data, this is perhaps a task better left to users of our system.Several other systems are available which have similar goals and provide good solutions to the problem of data integration. We have chosen to discuss Atlas in the context of three other systems: Entrez , SeqHounSeqHound  is a datEnsMart is a software system designed by EMBL-EBI  and the per se, but rather an engine that should be accessed 'in-house'. As with any locally-installable system of this nature, significant time and hardware resources are needed to make the system functional. The utility of the Atlas system will far outweigh the setup time required to get it up and running. Currently, API access to Atlas is limited to the users at the UBC Bioinformatics Centre, University of British Columbia, however the web tools are available worldwide.The Atlas system is designed to be locally installed and is not a data provider When working with sources of data from different data providers (for example UniProt and RefSeq), it is advantageous to create mappings from one data source to the other to prevent redundancy and to make associations between proteins to map annotations from one source to the other. We are investigating the idea of an identifier consolidation that can resolve mRNAs and proteins from different sources that are referring to the same protein product to a single identifier.We will constantly monitor and adjust any change of the data sources. In the near future, we will provide support for a PSI-MI level 2 release, and complete the migration of LocusLink to Entrez Gene. In addition, we are expanding Atlas to include other sources of data. We are currently adding MEDLINE, dbSNP and pathway data to support an integrative genomics and clinical informatics initiative, currently underway in our laboratory. With Atlas in hand, we are also working on an integration project that superimposes co-expression networks derived from microarray experiments and protein-protein interaction networks, to estimate the utility of co-expression networks in inferring protein interactions.Atlas is a data warehouse that enables high-throughput, flexible and complex queries on biological data. The system integrates sequences, molecular interactions, taxonomy and homology, and functional annotations on genes. The system functions as data infrastructure to support bioinformatics research and development. Atlas is currently being used in genome annotation projects, disease-gene association projects and inference of molecular interactions. We are releasing Atlas to the scientific community in the hope that it will foster creative ideas for how to make novel associations between disparate sources of data using existing public data sets.Atlas is available from the UBC Bioinformatics Centre, University of British Columbia. The Atlas package can be downloaded from the Atlas website at: The Atlas package contains the Atlas source code and represents the core of the project. The package is distributed under the GNU General Public License. Atlas is designed to run on Unix based systems. Please consult the user manual (available with the distribution) for detailed configuration, compilation and installation instructions. Additional packages are also provided at the website listed above. These packages include a snapshot of the NCBI C++ Toolkit (CVS version 20040505), a MySQL dump of sample data, and additional documentation. The NCBI C++ Toolkit, that is provided, is required only for those users who wish to build the loader applications or for those that require the utilities that convert ASN.1 format to GBFF, EMBL, and XML formats, etc. Those setting up the database will need to install MySQL Server 4.x. Atlas has been tested, specifically, with MySQL Server versions 4.0.9, 4.0.18 and 4.0.20, running on either Linux or AIX.The Atlas sequence-related binaries (toolbox applications and loader applications) are developed in C++ and therefore a C++ compatible compiler, such as the one included with the GNU GCC suite of tools, should be installed before attempting to build these binaries. We have tested the build process with GNU GCC versions 2.95.3, 2.96, 3.1 and 3.2. In addition, MySQL Client version 4.x and particularly its runtime library, libmysqlclient.a(so), is required. MySQL Client versions 4.0.14 and 4.1.0-alpha were tested. Details on the configuration and use of this library are outlined, in more detail, in the Atlas manual.For users that require Atlas tools that are based on Java, such as the loading and retrieval tools for LocusLink, BIND, HPRD, and HomoloGene datasets, a compatible Java interpreter must be installed. The API has been tested with J2SE 1.4.1 and J2SE 1.4.2. The Atlas Java API also requires BioJava version 1.4pre, or higher.For those using the Perl based Atlas tools, a compatible Perl interpreter must be installed. BioPerl version 1.4 must also be installed. Perl version 5.6.1 has been tested.Each of the packages have their own minimum system requirements. Specific memory, hard disk space and CPU requirements for each package are listed in the manual. As a general guideline, it is essential to have a generous amount of available memory, especially if one anticipates processing large sequences in memory. Another important factor is the amount of available hard disk space. The amount of sequence data to be loaded into Atlas will largely determine your disk space requirements. The Atlas database requires a minimum of 50 GB (RefSeq), plus adequate space for satellite databases. The satellite databases include such things as GO, LocusLink, HPRD, BIND, MINT, and DIP, which are relatively smaller datasets. Note that sequence data can greatly exceed these minimum estimates and the requirements should be carefully planned.SS was the architect of the system, developed the C++ APIs and wrote the first draft of this manuscript. YH was the database administrator responsible for schema design, data integrity and maintenance. TX contributed the Java APIs. MMSY contributed the PERL APIs. JL developed the C++ APIs, the toolbox and the user manual. BFFO was the principal investigator, conceived of the project and guided its development. JL, YH, MSSY and BFFO all contributed to the writing of this manuscript.


