Fish gonadal sex differentiation is affected by sex steroids treatments providing an efficient strategy to control the sexual phenotype of fish for aquaculture purposes. However, the biological effects of such treatments are poorly understood. The aim of this study was to identify the main effects of an androgen masculinizing treatment  on gonadal gene expression profiles of an all-female genetic population of trout. To characterize the most important molecular features of this process, we used a large scale gene expression profiling approach using rainbow trout DNA microarrays combined with a detailed gene ontology (GO) analysis.2,474 genes were characterized as up-regulated or down-regulated in trout female gonads masculinized by androgen in comparison with control male or female gonads from untreated all-male and all-female genetic populations. These genes were classified in 13 k-means clusters of temporally correlated expression profiles. Gene ontology (GO) data mining revealed that androgen treatment triggers a marked down-regulation of genes potentially involved in early oogenesis processes , an up-regulation of the translation machinery (GO 'ribosome') along with a down-regulation of proteolysis . Genes considered as muscle fibres markers (GO 'muscle contraction') and genes annotated as structural constituents of the extracellular matrix (GO 'extracellular matrix') or related to meiosis (GO 'chromosome' and 'meiosis') were found significantly enriched in the two clusters of genes specifically up-regulated in androgen-treated female gonads. GO annotations 'Sex differentiation' and 'steroid biosynthesis' were enriched in a cluster of genes with high expression levels only in control males. Interestingly none of these genes were stimulated by the masculinizing androgen treatment.This study provides evidence that androgen masculinization results in a marked dysregulation of early gene expression profiles when compared to natural testicular or ovarian differentiation. Based on these results we suggest that, in our experimental conditions, androgen masculinization proceeds mainly through an early inhibition of female development. In fiewed in . Many stiewed in . HoweverOncorhynchus mykiss, has a male heterogametic XY genetic system and we experimentally produced XX and YY males allowing the production of genetically all-male and all-female populations [a priori. Using the extensive collection of expressed sequenced tags (ESTs) obtained through sequencing projects in trout as a resource [Rainbow trout, ulations . These aresource ,8, we deresource ,10 is abUsing this genome-wide approach we characterized 2,474 genes  with a clear differential temporal expression profile in females masculinized by androgen. We classified these genes in 13 different clusters of correlated temporal expression profiles, and searched within these clusters for significant enrichment in Gene Ontology (GO) terms. This strategy allowed us to define a few very clear biological trends potentially explaining how androgen induces masculinization of female fish. Our results clearly demonstrate that masculinization with androgen proceeds through a marked dysregulation of gene expression profiles, including a quick down-regulation of the ovarian pathway. Surprisingly, most of the genes over-expressed during natural testicular differentiation were not restored by the androgen-induced masculinization suggesting that the inhibition of female gonadal development is the main required step sufficient for building a testis.The complete dataset is available through the National Center for Biotechnology Information (NCBI), in the Gene Expression Omnibus database  under thThis analysis was carried out on fish sampled at several stages of development from the onset of the free swimming period (Day 0 = D0), when fish first started to be fed with the androgen treatment until 110 days after the beginning of the treatment (D110). Unsupervised hierarchical clustering of samples Fig  reveals The 2,474 expression profiles were analyzed using a k-means clustering (with k = 13) in order to individualize clusters of genes with similar expression profiles  terms significantly enriched in these groups of correlated expression profiles compared to the overall GO terms found. Among the 1,805 genes with an annotation we selected 1,276 unique genes annotated with an official name, and 1,133 were found to be associated with at least one GO category. The top 5 significantly enriched GO terms for each of the clusters are described in Table -3). Among clusters 1 to 5, only genes from cluster 5 are also highly expressed in the androgen-treated group. The main GO theme of cluster 5 is related to translation, with over-representation of GO 'ribosome' , tropomyosins  and some muscle markers . The GO term 'extracellular matrix' . Many of these genes , are 'structural constituents of the extracellular matrix' (ID 5201), or are involved in 'cell adhesion' (ID 7155) . The expression profiles of some of these genes related to the extracellular matrix are shown in figure tgfb1 expression profile that also shows a slight up-regulation from D60 to D110 following androgen treatment in females. Cluster 8 contains genes related to 'chromosome' (ID 5694), 'condensed chromosome' (ID 793) but also, albeit only a few (2 out of 6), to the 'progression through the first phase of meiosis' . Cluster 9 is characterized by enrichment in the GO terms 'organ morphogenesis' (ID 9887), 'development' (ID 48513) and 'structural constituent of cytoskeleton' (ID 5200). Cluster 10 contains genes involved in 'urogenital system development' (ID 1655) and 'lipid binding' (ID 8289). Clusters 11 and 12 contain genes that are also assigned with the GO term 'extracellular matrix' but more specifically in relation with degradation of this extracellular matrix i.e., GO term 'metallopeptidase' (5.4 and 3.8 fold enrichment respectively) with at least 3 matrix metallopeptidases  that are directly involved in 'collagen catabolism' (ID 30574). Cluster 13 contains genes related to 'sex differentiation' (ID 7548) , and 'steroid biosynthesis' (ID 6694) . According to these annotations this cluster is likely to contain other important genes also involved in testicular differentiation or in steroidogenesis regulation. Several expression profiles for such potential genes are shown in figure As gene expression in clusters 1 to 5 displayed similar expression profiles in the control female group, these clusters have been analysed both all together and separately Table  are the tfa (transferrin), timp2  and bzrp  expression profiles did not significantly correlate. As a result, among the 13 k-means groups, at least 10 groups  contained genes with significant correlation between real-time RT-PCR and DNA microarray measurements.Expression profiles of 102 genes, involved in early gonad development, were measured by real-time RT-PCR. These genes all belonged to the 13 distinct k-means clusters , the androgen masculinization does not induce a natural physiological response since the transcriptome of testicular trans-differentiating gonads is quite different from the one observed during natural testicular differentiation. These differences might be due to the non physiological dosage of androgen used in our experiment. A similar study using a lower dosage may help to clarify this issue, but this study was first designed with an androgen dosage that is commonly used in rainbow trout aquaculture conditions. Whether the observed gene expression dysregulations are the reflection of a direct action of the androgens on the gonad, an indirect retro-control on the hypothalamus-pituitary axis, or a conjunction of both, remains to be elucidated. However, the synthesis of Gonadotropin Releasing Hormone (GnRH) and of pituitary hormones is established very early during rainbow trout ontogenesis  with at Due to the lack of specific Gene Ontology (GO) annotation for rainbow trout we linked the best blast hits of each clone sequence with a cross-species GO annotation. This strategy relies on the accuracy of the blast homology search and also on the resulting accuracy of the GO annotations with regards to their use in a fish species. However, even if this could lead to potential errors on a gene per gene scale, the global analysis and stringent statistical screen that we carried out enabled us to unambiguously assign most clusters with a clear biological theme. Among these GO categories, some were considered as biologically informative – i.e., not too general like for instance GO "physiological process" – and robust as they contain a sufficient number of different genes to support a potential biological meaning. We focused our analysis on these biologically informative GO categories.foxl2a and foxl2b, cyp19a1, fst, inha) were introduced in our analysis and all these genes were strongly and quickly inhibited by the masculinizing androgen treatment. But when pooled with our DNA microarrays dataset they did not form a tight cluster. This is probably because no additional similar expression profile was found within the DNA microarray dataset. This very small number of early female-specific genes is in agreement with the small number of candidate genes known to be involved in the ovarian differentiation pathway [amh, sox9, dmrt1, gata4, lhx9) [With regards to the effects on the gonad, our analysis first reveals that female development is highly affected by the androgen treatment, with a down-regulation of most of the genes involved in early oogenesis stages. However within this analysis we did not characterize any cluster of early female-specific up-regulated genes potentially involved in ovarian differentiation. Expression profiles of some female-specific candidate genes    and sometgfb1) in gonads of androgen-treated animals. In rat, TGFβ induced morphological changes in Leydig cells, accompanied by an increased secretion of fibronectin, laminin and collagen IV [tgfb1 in trout gonads triggers a fibrotic response. Surprisingly, these effects are detected transiently and rather late after the application of the androgen treatment (but concomitantly with tgfb1 up-regulation). Whether this reflects a total dysregulation or an exacerbation of a testicular-specific event remains to be analyzed. However, extracellular matrix deposition is known as a major event for the testicular organization. For instance, LAMA5 (Laminin α5) has been characterized as a structural protein involved in the formation of the basement membrane of the testicular cords [amh expression is not restored to male levels in androgen-treated females. This may produce a disrupted expression of some structural proteins, like lama5. In the same manner, sparc is highly up-regulated in