AEBP1/−− females display lactation defect, which results in the death of 100% of the litters nursed by AEBP1/−− dams. Mammary gland development during pregnancy appears normal in AEBP1/−− dams; however these mice exhibit expansion of the luminal space and the appearance of large cytoplasmic lipid droplets (CLDs) in the mammary epithelial cells at late pregnancy and parturition, which is a clear sign of failed secretory activation, and accumulation of milk proteins in the mammary gland, presumably reflecting milk stasis following failed secretory activation. Eventually, AEBP1/−− mammary gland rapidly undergoes involution at postpartum. Stromal restoration of AEBP1 expression by transplanting wild-type bone marrow (BM) cells is sufficient to rescue the mammary gland defect. Our studies suggest that AEBP1 is critical in the maintenance of normal tissue architecture and function of the mammary gland tissue and controls stromal-epithelial crosstalk in mammary gland development.Adipocyte enhancer binding protein 1 (AEBP1) is a multifunctional protein that negatively regulates the tumor suppressor PTEN and IκBα, the inhibitor of NF-κB, through protein-protein interaction, thereby promoting cell survival and inflammation. Mice homozygous for a disrupted AEBP1 gene developed to term but showed defects in growth after birth.  The mammary gland is a self-renewing tissue in which morphological changes and differentiation occur cyclically during menstruation, pregnancy and lactation. In prepubescent mice, the gland consists of a small ductal tree that emanates from the nipple into the proximal part of the fatty stroma, the mammary fat pad. Upon initiation of ovarian hormone secretion, the mammary epithelium enters an accelerated growth phase that leads to extension and branching of the ducts until they reach the limits of the fat pad. At the onset of pregnancy, extensive epithelial cell proliferation occurs, leading to the formation of lobulo-alveolar structures and secretory epithelial differentiation for lactation. At parturition, a dramatic increase in the expression of milk proteins and lipid biosynthetic enzymes has been observed in situ hybridization experiments showed that ACLP is expressed in the smooth muscle cells of the aorta, but not in skeletal muscle cells Adipocyte enhancer-binding protein 1 (AEBP1) is a transcriptional repressor that was initially shown to be involved in adipocyte differentiation TGAEBP1) mice, which over-express AEBP1 transgene in the adipose tissues and macrophages AEBP1/−− mice in vivo modulation of adiposity through its negative regulation of PTEN. PTEN has also been shown to play an important role in mammary gland development and tumorigenesis AEBP1 has been found to interact with mitogen-activated protein kinase (MAPK) in vivo data clearly demonstrated that macrophage AEBP1 plays critical regulatory roles in atherogenesis AEBP1 is also a pivotal player in macrophage cholesterol homeostasis and macrophage inflammatory responsiveness; key events involved in atherogenesis In this study, we explore the physiological role of AEBP1 in the lactation defect observed in AEBP1-null mice. Our results highlight the role that AEBP1 plays in signaling physiological maintenance of normal tissue architecture and function of the mammary gland tissue. BM transplantation experiments reveal that stromal expression of AEBP1 is required for normal mammary gland development. Our results presented here show that AEBP1 is a critical stromal factor that mediates stromal-epithelial crosstalk in the regulation of mammary gland development.All animal protocols used in these investigations have been reviewed and approved by the Dalhousie University Animal Care committee.AEBP1/−− mice was previously described ad libitum. Mice were sacrificed by euthanasia using an overdose of sodium pentobarbital (Somnitol), and mammary gland tissues were isolated for histological, whole mount and Western blot analyses.Generation of For the whole mount preparation of mammary glands, mice were sacrificed by cervical dislocation at different ages of virgin, at the appropriate time points during pregnancy, and at parturition and postpartum. The first inguinal and thoracic glands were removed and fixed in Carnoy's solution  for 4 hr. Subsequently, the glands were hydrated, stained with carmine-alum overnight, dehydrated, cleared in xylene, and mounted with permount. For histological analysis, mammary gland tissues were fixed in 10% acetate buffered formalin, paraffin embedded, sectioned at 5 µm and stained with hematoxylin and eosin. Histologic sections of mammary glands were examined in each instance.Beverly, MA, USA), anti-cleaved caspase 3 , or anti-C/EBPδ . The immunoperoxidase staining was performed using Vectastain ABC kit  according to the manufacturer's protocol. The signal was visualized using DAB peroxidase substrate (Sigma-Aldrich Co.).Mammary gland sections were deparaffined, rehydrated and treated in 10 mM sodium citrate (pH 6.0) by boiling for 10 minutes in microwave oven for antigen unmasking. The slides were treated with 3% hydrogen peroxide to inactivate endogenous peroxides and then treated with normal rabbit serum. The sections were treated with anti-p-STAT3  were isolated from an adult donor mouse (≥ 6 weeks old) and transplanted into the inguinal (#4) CFP of a 3 to 4 weeks old female mouse. The transplanted mammary gland tissue grew to form chimeric