Prophylactic treatment with N-acetylcysteine (NAC) for 3 months or more is associated with a reduction in the frequency of exacerbations of chronic obstructive pulmonary disease (COPD). This raises the question of whether treatment with NAC during an acute exacerbation will hasten recovery from the exacerbation.1 ≤ 60% predicted, FEV1/VC ≤ 70% and ≥ 10 pack year smoking history. Subjects with asthma, heart failure, pneumonia and other respiratory diseases were excluded. All subjects received concurrent treatment with prednisone 40 mg/day, nebulised salbutamol 5 mg q.i.d and where appropriate antibiotics. FEV1, VC, SaO2 and breathlessness were measured 2 hours after a dose of nebulised salbutamol, at the same time each day. Breathlessness was measured on a seven point Likert scale.We have examined this in a randomised, double-blind, placebo controlled trial. Subjects, admitted to hospital with an acute exacerbation of COPD, were randomised within 24 h of admission to treatment with NAC 600 mg b.d. (n = 25) or matching placebo (n = 25). Treatment continued for 7 days or until discharge (whichever occurred first). To be eligible subjects had to be ≥ 50 years, have an FEV1 (% predicted) was 22% in the NAC group and 24% in the control group. There was no difference between the groups in the rate of change of FEV1, VC, SaO2 or breathlessness. Nor did the groups differ in the median length of stay in hospital (6 days for both groups).At baseline FEVAddition of NAC to treatment with corticosteroids and bronchodilators does not modify the outcome in acute exacerbations of COPD. Exacerbations are an important cause of morbidity in Chronic Obstructive Pulmonary Disease. Seemungal et al found that exacerbations were an important determinant of quality of life in COPD . In addi1 ≤ 60% predicted and FEV1/VC ≤ 0.7 at time of inclusion. Patients were excluded if they had any of the following conditions: asthma (as the primary diagnosis), heart failure, bronchiectasis, bronchial carcinoma, interstitial lung disease, pneumonia. They were also excluded if they were unable to comply with the study procedures because they did not speak English or were demented or if they had any other medical problems that in the opinion of the investigator would interfere with the conduct of the study.Patients were eligible for inclusion in the study if they had a physician diagnosis of COPD, were ≥ 50 years of age, had a smoking history ≥ 10 pack years and had been admitted to hospital with an acute exacerbation of their COPD in the previous 24 hours. In addition they were required to have FEVSubjects were treated with N-acetylcysteine 600 mg twice daily (b.d.) or matching placebo. Treatment was continued for 7 days or until discharge whichever occurred first. Effervescent N-acetylcysteine tablets were purchased from Zambon pharmaceuticals  and were repackaged in size 0 gelatine capsules. Each capsule contained 300 mg of N-acetylcysteine. Placebo capsules were prepared containing lactose as a filler. A randomisation schedule was drawn up by the hospital pharmacy and patients were allocated sequential randomisation numbers as they entered the study.In addition to N-acetylcysteine the patients received standard treatment for their exacerbation as specified by the hospital guidelines. This was oxygen therapy, prednisone 40 mg o.d. for one week and nebulised bronchodilators i.e. salbutamol 5 mg four times daily (q.i.d.) and ipratropium 0.5 mg q.i.d. Antibiotics were prescribed if the patients had increased volume and/or purulence of sputum. Mucolytics were not permitted during the trial except as study medicine.1, oxygen saturation and length of hospital stay. The study assessments were performed at the same time each day and two hours after the last dose of nebulised bronchodilator. Breathlessness was assessed prior to spirometry. Spirometry was performed according to American Thoracic Society criteria using a Vitalograph spirometer. Oxygen saturation was measured using a Nelcor N-20 pulse oximeter. Supplemental oxygen was stopped for 10 minutes before any of the measurements were performed. In addition the subjects were interrogated on each occasion about any possible adverse effects.The primary endpoint was breathlessness measured on a seven point Likert scale Table . SecondaThe study was conducted at Auckland Hospital between June 2001 and October 2001. The study was approved by the Auckland Ethics Committee and all participants provided written informed consent.The power calculation was based on a previous study where we treated subjects for acute exacerbations of COPD with theophylline for up to seven days . In thatThe two treatment groups were compared at baseline using Student's t-test for normally distributed variables, Wilcoxon test for non-parametric data and Fisher's exact test for categorical data.The effect of N-acetylcysteine on breathlessness, lung function and oxygen saturation was analysed by fitting the patient data to a random coefficient model using a mixed linear model approach (least squares regression). The baseline (Day 1) measurements were used as co-variates in the analysis. This allowed individual slopes and intercepts to be formed for each patient and their random variation incorporated in the model. This model adjusted for the varying number of observations available on the different patients. Non-normal dependent variables were rendered normal by transformation. Significant and main interaction effects were investigated by the method of Tukey.All tests were two-tailed and a 5% significance level was maintained throughout these analyses. The analyses were carried out using SAS Version 8.0 The life test procedure of SAS was used to compute nonparametric estimates of the length of stay function by the Kaplan-Meier method. Comparison between these functions was made using the Wilcoxon and log rank tests.Two hundred and ten patients who had been admitted to hospital with an exacerbation of COPD were screened for the study. Fifty subjects were randomised to treatment with 25 subjects receiving N-acetylcysteine and 25 subjects receiving placebo. The commonest reasons for exclusion were concomitant heart failure (n = 41) or pneumonia (n = 35).The two groups were similar at baseline in terms of age, smoking history, lung function, oxygen saturation and breathlessness Table . There w1, and SaO2 the rate of change was greater with placebo than with N-acetylcysteine but none of these differences were statistically significant. Table 1 and