androgen-treated females. In mouse Sparc gene expression has been identified in pre-Sertoli cells at the time of sex differentiation [amh or tgfb1.Among the gene clusters specifically up-regulated in females following masculinization with androgens, extracellular matrix, muscle markers/cytoskeleton and meiosis were characterized as the 3 main gene annotations. Simultaneous up-regulation of extracellular matrix protein genes expression and down-regulation of matrix proteinase genes was detected in gonads of androgen-treated females. At the same time the histological analysis of these gonads showed that they contain a predominant stroma of conjunctive tissue with fibroblast like cells. Matrix protein synthesis and the concomitant decrease in matrix proteinase activity have been well described as a characteristic fibrotic response of an excessive Transforming Growth Factor-beta (TGFβ) production ,19. Of slagen IV . In fibrlagen IV , that wear cords  and thisar cords . In trountiation  and thisntiation . Structucnn1, myh11, myl6, tagln) are even considered as characteristic smooth muscle markers. This expression of muscle markers in the testis is likely in relation with the peritubular myoid cells that surround the seminiferous tubules [tpm1  [We also detected a high number of genes associated with cytoskeletal reorganization and muscle development that were up-regulated by the treatment. Some of them  , smooth , alpha) , and smo, alpha) . Differe, alpha)  and they, alpha)  along wi, alpha) . It is tin vitro in some species [In our experiment, the androgen treatment also induced a precocious spermatogenesis as revealed both by the histological analysis and by the increased expression levels of some genes involved in testicular meiosis. In fish, androgens and particularly, 11-oxygenated androgens, are strongly involved in spermatogenesis regulation  and they species . Similar species -36.This study gives a first comprehensive survey of gene expression during androgen-induced masculinization in female rainbow trout. Our data provide supportive evidences that this treatment results in a marked dysregulation of gene expression levels when compared to natural testicular or ovarian differentiation. In our experimental condition the androgen treatment induces the complete down-regulation of female specific genes, but not the complete restoration of the male-specific gene expression patterns. Instead, some disturbed responses were characterized by an exacerbation of extracellular matrix synthesis and muscle type cell differentiation and proliferation (myoid cells) followed by a precocious meiosis of germ cells. All together, we suggest that androgen masculinization acts mainly through an early inhibition of female development rather than through a direct induction of testicular differentiation.Research involving animal experimentation has been approved by the authors' institution (authorization no. 35-14). It conforms to principles for the use and care of laboratory animals and is in compliance with French and European regulations on animal welfare . All-male and all-female rainbow trout populations were obtained at the INRA experimental fish farm  as previously described . Treatme® kit  according to the manufacturers' instructions.Total RNA was extracted using TRIzol reagent  as previously described . The tot2 membranes  attached to glass slides using an 8-pin print head (Pin-and-Ring™ technology) on the GMS 417™ . Spotted DNA was then denatured and UV – cross-linked onto nylon filters. All DNA microarrays used in this study were made at the same time and under the same conditions. These trout microarrays contained 9,216 DNA spots representing 9,120 trout cDNA clones and a set of 96 controls. Among these cDNA clones, 7,584 were issued from a pooled-tissues library and 1,536 from a testis library [Arabidopsis thaliana cytochrome c554 clone which is devoid of similarity with trout DNA sequences, 8 spots of poly(dA)80 and 8 spots of PCR reaction without template.Gene expression analyses were carried out using home-made Nylon DNA microarrays using a previously described technology . These DMicroarrays were hybridized with two types of 33P-labeled probes. The first one was an oligonucleotide with a sequence common to all spotted PCR-products (vector hybridization) in order to determine the amount of target DNA accessible to hybridization in each spot. After stripping, a second hybridization was performed with complex probes made from 1 μg of retrotranscribed total RNA -42. Protef1a), which was used as an internal standard [In order to validate and enrich the DNA microarray dataset, expression of 102 genes involved in early gonad development  was measstandard . Data weFirst, non-linear effects such as background, print-tip effects or saturation were corrected by LOWESS , using aA triple filtering procedure was then applied to the microarray dataset. The first consisted of filtering background signals due to low amount of spotted DNA. When a "V" spot signal was too weak , the data of the corresponding cDNA clone was discarded (missing data). The second filtering procedure was applied to eliminate non informative genes that were not measured  in more than 20% of the samples. Finally, genes exhibiting little variation (coefficient of variation < 0.1) across all arrays were excluded from the analysis ,49. AfteAll data  were then log2-transformed and were analyzed by unsupervised and supervised clustering methods. Hierarchical clustering (Cluster program ) investiThe author(s) declares that there are no competing interests.DB and JM designed and spotted this rainbow trout microarray. DB carried out the microarray experiments and analysis with substantial help from RH for the design of the experiment and the analysis. YG and AF conceived the study, participated in its design and coordination. YG and DB drafted the manuscript. All authors read and approved the final manuscript.Median expression profiles of the 2,474 genes differentially expressed in androgen-treated masculinized rainbow trout females. Median expression profiles of the 2,474 genes that display significant changes between male, female and female treated with an androgen masculinizing treatment. This dataset has been subjected to normalization . The K-means cluster (KM) is given for all clones with their clone name, corresponding gene annotation and their median expression levels in females, females treated with androgen and males from D0 to D110. Duplicates samples are labelled -1 and -2.Click here for fileClone names and annotations of the 2,474 genes differentially expressed in androgen-treated masculinized rainbow trout females. Annotation of 2,474 genes differentially expressed in females, males and androgen-treated masculinized rainbow trout females. Information provided for each clones (or for each accession number of the sequence if the data has been obtained by RT-PCR): the corresponding contig (that can be found at the following website ), the best blast hit description along with the corresponding database (Prodom or Swissprot), the score, the e-value and the percentage of identity.Click here for fileComplete Gene Ontology (GO) analysis with significantly enriched GO in clusters of correlated expression. Significant  GO terms and their definition for all K-means clusters (KM 1 to 13). N KM: number of genes within the corresponding KM cluster. Total: total number of genes within the selected GO term. P-Value: p-value of the significant enrichment. RE: relative enrichment. GO ID: gene ontology identification number. Term: GO terms.Click here for file


Plasmodium and other human pathogens through an unknown mechanism. We have undertaken a comprehensive functional genomic approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito's microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes' antimicrobial immune responses, plausibly through activation of basal immunity. We show that the microbiota can modulate the anti-Plasmodium effects of some immune genes. In sum, the microbiota plays an essential role in modulating the mosquito's capacity to sustain Plasmodium infection.Malaria-transmitting mosquitoes are continuously exposed to microbes, including their midgut microbiota. This naturally acquired microbial flora can modulate the mosquito's vectorial capacity by inhibiting the development of  Anopheles gambiae mosquito that transmits the malaria-causing parasite Plasmodium has an intestinal bacterial flora, or microbiota, which comprises a variety of species. Elimination of this microbiota with antibiotic treatment will render the Anopheles mosquito more susceptible to Plasmodium infection. In this study we show that these bacteria can inhibit the infection of the mosquito with the human malaria parasite Plasmodium falciparum through a mechanism that involves the mosquito's immune system. Our study suggests that the microbial flora of mosquitoes is stimulating a basal immune activity, which comprises several factors with known anti-Plasmodium activity. The same immune factors that are needed to control the mosquito's microbiota are also defending against the malaria parasite Plasmodium. This complex interplay among the mosquito's microbiota, the innate immune system, and the Plasmodium parasite may have significant implications for the transmission of malaria in the field where the bacterial exposure of mosquitoes may differ greatly between ecological niches.The  Plasmodium's development occurs during the ookinete invasion of the midgut epithelium, prior to the development of oocysts on the basal lamina The malaria parasite has to go through series of complex developmental transitions within the mosquito vector before it can be transmitted to the human host. The major bottleneck for Plasmodium parasites in the mosquito midgut Large communities of diverse microorganisms reside in insects with a major concentration in the intestinal sections Plasmodium ookinetes and the midgut epithelium . Similarly to previous studies, the majority of the isolated bacteria were Gram-negative suggesting that the midguts of mosquitoes have more optimal growth conditions for this type, especially those from the Enterobacteriaceae family. This strong bias is also likely to have been attributed, to some degree, to the LB agar– based aerobic culturing method that was used for these assays. Sequence analyses of the 16s ribosomal genes from morphologically distinct bacteria colonies identified the following five different species as dominant in all assayed generations: Enterobacter asburiae (98%), Microbacterium sp. (98%), Sphingomonas sp. E-(s)-e-D-4(2) (100%), Serratia sp. (99%) and Chryseobacterium meningosepticum (100%). The C. meningosepticum and Serratia sp. species