secondary mammary glands consisting of donor-derived epithelial component and recipient-derived stroma. Three types of chimeras were generated: AEBP1+/+ donor tissue into AEBP1+/+ recipient mice, AEBP1+/+ into AEBP1−/− recipient mice, and AEBP1−/− donor tissue into AEBP1+/+ recipient mice. The thoracic #3 mammary glands of the recipients served as controls. The mice were allowed to recover for at least 3 weeks and were subsequently mated with wild-type males. Following delivery (parturition), the mice were euthanized and their #3 normal and #4 chimeric mammary glands were dissected out. Half of each gland was immediately frozen in liquid N2 for protein analysis while the other half was prepared for histological analysis. Rat mAb TROMA-1  was used to detect keratin 8; an epithelial marker.Epithelial cell transplants were used to determine if AEBP1 protein is expressed in stromal or epithelial compartments of the mammary gland. Surgical removal of the parenchyma results in a gland-free mammary fat pad, referred to as cleared fat pad (CFP), which is suitable for receiving donor tissue. Briefly, portions of mammary gland (<2 mm6) through tail vein. Two weeks after BM transplantation, mice were placed on high-fat diet (HFD) for 20 weeks or mated at 8 weeks post BM transplantation prior to whole mount analysis. Donor cell engraftment was evaluated by qRT-PCR analysis of SRY expression in the splenocytes and BM cells.Recipient mice at the age of 8–10 weeks were provided with acidified water with antibiotic  for 2 weeks before BM transplantation procedure. BM cells were collected from the femurs and tibias of donor mice (6–8 weeks old). Total T cells were depleted using CD90.2 microbeads . Recipient mice were lethally irradiated (900 Rad), and then injected with BM cells . Mammary gland tissue was also digested in collagenase/dispase (Sigma) solution for 2 hr at 37°C. Separation of macrophages and epithelial cells were performed using magnetic EasySep Mouse Monocyte Enrichment Kit and EasySep Mouse Epithelial Cell Enrichment Kit , and cell purity was confirmed by flow cytometry. For nuclear protein extraction, the Active Motif nuclear protein extraction kit (Active Motif) was used according to the manufacturer's recommendations. Cell lysis was performed on ice for 30 min. Clear protein extracts were obtained by centrifugation for 30 min at 4°C. Protein separation and immunoblotting, followed by ECL immunodetection system (Amersham), were performed as previously described Mouse mammary gland whole cell protein extracts were prepared by homogenization in high-salt buffer 5 via prolactin (Prl) signaling, followed by the synthesis and secretion of their corresponding proteins (β-casein and WAP) AEBP1/−− mice during late pregnancy (Pg) and postpartum (PP), suggesting that AEBP1 is an obligate mediator of normal mammary gland function. Interestingly, both β-casein and WAP levels are significantly increased in AEBP1/−− mammary gland at PP  can be seen within luminal mammary epithelial cells. By the end of pregnancy, the CLDs increase dramatically in size. Following parturition, CLDs are smaller and localized to the apical surface of the alveolar epithelial cells AEBP1/−− mammary glands also exhibit greatly enlarged alveoli with extended lumina containing many large CLDs within luminal mammary epithelial cells at 1-day PP  status AEBP1/−− mammary glands at PP is evident based on p-STAT3 induction that usually peaks at day 1 of weaning . These results suggest that AEBP1 is critical in signaling the initiation of physiological apoptosis during mammary gland involution.It is interesting that an excessive number of exfoliated cells were detected in AEBP1/−− dams, it is plausible to predict that AEBP1 expression is regulated during the transition from pregnancy to lactation when secretory activation occurs To further substantiate the role of AEBP1 in mammary gland development, we examined the expression profile of AEBP1 in mammary gland tissue at various stages during postnatal development. Based on the mammary gland defect found in −/−AEBP1 mice could be due to defects in stromal cells and/or a cell-autonomous defect localized to the epithelial cells. We have previously shown that AEBP1 is expressed in preadipocytes, but not in mature adipocytes +/+AEBP1 or −/−AEBP1 nulliparous mammary epithelial tissues were transplanted into cleared mammary fat pads of nulliparous +/+AEBP1 or −/−AEBP1 females. After formation of chimeric secondary mammary glands, which consist of donor-derived epithelium and recipient-derived stroma, mice were mated with wild-type males. Since −/−AEBP1 mammary glands display alveolar collapse and epithelial cell hypoplasia at PP2d, the chimeric mammary glands were examined at parturition, exhibiting normal lobuloalveolar structure (+/+AEBP1 stroma (lanes 4 and 5), but not −/−AEBP1 stroma (lane 3). Since AEBP1 expression was analyzed at PP, it is likely that the slow migrating form of AEBP1 found exclusively in the PP stage , and an overall smaller size. Layne and coworkers never described the phenotypes of the surviving adult ACLP-knockout mice, except the deficiency in wound healing ACLP/−− fibroblasts observed in their studies is most likely due to AEBP1 disruption. We have previously shown that AEBP1 stimulates cell growth and survival and inhibits apoptosis through positive regulation of MAPK To gain insight into the physiological activity of