SaO2 from the beginning to end of the study.All of the subjects completed the study with none being withdrawn early. The rate of change in the Likert scores, lung function and oxygen saturation is shown in Table The Kaplan-Meier analysis showed a similar time course until discharge for the treatment and placebo arms Figure . NeitherThree subjects reported adverse events in each group. One of the subjects treated with N-acetylcysteine reported nausea compared with two of the subjects treated with placebo. There were no serious adverse events.N-acetylcysteine has been consistently shown to reduce the number of exacerbations of COPD when it is taken for 3 months or more. In contrast we failed to show any benefit when N-acetylcysteine was administered as a treatment for acute exacerbations of COPD. There are a number of possible explanations for the failure to see any benefit.We cannot exclude the possibility that there was a Type II error and that there is indeed a beneficial effect of N-acetylcysteine in the treatment of acute exacerbations. A larger study would be needed to rule out this possibility but there was less improvement in breathlessness, lung function and oxygen saturation with N-acetylcysteine than with placebo that argues against this explanation.Another possibility that needs to be considered is that we used too low a dose of N-acetylcysteine and that the concentrations of N-acetylcysteine in the lung were not high enough to exert adequate antioxidant or anti-inflammatory effects. N-acetylcysteine is metabolized to cysteine and this in turn acts as a precursor of reduced glutathione which is an antioxidant . BridgemIn contrast to N-acetylcysteine, prednisone and prednisolone are effective treatments for acute exacerbations of COPD. Davies et al studied 56 patients admitted to hospital with an exacerbation of COPD and found that prednisolone led to a greater improvement in lung function and shortened the hospital stay . Other s1 and VC. Sputum volumes, however, were greater with the combination of the mucolytic and antibiotic. Ricevuti et al treated 24 patients with an exacerbation of chronic bronchitis [There have been a number of other studies looking at the effect of mucolytics in acute exacerbations of chronic bronchitis although none of these studies used N-acetylcysteine. Each of these studies has limitations. Langlands treated 27 patients, who had been admitted to hospital with an exacerbation of chronic bronchitis, with bromhexine 8 mg t.i.d. for two weeks (13 patients received bromhexine and 14 received placebo) . In thisonchitis . Half ofOur study does not suggest that 600 mg b.d. of N-acetylcysteine is effective in the treatment of patients who are admitted to hospital with an acute exacerbation of COPD and who receive concurrent treatment with corticosteroids. In future studies it may be appropriate to use a higher dose of N-acetylcysteine and to compare N-acetylcysteine with placebo in patients with mild exacerbations who do not require treatment with corticosteroids. Consideration could also be given to comparing the effects of N-acetylcysteine with prednisone or prednisolone in patients who would usually be treated with oral corticosteroids.NAC N-acetylcysteineCOPD chronic obstructive pulmonary disease1 forced expiratory volume in one secondFEVVC vital capacity2 oxygen saturationSaOThe author(s) declare that they have no competing interests.PNB conceived the idea for the study and was responsible for the study design and writing the manuscript. He was also was involved in the conduct of the study and the analysis of the data. AM-D was involved in the conduct of the study, in the analysis of the data and with writing the manuscript. PJP was involved with the design and conduct of the study. TEM and RPY were involved with the conduct of the study. All of the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs.Sphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium activity of a series of ceramide analogs containing different linkages  between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP . This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite.The anti-Plasmodium activity (IC50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target.Analogs containing methylene linkage showed a considerably higher anti-P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.It has been shown that ceramide analogs were potent inhibitors of  Sphingolipids are essential components of eukaryotic cell membranes, predominantly found in the outer leaflet. Sphingosine and ceramide Figure  are the Until recently, sphingolipids were primarily considered to be structural components of membranes. However, data accumulated during the last decade have expanded the view of their biological functions. They are now also considered to be key molecules which regulate many functions essential to eukaryotic cells -5. They Ceramide plays a central role in sphingolipid metabolism . It can Moreover, ceramide can be phosphorylated by a distinct kinase and can also be produced by enzymatic hydrolysis of complex sphingolipids. In turn, ceramide can be hydrolyzed to sphingosine and fatty acid by ceramidases.Plasmodium falciparum, the causative agent of malaria, is still limited. Gerold et al.  hypoxanthine  per well. After a further incubation of 24 hour, plates were frozen and thawed. Cell lysates were then collected onto glass-filter papers and counted in a liquid scintillation spectrometer. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated in the treated culture with that in the control culture  maintained on the same plate. The concentration causing 50% growth inhibition (IC50) and 90% growth (IC90) were obtained from the drug concentration-response curve and the results were expressed as the means ± the standard deviations determined from several independent experiments. The DMSO concentration never exceeded 0.1% (v/v) and did not inhibit the parasite growth.Drug susceptibility assays were performed using a modification of the semi automated microdilution technique of Desjardins et al. . Stock sper well in 100 μl. After 24 hours, the cells were washed and two-fold dilutions of the drug were added in 200 μl standard culture medium  and maintained for five days under 5% CO2 atmosphere. The final DMSO concentration in the culture remained below 0.1%. Untreated cultures were included as controls. The cytotoxicity was determined using the colorimetric MTT assay according to the manufacturer's recommendations  and