were dominant within all five generations and the former was the most abundant, especially within the second generation. Other bacteria identified from different generations were: Asaia bogorensis (99%), Bacillus subtilis (99%), Enterobacter aerogenes (98%), Escherichia coli (91%), Herbaspirillum sp. (99%), Pantoea agglomerans (98%), Pseudomonas fluorescens (99%), Pseudomonas straminea (99%), Phytobacter diazotrophicus (97%) and Serratia marcescens (99%). Interestingly, when C. meningosepticum became the dominant bacterium of the midgut flora, the growth of other bacterial species, that could be cultured on LB agar, was usually limited suggesting that this species may possess some competitive advantages in the gut environment.To gain a better understanding of the potential fluctuations in microbial load and species composition between laboratory reared mosquitoes of different generations and within the same generation, we monitored the bacterial loads and species composition in individual five-day-old female Our LB agar –based culture assays have some limitations in providing the complete picture of the composition of the mosquito midgut microbiota since a large fraction of bacteria are likely to be un-culturable, similarly to the human intestinal microbiota P. falciparum's capacity to establish infection through the removal of bacteria with antibiotic treatment, according to the established methodology 4 CFU, and those fed on blood contained as many as 106 CFU   .Plasmodium action, we compared infection intensities between the septic and aseptic mosquitoes at two time points after ingestion of infected blood: at 28 hrs when ookinetes are still invading the midgut epithelium and at 10 days when all viable parasites have developed into oocysts on the basal side of the midgut epithelium. A significant larger number of ookinetes were found in the midgut epithelium of aseptic mosquitoes compared to the septic at 28 hrs after ingestion, suggesting that the bacteria-mediated anti-Plasmodium action has already taken place at pre-oocyst stages (p<0.01) (To gain a better understanding on the infection stage– specificity of this anti-(p<0.01) . ParasitPlasmodium , in contrast to only 40% of the aseptic group despite an approximately 5-fold higher infection level . Interestingly, malaria-infected aseptic mosquitoes in which the midgut bacteria had been re-introduced exhibited reduced levels of mortality compared to untreated septic mosquitoes, possibly due to the presence of residual antibiotic in the tissues of these mosquitoes , and a 2.2-fold fewer oocysts developed in mosquitoes that had co-fed on heat-inactivated bacteria (HIA) (p<0.05) compared to the control mosquitoes. These and the previously described results suggest that the bacteria in the midgut lumen exert an anti-Plasmodium effect that could either involve a mosquito response or a direct interaction with the parasite. The frequency distribution of oocysts demonstrated that co-feeding with either live or heat-inactivated bacteria and pre-injection of live bacteria (discussed below) resulted in an over dispersion of oocysts, with the majority of mosquitoes having very few oocysts .The decreased numbers of developing oocyst on the midguts of mosquitoes that had been exposed to bacteria suggested that the bacteria-mediated inhibitory activity on the parasite is acting prior to the oocyst stage. To test whether the negative effect of bacteria on malaria parasite development was to some degree attributed to a direct interaction by which the bacteria kill P. falciparum occurs during ookinetes invasion, most likely through a mosquito response to the bacteria challenge which is likely to entail components of the mosquito innate immune system. Previous studies have indeed shown that the mosquito uses some of the same immune factors to combat bacteria and Plasmodium parasite infection The prevalence of ookinetes in the blood-meal at 24 hrs after ingestion showed no significant difference between the four cohorts, suggesting that the bacteria had no effect on the pre-invasive stages. However, the number of ookinetes observed within the midgut epithelium was significantly higher in the aseptic mosquitoes, by approximately a 2.5-fold compared to the cohorts that contained bacteria  while injection of heat inactivated bacteria had an insignificant effect on Plasmodium infection (p>0.05), compared to the PBS injected controls  and Gram-positive (Staphylococcus aureus) bacteria and control mosquitoes that had fed on uninfected blood with PBS. These treatments resulted in a much broader response. The ingestion of these bacteria triggered the regulation of as many as 656 and 520 genes in the midgut and carcass, respectively , while gene silencing of Cec3 and PGRP-LB resulted in an increased susceptibility to P. falciparum only in the aseptic mosquitoes . In contrast, when ClipA9 was silenced in aseptic mosquitoes it had no significant effect on susceptibility to the parasite , and the slope of the regression line (m = 1.247) demonstrated a high degree of correlation of the magnitude of regulation between the two assays (Probe sequence design and microarray construction were kept the same as described in o assays .http://frodo.wi.mit.edu/). All the primer sequences were listed in Primers' sequences for validation of microarray hybridization data were as described in dsRNA mediated gene silencing was done according to dsRNA as a control group or with target gene–specific dsRNA for the experimental group. Gene silencing in the whole mosquitoes was verified 3 to 4 d after dsRNA injection by qRT-PCR, done in triplicate, with the A. gambiae ribosomal S7 gene as the internal control for normalization. Gene silencing efficiency were listed in dsRNA–injected) and 50 experimental (gene dsRNA–injected) mosquitoes were fed on the same P. falciparum NF54 gametocytes culture at 3–4 d after the dsRNA injection. 24 hrs post blood feeding (pbf), the unfed mosquitoes were removed and the fed-mosquitoes were dissected at 7–8 d after feeding and midguts were stained with 0.2% mercurochrome dsRNA–injected control mosquitoes. The results for equal numbers of midguts from all three independent biological replicates were pooled. The dot plots of the oocysts number in each midgut within each treatment were presented by MedCalc software with the median value of the oocysts indicated. The Kruskal-Wallis (KW) test and Mann-Whitney test were used to determine the significance of oocysts numbers (p<0.05).Sense and antisense RNAs were synthesized from PCR-amplified gene fragments using the T7 Megascript kit (Ambion). The sequences of the primers are listed in E. coli and 60,000 S. aureus, or a mixture of heat-inactivated bacteria with the same number as the live ones. 24 hrs or 48 hrs after injection, mosquitoes were fed with P. falciparum NF54 gametocytes culture which were carried out according to our establish protocols About 80 4-day-old mosquitoes were first injected with PBS as control, or a mixture of live bacteria with approximately 30,000 dsRNA injection, and CFU were determined by plating the homogenate of the midguts with series dilutions on LB agar plates and incubating the plates at 27°C for 2 days. Each assay was performed with one midgut and at least 10 independent replicates were included for each gene. The species of the isolated bacteria were determined by amplifying a region of the 16s rDNA as described by using primers 27f and 1492r Isolation and colony forming units (CFU) enumeration of bacteria from midguts of untreated control, antibiotic-treated mosquitoes and gene-silenced mosquitoes were done essentially according to P. falciparum development within untreated, antibiotic-treated and bacteria co-feeding mosquito midguts were compared by using the immuno-staining of ookinetes with anti-Pfs25 antibody . Preparation of samples for immuno-fluorescence microscopy of malaria parasite within the bloodmeal was carried out based on The early stages of Figure S1A. gambiae Keele mosquitoes after P. falciparum infection. At least 40 mosquitoes were in each replicate, and three replicates were included with standard errors shown. Non-treated: septic mosquitoes harbor natural microbiota; Antibiotic-treated: mosquitoes treated with antibiotics, referred as aseptic mosquitoes; Rechallenged: aseptic mosquitoes co-fed with bacteria and P. falciparum infected blood.Survival rates of (0.02 MB PDF)Click here for additional data file.Figure S2Validation of microarray-assayed gene expression with qRT-PCR. The values for the expression data obtained by microarray analysis (log2 ratio) for six genes were plotted against the corresponding expression values obtained with qRT-PCR  from two biological replicates of each experiment. Only the comparisons between the whole septic and aseptic mosquitoes which fed on sugar or uninfected blood were shown here.(0.01 MB PDF)Click here for additional data file.Figure S3dsRNA injection. dsGFP-injected mosquito midguts were used as controls, and 10 midguts were included in each replicate and at least two replicates were done with only one replicate shown here. Def1: defensin 1, Gam: gambicin; Cec: cecropin.Verification of gene silencing in the mosquito midgut tissue 4-d post (0.09 MB PDF)Click here for additional data file.Table S1dsRNA synthesis, qRT-PCR validation of RNAi-mediated gene silencing and the efficiency of gene silencing.Primers used for (0.01 MB PDF)Click here for additional data file.Table S2A. gambiae Keele mosquitoes fed with sugar (7-day-old whole mosquitoes minus head).List of genes identified from microarray analysis exhibiting significant differential expression between untreated septic and antibiotic-treated aseptic adult female (0.06 MB XLS)Click here for additional data file.Table S3A. gambiae Keele mosquitoes fed on uninfected blood (7-day-old whole mosquitoes minus head).List of genes identified from microarray analysis exhibiting significant differential expression between septic and aseptic adult female (0.06 MB XLS)Click here for additional data file.Table S4A. gambiae Keele mosquitoes 12 hrs after feeding on uninfected blood supplemented with E. coli and S. aureus, PBS substitution of bacteria as control.List of genes identified from microarray analysis exhibiting significant differential expression in the midguts of 7-day-old female (0.17 MB XLS)Click here for additional data file.Table S5A. gambiae Keele mosquitoes 12 hrs post feeding on uninfected blood supplemented with E. coli and S. aureus, PBS substitution of bacteria as control.Table S5. List of genes identified from microarray analysis exhibiting significant differential expression in the carcass of 7-day-old female (0.15 MB XLS)Click here for additional data file.