scored as a percentage of reduction in absorption at 540 nm of treated cultures versus untreated control cultures. IC50 values were obtained from the drug concentration-response curve. The results were expressed as the mean ± the standard deviations determined from several independent experiments. The index of selectivity was defined as the ratio of the IC50 value on MRC-5 to that of P. falciparum.A human diploid embryonic lung cell line  was used to assess the cytotoxic effects towards eukaryotic host cells. MRC-5 cells were seeded at 5,000 cells 50 values. Aliquots were removed at the indicated times, washed three times with culture medium and maintained in culture in the absence or in the presence of a given drug. Parasite morphology was determined on Giemsa-stained smears defined according to the following criteria: the ring stage, when parasites exhibited a peripheral cytoplasm stained by Giemsa and a unstained intraparasitic vacuole; the trophozoite stage, when parasites showed a fully stained cytoplasm, haemozoin crystals and one nucleus; the schizont stage, when parasites presented several distinctive nuclei. Parasitaemias were determined by counting 3,000 cells for each sample. Controls consisted of parasites incubated with DMSO instead of drugs processed in the same way.Synchronized cultures (1–2% parasitemia) at the ring stage (0–10 hours old parasites), the trophozoite stage (25–35 hours old parasites) and the schizonte stage (40–48 hours old parasites) were maintained in the presence of drug concentrations in the vicinity of ICP. falciparum cultures at the trophozoite stage (20–30 h old parasites). 400 μl of culture (1 × 108 parasites) were incubated for 60 min at 37°C with 10 μM NBD-C6-ceramide and 0 to 500 μM PPMP or AD2646. Cells were then lysed by freezing and thawing of the culture. Lipids were extracted by a modification of the method of Bligh and Dyer [3OH/CHCl3 mixture  were added and the mixture vortexed for one min. Organic and aqueous phases were separated by centrifugation  and the organic phase was dried. Lipids were dissolved in 15 μl ethanol and analysed by thin layer chromatography on HPTLC plates  in CH3OH/CH3Cl3/NH4OH . For qualitative analyses, the fluorescent lipids were detected under UV and for quantitative analyses, the fluorescent lipid spots were scraped, eluted in one ml methanol and quantified at an excitation of 470 nm and an emission of 530 nm in a spectrofluorometer. The percentage of SPM synthase activity for each drug concentration was determined by comparison of the fluorescence quantified in the analog-treated culture with that in the control culture (without drug).SPM synthase activity was measured as described by Haldar et al. . Brieflyand Dyer . To each5-ceramide, washed three times with culture medium without serum and fixed overnight, at 4°C, in 3.7% formaldehyde/0.05% glutaraldehyde. Cells were mounted on poly-L-lysine coated slides and viewed using a Nikon Eclipse TE 300 DV inverted microscope with an 100X oil objective mounted on a piezzo electric device using appropriate fluorescence emission filters. Image acquisition (z-series) was performed with a back illuminated cooled detector  using a 0.2 μm step. Data acquisition and image deconvolution process were performed with Metamorph software . The images presented correspond to the maximum intensity projection of the deconvoluted z-series.Infected erythrocytes treated with or without ceramide analogs were incubated for 30 min, at 37°C, in culture medium containing 10 μM BODIPY-FL-C2-NH) or a thiourea (CS-NH) linkages  A pan cells  and did lciparum .50 values obtained for the different compounds on the development of the chloroquine-resistant strain FcB1 of P. falciparum in culture . Interestingly, the nature of the linkage considerably influences the anti-Plasmodium activity. Analogs with amide linkage were found to inhibit parasite growth with IC50 values in the micromolar range . However, this IC50 value for PPMP differed from the previously reported value (IC50 = 0.85 μM) [50 values in the nanomolar range  . The dis0.85 μM)  and on aD-threo nitro phenyl analogs of series A, no particular increase of the inhibitory activity was observed with the increase of the N-acyl chain length , F32 (IC50 = 21 nM) and W2 (IC50 = 28 nM), suggesting that the drug is not restricted to a specific strain and acts through a conserved mechanism in malarial parasites. Furthermore, analysis of drug combination with antimalarial drugs showed that AD2646 has a non-synergistic and non-antagonistic effect with CQ on the CQ-resistant strain K1, and with mefloquine and with artemether on the FcB1 strain (data not shown). Compound AD2646  nor hydroxylation on carbon 1 (compare compounds AD2730 and AD2724). In contrast, hydroxylation of carbon 3 is important for anti-Plasmodium activity since removal of the hydroxyl group reduced the activity 13.5 times (compare compounds AD2730 and AD2729).Structure-activity relationship around AD2646 showed that the presence of a nitro group linked to the phenyl is not essential for anti-50 values in the micromolar range, from 5 to 8 μM (except for AD2619), which are similar to the IC50 value of PPMP. No major difference of toxicity was observed between D- and L-threo enantiomers (compare AD2646 and AD2645). In contrast to what was observed for P. falciparum, hydroxylation of the sphingosine carbon 3 does not seem important for cytotoxicity since similar IC50 values were measured for AD2646 and AD2729, suggesting different mechanism(s) of action for AD2646 on MRC-5 cells and P. falciparum. AD2646 and 4 derivatives show high selectivity for P. falciparum as illustrated by the high index of selectivity of these compounds ranging from 160 to 624. The index of selectivity was defined as the ratio of the IC50 value on MRC-5 cells to that on P. falciparum. It can be noted that no selectivity was observed for PPMP. A similar range of growth inhibition was measured on P. falciparum  was evaluated Table . Derivat50 values than that observed with P. falciparum.It must be emphasized that the amide linkage of ceramide analogs is not required for activating apoptosis in cancer cells . An incrTo investigate the cytostatic or cytotoxic effects of AD2646 on the parasite development, cultures at the ring stage (0–10 hours), the trophozoite stage 25–35 hours) and the schizonte stage (40–48 hours) were incubated with 30, 100 or 250 nM of AD2646 for 24.5 hours for the ring stage, for 11 hours for the trophozoite stage, and for 14 