Beyond the well-characterized model nematode C. elegans, over 500,000 expressed sequence tags (ESTs) and nearly 600,000 genome survey sequences (GSSs) have been generated from 36 nematode species as part of the Parasitic Nematode Genomics Program undertaken by the Genome Center at Washington University School of Medicine. However, these sequencing data are not present in most publicly available protein databases, which only include sequences in Swiss-Prot. Swiss-Prot, in turn, relies on GenBank/Embl/DDJP for predicted proteins from complete genomes or full-length proteins.Nematode.net C. elegans counterparts, subject matches in KEGG's gene database, and also KEGG Ontology (KO) identification.Here we present the NemaPath pathway server, a web-based pathway-level visualization tool for navigating putative metabolic pathways for over 30 nematode species, including 27 parasites. The NemaPath approach consists of two parts: 1) a backend tool to align and evaluate nematode genomic sequences (curated EST contigs) against the annotated Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database; 2) a web viewing application that displays annotated KEGG pathway maps based on desired confidence levels of primary sequence similarity as defined by a user. NemaPath also provides cross-referenced access to nematode genome information provided by other tools available on Nematode.net, including: detailed NemaGene EST cluster information; putative translations; GBrowse EST cluster views; links from nematode data to external databases for corresponding synonymous . The nematode source sequences used for the metabolic pathway mappings are available via FTP , as provided by the Genome Center at Washington University School of Medicine.The NemaPath server hosts metabolic pathway mappings for 30 nematode species and is available on the World Wide Web at  Caenorhabditis nematode species [Nematodes or roundworms are one of the most common phyla of animals, with over 20,000 different described species , ubiquit species , and 32  species .We expect that complete annotated genomes are approximately 3–4 years away. Therefore, to empower the broader scientific community the available EST and GSS sequences from parasitic nematodes require organization in a functional context, a need underscored by their absent from the majority of publicly available protein databases such as Pfam  and KyotCaenorhabditis elegans and other related Caenorhabditis species, and Pristionchus.org [Pristionchus pacificus used in studying evolutionary developmental biology. NEMBASE3 [Currently, there are four comprehensive web-based databases available providing tools for exploring nematode sequences: two model-species-specific databases and two others encompassing parasitic and free-living nematode sequences. The model-specific databases include WormBase , a modelchus.org , a resouNEMBASE3  and NemaNEMBASE3  are dataHere we present NemaPath (made available as a component of Nematode.net) that allows for systematic study of nematode transcriptomes via enzyme pathway associations. NemaPath compares relatively refined nucleotide sequences  to the KEGG genes database of curated protein sequences. Sequences in the KEGG database have known, annotated Enzyme Commission (EC)  system nC. elegans, Brugia malayi, Ancylostoma caninum and Ascaris suum). Cluster sequence reports may be viewed online by reverse lookup using constituent contig IDs from NemaPath's pathway map hit table or by viewing the cluster in Nematode.net's implementation of the GBrowse [To eliminate data redundancy, ESTs have been assembled by identity into NemaGene contigs and further organized into clusters. ESTs within a contig derive from nearly identical transcripts, whereas contigs within a cluster may represent splice isoforms of a gene or transcripts from multi-gene families with extremely high sequence identity . NemaPat GBrowse  genome vFinally, summaries of mappings are provided as extendable tree-views, which organize mappings by identified EC numbers. The summary of pathway mappings when coupled with statistical tools  can provAlthough other excellent KEGG pathway mapping software platforms exist for EST-related data – such as ESTExplorer  and PathThe web-based NemaPath application  grouping of the ESTs into contigs based on sequence similarity; 2) elimination of chimeric ESTs; 3) accommodation of multiple splice-isoforms; 4) persistence of cluster names when new ESTs are added. For each contig with multiple EST members, the consensus sequence is longer and of higher quality than each stand-alone EST read. The KEGG genes file (used as a database against which we BLAST our queries) is comprised of concatenated FASTA protein sequences from all of the species in the KEGG genes database. As of this writing, the KEGG genes file can be retrieved for academic use by download via FTP from KEGG . Each FASTA entry contains metadata in the header line: species gene name, protein matched, EC id, and KEGG Orthology (KO) information. As the metadata represents information arrived at by KEGG's annotation process, we can make inferred relationships by identity using our nematode sequences.The initial step in the NemaPath pipeline involves analyzing clustered ESTs in context of the latest version of KEGG's high-quality genomes including manual assignment of orthologies. NemaGene EST contigs [, Materia.WU-BLAST  alignmenKEGGscan report information is cross-referenced with corresponding KEGG pathway image map information in the NemaPath database. EC ids have representative nodes within KEGG-supplied pathway bitmap images. Associations between NemaGene contig sequence and KEGG genes are pre-compiled on a per build basis for each NemaPath release. However, a user has the ability to narrow or broaden the pool of viewable matches by supplying a threshold for E-value, which is a statistical value of the quality of the BLAST alignment. To aid the user in this endeavor, NemaPath supplies an E-value hit distribution graph for each nematode species.C. elegans entry is listed and a link provided to the corresponding elegans gene page in WormBase.Once an E-value threshold is chosen, a user is presented with a table of pathways available for the chosen species; only pathways with matches based on the threshold the user has selected are presented. Choosing a pathway will present an annotated KEGG pathway image map wherein EC number nodes will be highlighted (colored) based upon number of corresponding matches to the node (darker colors represent enzymes with more matches over those of lighter color); the highlighted nodes are interactive in that a user may position their mouse pointer over the node to reveal a hit summary table  for which complete or partial gene sets are available based on finished or low coverage genome sequence. The C. elegans associations are supplemented with the information on RNA interference (RNAi) knockdown [C. elegans [C. elegans can be further extrapolated, to an extent, to orthologous genes in other nematodes where high-throughput screening is not yet practical [C. elegans associations by labeling enzymes within pathways  to each gene belonging to a clade or host category. Sequence data are currently available for four  of the five nematode phylogenetic clades. All five clades include parasitic species and parasitism is hypothesized to have arisen independently multiple times [Other multi-species views include clade-specific and host-specific aggregations that incorporate only the best matches ; functional classifications of many of the 70% of genes with homology to genes in other species were possible based on gene ontology and KEGG placement [As a step towards a full-scale genome project of lacement .Every effort has been made to improve the quality and fidelity of EST sequences by NemaGene clustering. However, final cluster products are putative, partial gene representations built on the most current information available. Enzyme Commission numbers are assigned to NemaGene contigs in the NemaPath database by primary sequence similarity. As such, associations include the caveats pertaining to any automated, high throughput alignment analysis.C. elegans was compared to the metabolic pathways in C. elegans in the KEGG database. This validation screen was performed using two cutoffs (1e-30 and 1e-100) and identified a number of unique EC ids by the original KEGG associations and also our NemaPath associations. Using 1e-30 and 1e-100 as a cutoff, we identified that 1e-100 gave a number of unique EC mappings closer to the original KEGG associations. Only in 4 out of 94 pathways were the number of EC ids identified by NemaPath were lower than in KEGG's C. elegans reference pathway. The revealed differences, consistently higher EC ids identified by NemaPath, are mainly based on the manual curation of the KEGG mappings compared to our automated associations guided by cutoff value; our associations do not account for the ancillary information included in KEGG Orthology numbers (KO) generation.To assess the accuracy of our methods, the NemaPath associations of the complete full-length gene set for a priori KO knowledge. By design, an association of a user's query sequence to a particular EC identifier will be highlighted in every pathway where the EC id exists, rather than exclude information from the end-user or enforce presumptive omissions. Partial EC numbers  are not excluded; care must be taken in their interpretation. The ambiguous nature of the partial EC number allows different enzymes that catalyze different reactions to share the same identifier within the same class, even though this does not necessarily mean they have the same activities [KO numbers incorporate ancillary information  that is not represented in the EC number annotation. KO is a further extension of this scheme (similarity-based automatic EC id association) based on computational analysis – as well as manual curation – of SSDB ortholog clusters in order to classify all gene functions and explore unknown pathways . NemaPattivities .Because we do not provide exhaustive curation, proper interpretation requires the user's cognizance of the metabolic pathways, as well as an understanding of KEGG annotation and vocabulary.All species-to-KEGG associations in NemaPath are re-compiled each time KEGG releases a new version of their genes database (as of this writing the associations are made using KEGG release 46). New nematode species are added post NemaGene clustering. Genome sequencing projects of several free-living and parasitic nematode species are underway or planned , and theNemaPath, the database described herein, provides the research community a unique resource for pathway visualization of multi-species nematode genomic data in terms of KEGG database vocabulary. NemaPath is part of the larger Nematode.net web resource and integrates well with the site's previous functionality, streamlining access to significant internal nematode sequence data, as well as information provided by off-site resources at NEMBASE3  and Worm on the World Wide Web. Direct access to the NemaPath pathway browser – as well as species-specific tree-views – can be found at  URL. Plaintext file versions of alignment information (as compiled by KEGGscan) are also available by species . The most current NemaGene EST cluster builds are available via FTP at  after completing a short access form. Please contact the authors concerning access to ancillary information associated with this resource.For accessing the Nematode.net web site, direct your web browser to TW developed the software and wrote the draft manuscript. JM, SA, and DM helped develop software and informatics approaches for the Nematode.net web site. YY and ZW contributed pathway analyses for specific nematode sets. JPM and MM initiated the parasitic nematode project at Washington University School of Medicine, St. Louis. All authors contributed to the writing and editing of this manuscript.