hours for the schizonte stage. Aliquots were then taken, washed and incubated in the absence or the presence of drug for a further 13 hours to 24 hours depending upon the parasite stage tested  was insensitive to this concentration of drugs ,19. A cyFigure In contrast, PPMP and AD2646 have completely different effects on the TVN formation for drug concentrations that block parasite growth. After 24 hours of incubation, ring development was totally inhibited by 5 μM PPMP and no TVN was observed as previously described  Figure . As in cPlasmodium activity of AD2646 does not correlate with its inhibitory activity on the SPM synthase. Although AD2646 and PPMP showed similar inhibitory activity on this enzymic activity in parasites in cultures, AD2646 is about 300 times more efficient in inhibiting parasite development than PPMP; 2) In contrast to PPMP which inhibits the parasite development preferentially and reversibly at the ring stage [These data do not support the hypothesis of parasite growth inhibition due to an inhibition of the parasite SPM synthase activity as was demonstrated for PPMP -22 : 1) ng stage , AD2646 ng stage -22. ThisBy their lipidic nature, these analogs might act through a detergent effect that could lead to lysis or modification of the integrity of infected-erythrocyte membranes. This apparently is not the case. No significant lysis of normal erythrocytes was observed after 48 hous of incubation with concentrations of analogs up to 10 μM (data not shown). Furthermore, no preferential lysis of infected-erythrocytes was observed on Giemsa-stained smears of infected cultures maintained 48 hours with 250 nM AD2646, a concentration inhibiting parasite growth totally.Plasmodium and are involved, at least, in the uptake of erythrocyte raft proteins and maintenance of the parasitophorous vacuole containing the parasite, inside the erythrocyte [P. falciparum growth is characterized by a setting up of new permeabilities of the infected-erythrocyte membrane [Interestingly, the absence of a fatty acyl carbonyl group (methylene linkage) in our ceramide analogs is a critical factor for the efficacy of their antiplasmodial activity. Sphingolipids preferentially interact with cholesterol in membranes, especially in detergent-resistant microdomains (DRMs or rafts). Rafts have been described in throcyte . This inthrocyte  and 2) hthrocyte . The amithrocyte . It coulmembrane . Althougmembrane ,43. The in vitro [P. falciparum. Parasites exposed to 1 μM AD2646 for up to 36 hours failed to exhibit characteristic apoptosis, as determined by terminal deoxynucleotidyl transferase DNA fragmentation assay and DNA fragmentation using both gel electrophoresis and fluorescence microscopy methods, although the nucleus appeared highly condensed . Apoptosis in P. falciparum is still controversial although some characteristics of apoptosis has been described in Plasmodium [Ceramide is at the parting of different ways of sphingolipid metabolism. Analogs have the potential to inhibit different ceramide-metabolizing enzymes and then might have a pleiotropic effect. Ceramide analogs in amide linkage were described as potent inhibitors of alkaline ceramidase in HL60 human myeloid leukemic cells ,45. Methin vitro . When apin vitro ,45. In cin vitro , prelimiin vitro inhibition assays on P. falciparum and MRC-5 cells. MG and MD performed the fluorescence microscopy and apoptosis investigations on P. falciparum, respectively. SE and ST carried out electrophysiological studies on the malaria-induced anion channel. AD, CW and SG participated in the design and synthesis of ceramide analogs. All authors read and approved the final manuscript.ML and PG carried out the


Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells.In yeast and Leihmania infection.Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) β-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed.LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help  Leishmania is an intracellular pathogen that causes Leishmaniasis, which remain an important medical problem in several countries. Protozoan parasites of the genus Leishmania result in a spectrum of human disease that range from self-healing cutaneous ulcers to potentially fatal visceral infection, depending primarily upon the species of parasite involved . In mameview in . SIRT1 ieview in . Also, teview in .Leishmania amastigotes, was able to promote parasite survival in in vitro culture systems  using the Superscript II RNase H-reverse transcriptase. PCR amplifications were performed using 1–2 μl of reverse-transcription of each product and 20 pmol of each primer, using Taq DNA polymerase. Each PCR cycle consisted of a denaturation step , an annealing step  and an elongation step (72°C 1 min). For the last cycle, the elongation step was extended to 10 min at 72°C. Reactions were carried out for 25–35 cycles in a thermocycler . PCR products were analyzed on 1.5% agarose gel and visualized with ethidium bromide. As an internal control, a housekeeping gene, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript, was amplified. LmSIR2 primers were: Sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG. GAPDH primers were: Sense GTC TTC ACC ACC ATG GAG and antisense CCA AAG TTG TCA TGG ATG ACC.Total RNA was isolated from wild type and transformed cells using the Rneasy Mini Kit following the manufacturer's instructions. One microgram of RNA was reverse transcribed to cDNA with an oligonucleotide [poly (dT)2 for 12 h with freshly prepared β-Gal staining solution pH 6.0, containing the 5-bromo-4-chloro-3-indoyl-b-D-galactosidase  chromogenic substrate which produces blue 5-bromo-4-chloro-3-indole upon hydrolysis. Cell preparation was stored at 4°C in 70% glycerol upon analysis.Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. . BrieflyLmSIR2 gene.The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure LmSIR2 gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione [leishmania's excreted material as previously demonstrated raised the question of its function during the infection process. Thus, heterologous expression in eukaryotic cells could represent a useful approach to give some insight into the biological activity of secreted parasite material. Immunolocalization of LmSIR2 shows that cells expressing LmSIR2 .In order to investigate the potential functions of LmSIR2 protein, when delivered intracellularly in L929 fibroblasts, we have determined the growth kinetic parameters of cells via two different methodologies. Using a counting method, we observed a high reduction in the saturation density of cells expressing LmSIR2 Figure . The redn figure , expressin vivo and in vitro [LmSIR2 gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector . As shown in Figure Leishmania infection since the mean percentage of infected fibroblast reach 70% after 24 hours of contact. However, once inside fibroblasts no differences in the capacity of parasites to survive and/or multiply was observed and the intracellular amastigote population was eliminated after three days of contact (data not shown).Fibroblasts at the lymph are used as safe target for long lasting residual parasite population . However8, Chang  demonstrrvations . Thus, wLeishmania infection. These results suggest that LmSIR2 has dual function according to the cell type in which it acts: in Leishmania parasites, LmSIR2 is able to promote cell survival while in the host fibroblast it induces a senescence phenotype. This effect could be beneficial for Leishmania since senescent cells are no longer capable of dividing yet remaining metabolically active. They could thus represent safe target for parasite. It is documented that senescent fibroblasts present an inherent higher capacity to resist to apoptotic death [in vivo relevance of our observation is rather difficult to appreciate because; (1) the level of the protein produced in fibroblasts transformed with the pcDNA is certainly far higher than the level achievable in vivo, and (2) the presence of excreted parasite material inside the fibroblast cytosol has yet not been reported. However, an action of the excreted form of LmSIR2 on he host cell could no be ruled out. Two series of observations support this possibility: (1) it has been shown that the Leishmania EF1-a could be translocated from the parasitophorous vesicle into the macrophage cytosol [Our study demonstrates that LmSIR2 is able to down regulate the proliferation of fibroblasts and to induce a senescence-like state in L929 fibroblast cell line. The most interesting finding is that a parasitic protein is able to modify the physiology of the host cell making them more permissive toward ic death . In this cytosol ; (2) the cytosol .in vivo infection process awaits further investigationsIn conclusion, although the effect of the protein, when delivered extracellularly, on the host cells have not being examined in the present study, our data support the notion that the excreted form of LmSIR2 is able to interfere with the host cell physiology, when delivered intracellularly. Unexpectedly the expression of the protein is associated with the appearance of a senescence biomarker. Relevance of such finding during the


Nasal strips are used by athletes, people who snore, and asthmatics to ease the burden of breathing. Although there are some published studies that demonstrate higher flow with nasal strips, none had directly measured the effect of the strips on nasal resistance using the airflow perturbation device (APD). The APD is an inexpensive instrument that can measure respiratory resistance based on changes in mouth pressure and rate of airflow.This study tested forty-seven volunteers (14 men and 33 women), ranging in age from 17 to 51. Each volunteer was instructed to breathe normally into the APD using an oronasal mask with and without nasal strips. The APD measured respiratory resistance during inhalation, exhalation, and an average of the two.20/Lps from an average nasal resistance of 5.5 cm H20/Lps.Results of a paired mean t-test comparing nasal strip against no nasal strip were statistically significant at the p = 0.05 level. The Breathe Right™ nasal dilator strips lowered nasal resistance by an average of 0.5 cm HNasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation. Nasal dilator strips (NDS) are used by athletes, people who snore, and asthmatics to ease the burden of breathing. The nasal strips are used as a mechanical means of reducing nasal airflow resistance . By loweThe size of the nostril limits the amount of air entering into the body. The NDS is placed along the nasal valve of the nose. The adhesiveness of the strip binds to the creases of the nasal valve to prevent the outer wall tissue of the nose from collapsing inward during nasal breathing. This mechanism thus dilates the nose and allows more air to flow into the nose .The primary effect of the NDS could be either to dilate the air passage of the nose or to stiffen the nasal wall. Either mechanism would reduce nasal resistance and allow higher flow of air, but they can be distinguished over a range of air flows. Stiffening the nasal wall would have its most profound effect at higher flows where the Bernoulli effect would decrease internal nasal pressures and tend to constrict nasal passage diameter. Air passage dilation would tend to decrease nasal resistance more uniformly over a range of air flows.Recent studies on the effectiveness of Breathe Right™ nasal strips tested participants under various rest and exercise conditions. Some found that the strips neither improve or diminish airflow ,4-9, whiThe Airflow Perturbation Device (APD) is a small, light weight, and easy to use instrument that measures respiratory resistance . A segmePeople breathe normally through the APD. No special breathing maneuvers are required. For this reason, the APD can be used with young children, older adults, unconscious patients, and animals. Respiratory resistance can be separated into inhalation and exhalation components, and resistance can be displayed against lung volume and air flow rate.Respiratory resistance is normally measured through the mouth, with a nose clip and hands pressed against the cheeks. An oronasal mask may be used to obtain combined mouth and nose resistance, or nose resistance by itself if the mouth is closed. The APD used with an oronasal mask should be an ideal instrument to assess the effect of NDS.This study is as much a test of the capabilities of the APD as it is a study of NDS. Objectives of this study were to: 1) determine if APD measurements of respiratory resistance measured with an oronasal mask matched those with breathing through a mouthpiece, and 2) measure the effects of NDS on respiratory resistance made with the APD. This is not a clinical study.This study tested forty-seven volunteers . Some had symptoms of nasal congestion such as asthma, allergies, and snoring. A written informed consent was obtained from each subject and the protocol was approved by the University of Maryland Institutional Review Board (IRB).The nasal strips used in this study were a commercial product called "Breathe Right" . According to the manufacturer's instructions, the nasal strips should be placed halfway down the nose along the nasal valve. The two end regions of the nasal strips should cover the left and right nasal creases.+, Laerdal Medical, Wappingers Falls, NY). The subject was instructed to press their face against the mask while he/she breathed normally. The size of the mask was large enough that it contacted only the hard tissue on the bridge of the nose and did not compress the soft nasal septum.This study contained three phases: 1) to determine the ability of the APD to measure oral resistance using either a mouthpiece or an oronasal mask, 2) to determine the ability of the APD to measure nasal resistance, and 3) to determine the effect of nasal strips on nasal resistances. Phase I consisted of two tests that measured oral breathing resistance. In the first test, the subject's nose was occluded with two layers of Durapore surgical tape  while the subjects were sitting in an upright position. The subject was instructed to breathe normally into a cardboard mouthpiece. The second test repeated the same procedure as the first test, except the subject was breathing into an oronasal mask y = 0.9624 + 0.1041 Rwhere y = mask value of resistance and x = mouthpiece value of resistanceThis equation was tested to be statistically equivalent to y = x. This indicates that the oronasal mask had no effect on the inhalation values.A similar comparison of exhalation resistances gave the following:2 = 0.8828    (2)y = 0.874bx + 0.4594 RThis equation did not pass the statistical test for equivalence y = x. The oronasal mask may have affected the measurement of respiratory resistance in the exhalation direction.2O/Lps, while nasal resistance with the NDS ranges from 3.39 to 7.33 cm H2O/Lps. This demonstrates the expected resistance reduction with NDS.Figure All subjects except three showed a decrease in nasal resistance when breathing with the NDS. Average value of nose breathing without NDS was 5.50; with NDS it was 5.00. These means were highly statistically significantly different.2O/Lps in resistance, and only six out of 47 subjects failed to demonstrate a decrease in resistance with NDS.The effect of NDS on resistance during exhalation was also statistically highly significant. There was an average reduction of 0.45 cm H20/dps. Again, six subjects failed to demonstrate a decrease in resistance with NDS. These were not the same subjects that increased resistance in the exhalation direction.The effect of NDS on resistance during inhalation tested to be statistically highly significant, as well. The average resistance reduction was 0.49 cm HResistance differences with and without NDS in the inhalation and exhalation directions were tested to determine if NDS had a larger effect while breathing in one direction or the other. Means of the differences for inhalation and exhalation directions were tested with a paired t-test, and found to be statistically nonsignificant. It appears, therefore, that NDS affect nasal resistances equally during inhalation and exhalation.This study confirmed the results of other studies that showed a reduction of about 10% in nasal breathing resistance, as well as supported the claim of the manufacturer that the nasal strips provide nasal relief. Several subjects who had nasal congestion reported some relief in nasal breathing when using the nasal strip. Exactly which subjects these were was not recorded.There was one surprise, though, in the results. The reduction in respiratory resistance due to NDS was a constant amount and not proportional to the resistance level present without NDS. This result was not expected, and no adequate explanation for it can be given at this time. It is not clear why this should be so, but we do not doubt that measurements made with the APD are correct, based on previous studies ,16.We cannot comment on the clinical significance of the resistance reduction with NDS use. It seems likely that some benefit could be obtained from such a resistance change, but whether it is actually detectable is not clear. Other reports in the literature ,18 have The APD has been shown to be able to measure respiratory resistance with either a mouthpiece or an oronasal mask. This may be a significant advantage of the instrument, especially because respiratory resistance measurement on unconscious or uncooperative patients would be much more easily made with a mask than with a mouthpiece. Equations (1) and (2) show the close correspondence between measurements made with both techniques, although the presence of an intercept and a slope different from unity indicate that the correspondence between mask and mouthpiece is not perfect. Resistances with a mask are both higher than resistances with a mouthpiece. The reason for this seems to be different mouth positions in both cases. We have laboratory experiences (not published) that demonstrate that tongue position can influence measured resistance. Breathing through the mask is probably done with the mouth closed more than when breathing through the mouthpiece. The measured difference between inhalation and exhalation resistances could reflect the effect of a pressure difference across the distensible smaller airways, which is greater inside than outside during inhalation, but smaller inside than outside during exhalation. This would lead to a dynamic compression of the small intrathoracic airways during exhalation. Another possible explanation is natural movement of the vocal chords such that they are closer during exhalation than during inhalation.This study was a good test of the capabilities of the APD measuring device. Testing confirmed that the APD can detect resistance changes, and that measurements are easy to obtain. Results in this study are generally more consistent than other studies using other techniques -11. The 2O/Lps. Thus, nasal strips do have a measurable effect on nasal resistance. The effect of NDS appears to be equal during exhalation and during inhalation.Nasal strips reduce nasal resistance by about 0.5 cm HThe APD can be used to measure nasal resistance, and can detect resistance levels.The APD can consistently measure oral resistance with either a mask or a mouthpiece.LW conducted the testing as an undergraduate student. ATJ provided the APD and mentored LW. All authors have read and approved this manuscript.