EdaTa and Edardl-J mutants. A detailed study of tooth morphology in mutants bearing losses of functions of these two genes thus appears necessary to test the pattern variability induced by the developmental modifications.The Eda-A1-Edar signaling pathway is involved in the development of organs with an ectodermal origin, including teeth. In mouse, mutants are known for both the ligand, Eda-A1 (Tabby), and the receptor, Edar (Downless). The adult dentitions of these two mutants have classically been considered to be similar. However, previous studies mentioned differences in embryonic dental development between EdaTa and Edardl-J mutants have then been compared in detail. Despite patchy similarities, our detailed work on cheek teeth in EdaTa and Edardl-J mice show that all dental morphotypes defined in Edardl-J mice resolutely differ from those of EdaTa mice. This study reveals that losses of function of Eda and Edar have distinct impacts on the tooth size and morphology, contrary to what has previously been thought.3D-reconstructions of the cheek teeth have been performed at the ESRF  by X-ray synchrotron microtomography to assess dental morphology. The morphological variability observed in Eda gene product but not that of Edar), a more complex connection than has been appreciated between Edar and another protein, or a ligand-independent activity for Edar. Further work is necessary to test these hypotheses and improve our understanding of the mechanisms of development.The results indicate that unknown mechanisms of the Eda pathway are implicated in tooth morphogenesis. Three hypotheses could explain our results; an unexpected role of the Xedar pathway (which is influenced by the  Eda gene, carried by the X chromosome, encodes the ligand (Eda-A1), and the Edar gene encodes its receptor (Edar). EdaTa (Tabby) and Edardl-J (Downless) mutant mice bear loss-of-function mutations for the Eda (ectodysplasinA) and the Edar (ectodysplasinA-receptor) genes, respectively Eda and Edar mutants in the histological structure of the submandibular salivary gland have been reported Eda mutants has been deeply investigated. These studies revealed a high morphological diversity of the cheek dentition, characterized by modifications in the number of teeth and in the number and arrangement of cusps for homozygous and heterozygous mice Edardl-J dental phenotype, which is usually supposed to display the same dental defects as in EdaTa mice EdaTa embryonic teeth are simply smaller in than those of WT mice Edardl-J mutant molars have a structure termed the ‘enamel rope’ which is composed of enamel knot cells that are extended across the tooth primordium due to a failure of cell condensation Eda and Edar losses of function are likely to have different consequences on mature dental morphology. This prompted us to study the cheek dentition in Edardl-J mutant mice to determine the extent to which EdaTa and Edardl-J phenotypes are similar and to discuss the implications of their putative differences and similarities.Genes of the Eda-A1/Edar signaling pathway are involved in the development of organs with an ectodermal origin, such as hair, glands, teeth Edardl-J mice (FVB background) have been bred at the PBES of IFR 128 (Lyon). These mice carry a G to A transition mutation causing a glutamate to lysine substitution in the death domain of the Edar protein (E379K) Edar mutant mice used either EdardlEdarsleekEdardl-J mice have been used in more recent studies comparing the dental development and functional morphology of Eda and Edar mutant mice Edardl-J and Edarsleek dental morphology showed no differences Eda and Edar mutant mice were identified according to external morphological criteria, such as the bald spot behind ears. Heterozygous Eda  mice were identified morphologically by the distinctive striping of the coat that gave these mutants their original name, ‘Tabby’. Heterozygous and wild-type Edardl-J specimens exhibit similar external traits and were genotyped through PCR amplification of a 306 bp fragment covering the point mutation (primers: 5′ GTCTCAGCCCCACCGAGTTG and 3′ GTGGGGAGGCAGGTGGTACA), followed by sequencing. The Edardl-J sample was composed of 20 heterozygous (Edardl-J/+), 47 homozygous (Edardl-J) and 5 control (WT) specimens. The Tabby sample used in comparison overlaps the sample studied by Kristenova et al. EdaTa null allele of the Eda gene, carried by the X-chromosome. EdaTa mice are on a mixed background (C57Bl6J+CBA), the sample included 60 heterozygous females (EdaTa/+), 23 homozygous (EdaTa/Ta) or hemizygous females  and 40 WT mice. Mice were killed by cervical dislocation. The experimental protocol was designed in compliance with recommendations of the EEC (86/609/CEE) for the care and use of laboratory animals.The x and Tx where T and x respectively symbolize the tooth and its rank within the row .The uncertain homology of teeth between WT and mutant mice led us to adopt a nomenclature using TCheek teeth were examined using a Leica MZ16 stereomicroscope. Morphotypes have been defined for lower and upper tooth rows on the basis of the number and arrangement of cusps. Occlusal surface areas of cheek teeth were measured from digitized pictures using Optimas software. It has been demonstrated that X-ray synchrotron microtomography brings very high quality results for accurate imaging of small teeth EdaTa and Edardl-J mice. This is a non-parametric method, which tests equality of population medians among groups. The non-parametric Wilcoxon rank-test is used to indicate which groups are statistically different from the others.Statistical tests have been performed to compare tooth size. The Kruskal-Wallis analysis of variance was used to verify the significance of observed differences between EdaTa and Edardl-J breeding colonies all display the normal dental morphology, without anomalies, and measurements show similar tooth size . Thus, molars from WT from both genetic backgrounds were included in a single WT sample.WT specimens from Eda is located on the X chromosome while Edar is on an autosome, morphological differences linked to the X-inactivation process were predictable. Accordingly, in both the upper and lower cheek dentitions, heterozygous Edardl-J/+ mice display dental morphologies very close to WT specimens  a T1 with a single lingual cusp (asterisk in (ii) a T2 with a lingual interconnection between mesial and distal cusps  The first morphotype, Dl1 (for Downless1), includes 45% of the studied material. It is characterized by a three-toothed row and a four-cusp T1. The three mesial cusps form a three-leaf clover shape, while the fourth cusp is either isolated in the distal part of the tooth  The Dl2 morphotype (12% of examined tooth rows) also exhibits three cheek teeth, but the T1 only displays three cusps. As in Dl1 morphotype, the cusps form a three-leaf clover shape connected in the centre of the occlusal surface (arrow in TaEda Ib morphotype (2 ((iii) The Dl3 morphotype (31% of tooth rows) is characterized by rows with two lower cheek teeth (1 of the Dl3 morphotype resembles this of the Dl1 morphotype (EdaTa IIb morphotype (EdaTa IIa and IIb morphotypes ((iv) The Dl4 morphotype (7% of tooth rows) is characterized by a small T1  Tek teeth . The T1 rphotype . Howeverrphotype  to a higrphotype . This larphotype . The Dl3photypes . (iv) Tharrow in , 57% of rphotype . HoweverEdaTa/+ and Edardl-J/+ mice can be explained by the fact that the Eda gene is X-linked and that Edar is carried by an autosome. Due to the X-inactivation effect in females Ta/+Eda mice are mosaics of cells with expression of a wild-type or a null Eda gene. This might induce a strong and random variability in the amount of Ectodysplasin-A1 protein available for dental development, explaining the higher morphological variability recorded in EdaTa/+. This phenomenon does not occur in the Edardl-J teeth since the gene is located on an autosome (chromosome 10).The many observed differences, and the higher variability of tooth rows, between EdaTa and Edardl-J mutants. However, none of the EdaTa and Edadl-J tooth rows are identical. The two mutants are supposed to display total losses of function of the ligand and the receptor of the Eda pathway, respectively. According to Tucker et al. Eda mice have small enamel knots while Edardl-J mice have disorganised “enamel ropes”. As enamel knots are orchestrating the final mineralized crown pattern, differences of size and morphology, differences in enamel knots are likely to explain differences of size and morphology between EdaTa and Edardl-J dentitions.The same general trends in the reduction of the cusp number and tooth size are observed in both (i) They could be linked to epistatic differences between the two different backgrounds of EdaTa and Edardl-J mice (respectively mixed C57Bl6J+CBA and FVB). However we consider this possibility to be unlikely to be the only explanation. Indeed, suppression of Eda action in embryonic CD-1 mice by addition of soluble forms of Edar to tooth explants results in the same phenotype as seen in EdaTa mice on NMRI and mixed CBAT6T6xNMRI backgrounds but differs from the Edardl-J on CD-1 background EdaTa and Edardl-J mice are not due to a difference in background but rather to an intrinsic difference between loss of Eda function and alteration of Edar function in Edardl-J. (ii)Eda codes for two proteins, Eda-A1, which binds to Edar, and Eda-A2, which binds to Xedar EdaTa mice but still present in Edardl-J mice. An effect on tooth development of the loss of the Xedar pathway in the case of Eda loss of function and not in the Edar one might explain some of the differences that we report. Though a study of Xedar-null mice indicated no requirement for this gene in the normal development of ectodermal organs (iii) The Edardl-J allele may not be a null mutation. Indeed, the Edardl-J mutation causes a substitution in the death domain of the Edar protein, which connects to the Edar adapter with death domain, Edaradd Edardl-J mutants EdarE379K protein may retain some interaction with Edaradd allowing transmission of a weak signal through this pathway. Assays in cultured cells have given conflicting results on the nature of the signal output of EdarE379K. Yan et al. (iv) A ligand-independent activation mechanism for Edar could also explain the morphological differences observed. Indeed, the finding that elevation of Edar expression can rescue the hair phenotype of EdaTa mutant embryos Edar. This hypothesis could be tested by crossing Edardl-J with EdaTa mice. Such a cross should result in an Edardl-J type phenotype. Another test could be a cross of Tabby mice with a loss-of-function mutant of the Edaradd gene that should also give rise to an Edar loss-of-function phenotype.Various hypotheses could explain these differences: Eda or Edar leads to different dental morphologies, presumably linked to the differences in their enamel knot morphologies. These differences indicate unknown mechanisms of the Eda pathway involved in tooth morphogenesis. These unknown mechanisms we hypothesize to be based on an unexpected role of the Xedar pathway in tooth development, a more complex connection than thought between Edar and Edaradd, or on a ligand-independent activity for Edar.To sum up, loss of function of


This study explores the feasibility of non-invasive evaluation of left ventricular (LV) flow-volume dynamics using 3-dimensional (3D) echocardiography, and the capacity of such an approach to identify altered LV hemodynamic states caused by valvular abnormalities.Thirty-one patients with moderate-severe aortic (AS) and mitral (MS) stenoses  and 10 healthy volunteers underwent 3D echocardiography with full volume acquisition using Philips Sonos 7500 equipment. The digital 3D data were post- processed using TomTec software. LV flow-volume loops were subsequently constructed for each subject by plotting instantaneous LV volume data sampled throughout the cardiac cycle vs. their first derivative representing LV flow. After correction for body surface area, an average flow-volume loop was calculated for each subject group.Flow-volume loops were obtainable in all subjects, except 3 patients with AS. The flow-volume diagrams displayed clear differences in the form and position of the loops between normal individuals and the respective patient groups. In patients with AS, an "obstructive" pattern was observed, with lower flow values during early systole and larger end-systolic volume. On the other hand, patients with MS displayed a "restrictive" flow-volume pattern, with reduced diastolic filling and smaller end-diastolic volume.Non-invasive evaluation of LV flow-volume dynamics using 3D-echocardiographic data is technically possible and the approach has a capacity to identify certain specific types of alteration of LV flow-volume pattern caused by valvular abnormalities, thus reflecting underlying hemodynamic states specific for these abnormalities. Measurement of left ventricular end-systolic (ES) as well as end-diastolic (ED) volumes and LV ejection fraction (EF) is a well-established procedure in the evaluation of cardiac function. Two-dimensional (2D) echocardiography is the most commonly used imaging modality in the diagnosis of heart diseases and the primary tool for the assessment of LV function and volumes. However, 2D echocardiography has limited reproducibility due to the fact that algorithms for the calculation of LV volumes and EF are based on certain geometrical assumptions that do no necessarily hold true in different clinical scenarios, as for example, in the presence of regional wall motion or structural abnormalities and dilated ventricles with changed geometry -3. NeverThree-dimensional echocardiography offers the possibility of a more accurate assessment of LV volumes and EF. Studies performed hitherto have consistently demonstrated improved accuracy of LV volumetry by 3D echocardiography -13 compaThe most reliable method for the evaluation of LV contractility is the simultaneous, real-time measurement of LV pressure and volume and the analysis of thus created pressure-volume loop diagram . HoweverThree-dimensional echocardiography provides possibility of a direct measurement of LV volumes and the first derivative of LV volume would describe blood flow in and out of the LV. Hence, by using volume and flow data extracted from the 3-D data set acquired throughout the cardiac cycle, left ventricular flow-volume loops can be constructed. The aim of the present study was to explore the feasibility of non-invasive evaluation of LV flow-volume dynamics using 3D echocardiography, and to assess the diagnostic potential of such flow-volume loops in two different types of hemodynamic pathologies, i.e. the obstructive disorder due to aortic stenosis and the restrictive state resulting from mitral stenosis.The study population consisted of 41 subjects. Thirty-one of them  were patients referred to the Department of Cardiology, Malmö University Hospital for echocardiographic evaluation of mitral or aortic stenosis, and 10 subjects  were healthy volunteers. In addition to standard 2D echocardiography, 3D echocardiography was performed in all subjects. The severity of the mitral and aortic stenosis was defined according to the clinical practise guidelines at Malmö University Hospital and in agreement with ACC/AHA guidelines  as mild,All 3-D echocardiographic examinations were performed by the same experienced sonographer with the patient in the left lateral position. 3D images were acquired over 4 consecutive cardiac cycles during a breath hold from the apical window using Sonos 7500 equipment and a full matrix array ultrasonographic (x4) transducer . The sampling frequency of 3D data was 15 – 24 frames/s. The acquired data sets were stored on CD-ROM disks and transferred to a standard PC for subsequent off-line analysis using commercially available software 4D LV-Analysis CRT 1.0 . The analysis started with the definition of long axis plane in apical 5-chamber view, whereupon 6 long axis image planes of LV were automatically generated. In each of the 6 image planes, the position of mitral valve, aortic valve and apex was marked at the end of systole  and diastole (largest LV cavity size). The semi-automated border detection algorithm provided subsequently a delineation of LV endocardial border with a preconfigured ellipse that was manually adjusted and the total endocardial border in all frames could be defined. The analysis program allowed then a reconstruction of LV volumes as a dynamic surface-rendered image in which LV wall motion was shown three-dimensionally. A graph describing LV volume changes throughout the cardiac cycle was also obtained . All volume variables were then adjusted for body surface area and plotted against its first derivative (describing LV flow) for each cardiac cycle to create LV flow-volume loops. Subsequently, an average flow-volume loop for each examined patient group was constructed. The created flow-volumes loops are limited to one cardiac cycle and lack time scale having LV volume as t-test for unpaired samples was used as appropriate.All values are presented as mean ± SD. The Student's 3D-echocardiographic data were acquired from all 41 subjects. However, in 3 patients with aortic stenosis (AS), the image quality was poor and their 3D data have been excluded from analysis.The clinical characteristics of the study population are presented in Table A schematic diagram of a LV flow-volume loop during one cardiac cycle is presented in Fig. The average LV flow-volume loops in patients with severe valvular abnormalities and in individuals without valvular pathology are presented in Fig. Analysis of invasively derived LV pressure-volume loops is a very useful and reliable method for monitoring of myocardial contractility (16) and an appreciated research tool whenever detailed information about the dynamics of LV performance is needed. However, the method suffers from its invasiveness that limits its general applicability in clinical cardiology. Therefore, there has been a long-felt need of most cardiologists for an alternative, non-invasive approach to the evaluation of myocardial contractility and it has been proposed that non-invasive measurement of changes in end-systolic pressure/volume ratio (systolic pressure by cuff sphygmanometer/end-diastolic volume by biplane Simpson's rule method) from rest to peak exercise could be used as a sensitive indicator of left ventricular function . Based oThe concept of LV flow-volume loop provides a new and different from pressure-volume loop approach to the estimation of the dynamics of cardiac performance. The approach was originally proposed by Söderqvist et al.  and was The hemodynamic information that can be retrieved from flow-volume loops describes the dynamics of flow-volume events. For this reason, the present approach is superior to the standard methods of functional LV assessment that use only end-diastolic and end-systolic volumes for calculation of LV ejection fraction. Incorporation of the dynamics of LV flow and volume in the same loop provides a possibility of a new and more detailed assessment of cardiac hemodynamics from different point of view. The continuous information about LV flow and volume expressed by flow-volume loop provides the prerequisites for hemodynamic pattern recognition in health and disease. In this process, the qualitative assessment of flow-volume loop form can be used as a tool for a rapid evaluation of cardiac hemodynamics and differentiation between normal state and different cardiac pathologies, as for example, significant valvular diseases that would change the normal pattern of LV filling and emptying. In the course of disease in any given patient, the sequential comparison of the respective loop forms and positions in the diagram can be easily performed and may provide valuable additional information and therapeutic guidance.The present results demonstrate that flow-volume loops based on 3D echocardiography measurements indeed have a potential to separate groups of different hemodynamic states. The characteristics of the average flow-volume loops obtained in normal subjects and in patients with AS an MS differed and were easily distinguishable. The patients with AS demonstrated a typical "obstructive" pattern with lower flow values during early systole and larger end-systolic volume as compared to normal pattern. On the other hand, in the patients with MS a "restrictive" pattern with reduced diastolic filling was observed. The prevailing hemodynamic state may thus be rapidly determined by recognition of a specific typical pattern of flow-volume relationship.The present methodological approach has, however, some limitations. The major limitation is caused by the relatively low 3D-echocardiographic sampling rate ranging from15 to 24 Hz. This would increase the variability of the flow-volume loops. As a consequence, although providing a typical pattern for a group of patients with a specific cardiac pathology, the loops may not yet be applicable on the individual basis. The other important limitation lies in the fact that the same data set is used for calculation of both volume and flow, the latter being calculated as the first derivative of volume. This limitation can be overcome by using integrated 3D colour Doppler in the LV outflow tract for flow measurements and 3D grey scale data for continuous measurement of LV volume. Finally, the image quality is also a limiting factor.3D echocardiography offers the possibility of non-invasive estimation of the dynamics of LV flow-volume relationship during cardiac cycle. The approach provides new information about LV hemodynamics and function, and has a capacity to differentiate between normal LV hemodynamics and specific types of alterations of flow-volume relationships caused by valvular abnormalities, such as aortic and mitral stenoses. The method has a potential to provide additional hemodynamic information in the evaluation of cardiac and valvular function.The author(s) declare that they have no competing interests.RW and LÅB initiated the study. RW supervised the study and participated in the interpretation of the results and manuscript preparation. KS performed measurements, made all data conversions, plots and calculations from ultrasound data, and participated in the preparation of the manuscript. PG participated in data collection and interpretation of the results. ES participated in data conversion, creation of plots and calculations from ultrasound data. JN performed statistical analysis and was responsible for final manuscript. All authors read and approved the final manuscript.