The disparity in the scientific output between developed and developing counties is dramatic, but, as the Americas show, this grim picture is improving Kofi Annan, the Secretary-General of the United Nations, recently called attention to the clear inequalities in science between developing and developed countries and to the challenges of building bridges across these gaps that should bring the United Nations and the world scientific community closer to each other . Mr. AnnNorth America and Europe clearly dominate the number of scientific publications produced annually.It is rather obvious that richer countries are able to invest more resources in science and therefore account for the largest number of publications. It is also likely that there is a statistical bias on the part of the SCI as a bibliometric database, since it represents North American and European publications far better than those of the rest of the world . But is Using the SCI databases produced by the Institute for Scientific Information (ISI), as well as data compiled by the Red Iberoamericana de Indicadores de Ciencia y Tecnología (RICYT), we examined the differences in the number and proportion of scientific publications between the developed world and the developing world from 1990 until 2000, focusing on the Americas as a case study. Not surprisingly, there was a huge disparity in the number of publications from 1990 until 2000, with the United States contributing the lion's share (84.2%), followed by Canada (10.35%). Latin America as a whole contributed only 5.45% to the total number of scientific publications in these ten years .The total number of publications, however, is not necessarily the best measure for assessing scientific productivity or technical advances . More reOther relative indicators of scientific productivity, such as the number of publications picked up by the SCI in relation to the number of scientists in a particular country, also demonstrate that such developing regions as Latin America are making substantial contributions to science, despite the fact that the average proportion of gross domestic product (GDP) invested in science in Latin America throughout this 10-year period was only 21% of the amount invested in United States . Indeed,Among Latin American countries, there is a high degree of variability in publication rate as well as in financial investment in science and technology. Some countries have performed particularly well. For example, Uruguay, Chile, Panama, and Cuba averaged, respectively, 6.8, 5.3, 5.2, and 3.4 publications per million dollars of research and development investment in the 10 years studied, which is notoriously high compared with United States (1.5) and even Canada (3.3) . Other cWhy has the number of publications per dollar invested in research and development been increasing in Latin America while decreasing in United States and Canada?One potential explanation for the increase in scientific productivity in Latin America is that scientific development during the 1990s was particularly strong for many countries of this region. Indeed, this would explain the rapid rise in the number of publications in Latin America compared with the relatively flat increases in the United States and Canada, which were publishing just as well at the beginning of the decade. A potentially more important question, however, is why the number of publications per dollar invested in research and development has been increasing in Latin America while decreasing in the United States and Canada. This pattern could be the result of a variety of factors, none of which are mutually exclusive. It is possible that publishing in international journals as a measure of scientific productivity is becoming more important in Latin America. Increased funding to the most productive scientists from the national science development programs might have been an important stimulus. International cooperation resulting in more scientific collaborations among scientists in Latin America, Europe, and the United States may also have increased the relative number of publications in Latin America. In contrast, the decreasing trends in the number of publications per investment dollar in Canada and United States could reflect a trend towards more costly research in larger scientific programs.Nature and Science; with impact factors of 27.96 and 23.33, respectively) and in the 20 top ecological journals (with impact factors of 10.51–2.47)  between 1990 and 2002 in both the two top general science journals (51–2.47) . We credFor the top 20 ecological journals, the American subcontinents of South, Central, and North America accounted for 62% of the publications worldwide. Within the Americas, however, Latin America represented only 6%, while Canada and United States accounted, respectively, for 13% and 82% of the top 20 ecological publications. When we examined the data as contributions to the top 10 ecological journals (impact factors 10.51–3.31) versus the top 11–20 (impact factors 3.28–2.47), the Latin American countries contributed nearly twice as many publications to journals in the second category (8% in the top 11–20 compared with 4% in the top 10). These findings suggest that publications from such developing regions as Latin America are falling short of reaching the top journals. In contrast, the United States contributed somewhat more publications to the top 10 journals (84%) than the top 11–20 journals (79%). The difference in the proportion of publications contributed by the United States to the top 10 and top 20 journals was even more pronounced when we examined it in respect to worldwide publications. In this case, the United States contributed 60% of the publications to the top 10 journals and only 40% of the publications to the top 11–20 journals.Science and Nature were nearly identical to those of the top 20 ecological journals. In Science and Nature, Latin America had 7% of the publications within the Americas versus 6% in the top 20 ecological journals, whereas the United States and Canada had 81% versus 82% and 12% versus 13%, respectively. These similarities suggest that the Latin American researchers are not shying away from the two top-ranked general science journals. However, publishing in Science and Nature was not enough to gain prominence, as evidenced by the number of citations of these researchers. The latest list of the 247 most-cited researchers in ecology and environmental sciences emphasizes the overwhelming contributions of authors from North America (73%) and Europe (21%)  . No resepe (21%)  and thusAlthough there are outstanding scientific researchers in the developing world who independently are making important contributions to the international scientific community, they are the exception. Why, in general, do Latin American scientists often fail to reach the top journals or become amongst the most cited researchers in their fields? One possibility is that the main research agendas between both regions are somewhat different and that the top journals, which are published in the developed world, respond more to the scientific mainstream of the developed regions. This is not to suggest any sort of conspiracy, but rather it implies that the perception of the most important science is linked to the region and that because the major funding agencies as well as most prominent journals share a similar economic region, they also share the same perception of what science is most interesting to them. Another consideration is that more local journals from developed regions are listed by the SCI than similar journals from developing regions . ConsequThe positive trends in scientific productivity in Latin America should not be misinterpreted as a reason to be unconcerned about the existing gap highlighted by Mr. Annan. There are many compelling reasons for the push to increase scientific input from the developing world . One is Climate change and biodiversity research urgently need the scientific input from those developing regions that are so important for global processes.Although the evidence presented here demonstrates that there is a long way to go before developing countries contribute a more equitable share to the international scientific community, there are also reasons to be optimistic. The relative increase in the number of publications, especially when corrected for the amount of money available in research and development, demonstrates that many developing countries are heading in the right direction. The extremely high scientific productivity of many developing nations, corrected for and despite the rather limited availability of funds, suggests that increased funding to the sciences will be an excellent investment by developing nations in terms of publications as a measure of scientific output, particularly if these publications can target the journals that have the greatest impact. Although there may still be a long road to travel, we feel optimistic that the bridges mentioned by Mr. Annan are slowly being built.