We have investigated the effect of the soybean isoflavone genistein on the growth and differentiation of human melanoma cells. Four human melanoma cell lines, either completely lacking or containing different levels of wild-type p53, were treated with genistein in vitro in culture. It has been found that genistein significantly inhibited cell growth and that the chemosensitivity might depend on cellular p53 content. Specifically, the data suggest that high levels of wild-type p53 expression make cells resistant to genistein's growth-inhibitory action. Further support for this observation came from the stable transfection studies in which p53 transfectants expressing high levels of wild-type p53 became resistant to genistein. With respect to cell differentiation, our study showed that genistein increased melanin content and tyrosinase activity and caused the cells to form dendrite-like structures. Cells lacking p53 responded more than cells with p53 to dendrite-like structure formation. We also observed that genistein-induced differentiation involved an increase in tyrosinase mRNA level; the mechanisms by which genistein increases tyrosinase transcripts remain to be elucidated. Genistein treatment of the melanoma cell lines resulted in cell cycle arrest at G2/M check point and no significant apoptosis was observed.


These in vitro results prompted us to examine the effect of Epo-signal withdrawal on tumours in vivo. RT-PCR analysis demonstrated the expression of mRNAs for Epo and EpoR in the transplants of uterine and ovarian tumours in nude mice. Then we injected locally anti-Epo antibody or soluble form of EpoR into the transplants. At 12 h, 1, 7 or 14 days after the injection, all transplants were resected and examined macro- and microscopically. Tumour size was reduced in Epo signal-deprived transplants. Immunohistochemical examinations revealed destruction of Epo-responding malignant and capillary endothelial cells through apoptotic death. The degree of tumour regression correlated well with the dose and frequency of the injections. Control xenografts with saline injection or needle insertion showed well-developed tumour masses. This Epo response pathway will have profound implications for our understanding of the development and progression of malignant tumours and for the use of Epo-signal deprivation as an effective therapy. © 2001 Cancer Research Campaign


A long term follow-up study has been undertaken in 33 patients with acute non-lymphoblastic leukaemia (ANLL) in order to establish whether a correlation exists between the clinical course and the immunologic pattern of lymphoid subpopulations. Peripheral blood lymphoid cells have been investigated longitudinally (each 1 to 4 months) during complete remission (CR), by morphologic, phenotypic and functional analyses. Particular attention has been paid to the evaluation of the natural killer (NK) cell compartment, by the detection of cells expressing an NK-related phenotype and by NK in vitro assay. Among the patients so far evaluable, 20 relapsed (R) and 10 are long survivors in CR 'off therapy' (LS). The most relevant finding was represented by statistically higher values of NK activity observed in LS vs. R patients (P less than 0.01). The removal of adherent cells before the NK assay, performed to investigate the possible inhibitory effect on NK function played by the macrophage component, abolished this difference, due to a selective increase of NK function in the R group. The longitudinal study revealed that NK activity tended to decrease in individual patients who subsequently relapsed. These data suggest a possible role of NK cells in the relapse control of ANLL, although it cannot be excluded that the low level of NK activity observed in the R group is the result of impending relapse rather than its cause.


Aiming to assess the role of individual molecular structures in the molecular mechanism of ligand-receptor interaction correlation analysis, the recent Spectral-SAR approach is employed to introduce the Quantum-SAR (QuaSAR) “wave” and “conversion factor” in terms of difference between inter-endpoint inter-molecular activities for a given set of compounds; this may account for inter-conversion (metabolization) of molecular (concentration) effects while indicating the structural (quantum) based influential/detrimental role on bio-/eco- effect in a causal manner rather than by simple inspection of measured values; the introduced QuaSAR method is then illustrated for a study of the activity of a series of flavonoids on breast cancer resistance protein. Such an approach will also be helpful for checking the chemical domain spanned by training molecules – a feature of the paramount importance also for further external tests.The present communication wishes to start filling this gap by deepening the modeling of inter-molecular activity through extending the main concepts of recent developed Spectral-SAR –40, deve2.N-molecules, one can chose to correlate their observed activities M-selected structural indicators in as many combinations as:k=1 to k=M.Paradoxically, the main problem for QSAR resides not in performing the correlation itself but setting the variable selection for it; the mathematical counterpart for such problem is known as the “factor indeterminacy” –45 and aC-combinations a correlation (endpoint) QSAR equation is determined, say N-molecules within the l- selected correlation. Now, the Spectral-SAR version of QSAR analysis computes these activities in a complete non-statistical way, i.e. by assuming the vectors for both observed (activities) and unobserved (latent variables) quantities while furnishing their correlation throughout a specific S-SAR determinant obtained from the transformation matrix between the orthogonal (desirable) and oblique (input) correlations. Yet, besides producing essentially the same results as the statistical least-square fit of residues the S-SAR method introduces new concepts as:endpoint spectral normallowing the possibility of the unique assignment of a number to a specific type of correlation, i.e. performing a sort of resumed quantification of the models [algebraic correlation factorviewed as the ratio of the spectral norm of the predicted activity to that of the measured one, giving the measure of the overall (or summed up) potency of the computed activities respecting the observed one rather than the local  molecular distribution of activities around the mean statistical yields; thus, it is a specific measure of the molecular selection under study, always with a superior value to that yielded from statistical approach [spectral path, with the distance defined in the Euclidian sense as:allows for defining complex information as path distances in norm-correlation space with norms computed from Yl corresponds to the measured activity Al defined as logarithm of inverse of 50%-effect concentration (EC50), see bellow, both modulus of Yl vectors and R values have no units so assuring the consistency of the least spectral path principle, formally shaped as:provides a practical tool in deciding the dominant {α,...}hierarchies along the paths constructed by linking all possible k-models (i.e. models with k correlation factors) from (1a) combinations selected one time each on a formed path – generating the so called “M-endpoints containing ergodic path on K-paths assembly” of (1b). However, the implementation of the principle (5) is recursively performed through selecting the least distance computed upon systematically application of C-models where connected on shortest paths; there was already conjectured that only the first M-shortest paths  are enough to be considered for a comprehensive (and self-consistent) mechanistic analysis [Basically, for each of the e models ;algebraapproach , howeveranalysis –40.inter-endpoint norm difference (IEND),▪ that accounts for norm differences of the models lying on the M-shortest spectral paths linking M-from the C-models of inter-endpoint molecular activity difference (IEMAD),▪ is considered from activity difference between the fittest molecules , in the sense of minimum residues, for the models  belonging to the shortest paths α1,..., αM for which the inter-endpoint norm difference is given by Nevertheless, for present purpose another two quantities are here introduced, namely:i, j) as reciprocally activated by the models  through the spectral path whom they belong; put in analytical terms, the difference between quantities of transition activity that turns the effect of i molecule on that of j molecule across the least spectral  path connecting the models l and l’:This way, we can interpret the two fittest molecules  and for special selected paths (the least for the M-ergodic assembly), being M and C related by ○ it involves specific transition or specific transformation of the effect of a certain molecule into the effect of another special molecule out from the N-trained molecules, paralleling the phenomenology of consecrated quantum transitions;○ it provides the quantum-SAR factor of an exponential form○ it has the amplitude of transformation driven by the so called identity○ it allows the “real” quantum-SAR transformation○ it has a “phase” with unity norm, in the same manner as ordinary quantum wave functions, allowing the inter-molecular multiple transformations take place across paths with multiple linked models, say , the inter-molecular transformation i→j→t is characterized by the overall quantum-SAR factor (10) written as product of intermediary onest or intermediated by j molecular effect transformations, respectively:M-contained k-endpoints giving an overall quantum-SAR (“metabolization power”) factor as:○ when um paths ; certainself-transformation as well, with the driven qua-SAR factor given by:i=j) is metabolized by activating certain structural features (l≠l’) though specific indicators (variables) in correlation (bindings with receptor site); this case resembles the stationary quantum case according which even isolated (or with free motion), the molecular structures suffer dynamical wave-corpuscular or fluctuant transformation along their quantum paths;○ With the present Qua-SAR methodology one can appropriately identify the molecular pairs that drive certain bio-/eco- activities against given receptor by means of selected descriptors in a “wave”-or “quantum” mechanistic formal way. The ultimate goal will be the computation of quantum-SAR factors along the least paths of actions that give the potential information of the conversion power of the fittest molecules in their specific bindings.However, in order to practically understand the actual Qua-SAR approach all steps above will be in next specialized through an application for identifying the most involved polyphenolic molecules for their activity related to mammalian breast cancer.3.Although in general considered beneficial for their protective role in many age-related diseases - flavonoids (see 50) of the maximum increase in mitoxantrone (MX) inhibitor substrate accumulation (interaction) with breast cancer resistance protein (BCRP), helping in reversing the multidrug resistance (MDR) mechanism of overexpressing MCF-7 MX100 cancer cells [For instance, recently, it was inferred that for certain flavonoids such as chrysin, nbiochanin A and apigenin a very low micromolar concentration is capable of producing 50% (ECer cells –54.TOT) Hantsch correlation variables [Therefore, in order to assess the molecular role and structural- related mechanisms for potential lead compounds in the drug design for anti-cancer treatment, a series of representative classes of flavonoids have been employed, see ariables , see Tabthe inter-endpoint molecular activity differences of quantum-SAR factor of Note that in inter-endpoint norm difference of Next, for computing the other influential activity difference in Qua-SAR, namely the TOT unfolds some statistically sensitive role in ligand (MX)-receptor (BCRP) binding (model Ic). The last assertion may also be sustained by going to the two-correlated parameters endpoint models, when one can see the confirmation of the stericity role through ETOT correlation variable: while combination LogP∧POL does not improve the statistical correlation of model IIa significantly over single-parameter LogP∨POL correlations, the total energy presence provides better and better correlation behavior as it is combined with LogP (the model IIb) and with POL (the model IIc), respectively. Instead, when all the Hansch structural variables are taken into account the model III is generated with appreciable statistical correlation respecting the other computed combinations.The first conclusion is that flavonoids have practically no exclusive or primarily role in drug transporting to BCRP site; still, the electrostatic influence through POL is practically missing as well (model Ib), while the stericity through ETOT correlation of model Ic as well as mixed correlations of bi-variable models IIb and IIc. Therefore, the mechanistic “alchemy” of structural features on molecular activity seems complex enough when all hydrophobicity, electrostatic and stericity influences combine as they are reciprocally activating one each other with a superior resultant in modeling ligand-receptor binding.Overall, it cannot be inferred that LogP and POL does have no influence on correlation only because when alone they do not correlate at all with flavonoids’ bioactivity, because their cumulative presence in model III highly improves the single ETOT in model III they considerably enrich the single ETOT correlation power of model Ic. Such behavior shows that orthogonal, i.e. independent, descriptors may provide better results when are combined than when considered apart due to the increase of the (inter) correlation space.Yet, the algebraic correlation factors in  ergodic paths with the spectral Euclidian lengths given by M = 3 paths with the dominant M-factors influence are selected by applying the above exposed recursive rule of least path principle resumed by Having performed the QSAR analysis, the specific Spectral-SAR stage can be unfolded by means of the Going now to the individual molecular level analysis, specific chosen structural variables . This is the case of the most fitted molecule on the most correlated endpoint (III) appeared to be no.3 (naringenin), with low activity on the observed range compared with the no. 25  in Such result tells us that the maximum recorded activity is not necessarily that one induced by TOT used in this study; it offers a visual way for assessing the almost no-correlation of LogP with other concerned variables, POL and ETOT, respectively. This lead with conclusion that LogP is almost orthogonal (independent) on (respecting) the other two Hantsch variables. Instead, when further performing the factor analysis, the TOT) interactions. Moreover, while PCA shows the POL factor influence equals that of ETOT, whereas their role in correlation is sensible different in Spectral-SAR analysis SAR results are to be compared with the consecrated Principal Component Analysis (PCA) . This wation see , from whIEND and IEMAD of per path is finally obtained by their couplings, according with multiplicative quantum rule of Nevertheless, going ahead with the Spectral-SAR results the Qua-SAR factors may be immediately recover by employing the molecular activity differences from This result may be quite important if such a behavior may be proven to hold in general since it would allow the effective quantification of paths according with their metabolization power. However, such endeavor exceeds the present communication purpose and will remain as a future challenge in Qua-SAR studies.M=)3 selected mechanistic paths of actions, see Finally, all QSAR, Spectral-SAR and Qua-SAR computational results may be collected and resumed by associate “spectral” scheme for evolution of the fittest molecular structures along the endpoint models for the (ToT) to IIb (LogP∧ETOT) corresponds to quantum free motion so that the null IEMAD for molecule no. 12  is carried; here, the quantum metabolization factor Going now to comment upon the “metabolization power” as indicated by the quantum-SAR factors on A somewhat different situation is met for β path in Even more metabolization “operations” take place along the γ path of ToT seems to mainly drive the mechanistic molecular transformation in MX-BCRP binding phenomenology, while the molecule no. 3 (naringenin) appears as the best fitted molecules belonging to the most relevant endpoint, in clear disjunction with the roughly molecular selection upon initial input observed activity data. That is, naringenin (no. 3) is shown to be the best adapted molecule for the actual LogP∧POL∧ETOT structural (independent) factors being metabolized from molecules as 6,2′,3′-7-hydroxyflavanone (no. 8), 4′-5,7-trimethoxyflavanone (no. 12), and flavone (no.13) by specific molecular mechanistically paths. However, there appears that these molecules are not linked even through the paths with the most active compounds of Overall, it is clear that the Qua-SAR scheme offers a quantification recipe along the most effective spectral paths combined with most fitted molecules for a trial basin of analogues compounds and structural variables. In the present case there was revealed that the energetic steric factor EOn the other hand, the present study affirms the position 7 of ring A and position 4′ of ring B respecting the pattern molecule no. 0 of Further Qua-SAR studies are necessary and will be developed for exploring other bio- and eco-active compounds for their interactions with organs and organisms; they may hopefully lead to a coherent analytical picture of chemical-biological bonding focused on selecting the most adapted molecules and of the most privileged molecular positions for delivering controlled structural based chemical reactivity and biological activity.4.in silico  chemical analysis respecting the bio- activity and availability of analogues substances, potentially beneficial or detrimental for specific interaction in organs and organisms, faces with a paradoxical dichotomy: if searching for the best correlation useful for prediction of specific molecular bio- or eco- activity QSAR models involving un-interpretable many latent variables may be produced, while always remaining the question of correlation factor indeterminacy (i.e. the assumed descriptors can be at any time replaced with other producing at least the same correlation performances); instead, when restricting the analysis to search for molecular design and mechanisms throughout performing SARs by means of special structural indicators for a given class of relevant molecules, arises the price of limiting the use of generated models for further prediction. The present communication is mainly devoted in developing the second (Q)SAR facet by extending the recent introduced notion of spectral-path-linking-endpoints and the associate least action principle to spectral path quantification, in terms of the best fitted molecules, along the contained computed models, by means of the introduced q(uantum)-SAR factor within the generally called Quantum-SAR (QuaSAR) methodology.The modern in vivo environment.As an application, for representative flavonoids’ inhibiting activities on breast cancer resistant protein there was clearly shown that the newly introduced q-SAR factor offers relevant analytical characterization of previously conceptually introduced spectral path hierarchy; moreover, the present QuaSAR may allow interpretation inter-conversion of concerned molecules’ towards receptor binding since belonging to the same class of analogs, while they certainly undertaking such transformation during their interaction with macromolecules, proteins and enzymes present on cellular walls or with Basically, the QuaSAR stands as the first step in assessing the quantum mechanically equivalent of wave function to the sample of molecules interacting with a specific organism site; it will eventually lead with the hyper-wave function with the help of which the associate hyper-density probability of binding (metabolization) is to be computed; the last information may provide the density probability map of the ligand-receptor interaction abstracted from the structural Spectral-Qua-SAR correlations; with this tool the molecular design of new chemical structures may be appropriately undertaken.However, the actual QuaSAR scheme and quantum factor carry the main features of quantum dynamical systems and may stimulate future computational and conceptual developments in molecular design for structurally controlled activity. Further generalization of the present QuaSAR method to modeling all potential inter-conversions of employed molecules involved in correlation as well as for establishing their quantum metabolization complete map  is actually in progress and will be reported in subsequent communications.