AEBP1, AEBP1-deficient mice were generated by homologous recombination. Mice homozygous for a disrupted AEBP1 gene developed to term but showed defect in growth after birth with no other macroscopic physical aberrations. AEBP1−/− females, the mammary epithelial compartment showed aberrant development at late pregnancy and female mice were unable to nurse their newborn pups, possibly due to an impaired secretory activation at parturition. The glands may have failed to switch to a lactation state resulting in a rapid involution postpartum. It appears that disruption of AEBP1 gene caused premature mammary gland involution, which is characterized by elimination of epithelial cells by apoptosis. AEBP1 expression may be critical in regulating the apoptotic signal in mammary gland involution. The mammary gland has emerged as a rich developmental model that depends on epithelial-stromal interaction. The stroma-derived mediator that signals epithelial cell proliferation and apoptosis is still elusive. Crosstalk between the mammary stroma and epithelium is crucial for proper patterning and functioning of the mammary gland. Disruption of stromal-epithelial communication can promote uncontrolled proliferation of mammary epithelial cells and promote mammary tumorigenesis AEBP1 in the control of mammary gland development, presenting AEBP1 as a novel developmental factor whose expression is vital for the maintenance of mammary gland homeostasis. Hence, AEBP1 is a critical stromal factor implicated in mammary epithelial cell growth. In addition, AEBP1 may function as an indispensable mediator of the communication signal between the mammary gland stromal and epithelial compartments, orchestrating the functional differentiation and involution of the mammary gland epithelium.AEBP1 may be very critical in the functional development of the mammary gland. In AEBP1 gene in only a few cell types may indicate combinatorial regulatory mechanisms that utilize shared components leading to unique responses in mammary gland tissue. Our results open up a new avenue of research directed towards further understanding of the regulation of mammary gland development. The availability of AEBP1-knockout and AEBP1-overexpressing transgenic mice will allow a more precise clarification of the role of AEBP1 in maintaining homeostasis in the mammary gland tissue.These recent discoveries and developments have changed our understanding of AEBP1 functions and have offered new insights into the mechanisms by which endogenous AEBP1 exerts its physiological effects. The specific lesions resulting from the absence of ubiquitously expressed


Effective proteome analyses are based on interplay between resolution and detection. It had been claimed that resolution was the main factor limiting the use of two-dimensional gel electrophoresis. Improved protein detection now indicates that this is unlikely to be the case. Using a highly refined protocol, the rat brain proteome was extracted, resolved, and detected. In order to overcome the stain saturation threshold, high abundance protein species were excised from the gel following standard imaging. Gels were then imaged again using longer exposure times, enabling detection of lower abundance, less intensely stained protein species. This resulted in a significant enhancement in the detection of resolved proteins, and a slightly modified digestion protocol enabled effective identification by standard mass spectrometric methods. The data indicate that the resolution required for comprehensive proteome analyses is already available, can assess multiple samples in parallel, and preserve critical information concerning post-translational modifications. Further optimization of staining and detection methods promises additional improvements to this economical, widely accessible and effective top-down approach to proteome analysis. The main obstacle to effective and comprehensive proteome analysis has ostensibly been resolution. A variety of methods have been investigated in order to resolve ever smaller quantities of protein and detect them quantitatively In conjunction with these methodological improvements, advances in protein detection have also occurred Here, we capitalize on the resolution afforded by a refined 2DE protocol and sensitive protein detection using an established fluorescent stain, to test whether significantly more proteins are detectable following excision of hyper-abundant spots. Thus, how much of the proteome is resolved using a standardized 2DE protocol? By now addressing another issue concerning protein detection, we visualize protein species of low abundance in a genuine, well established top-down analytical format, thereby also avoiding the use of multi-step depletion methods which affect sample integrity; in a sense this is an alternate and complementary approach to our original introduction of post-fractionation and third dimension resolution Rats were originally obtained as breeding pairs under the UWS School of Medicine Animal Facility Rodent Breeding Program with the approval of the UWS Animal Ethics Committee . The donated rat brain tissue used here was obtained from old breeding pairs that had had their fifth litter and were due to be culled (Section 1.26). The method of sacrifice was carbon dioxide asphyxiation. All animals were handled in strict accordance with the UWS Animal Ethics Committee guidelines.All materials were of electrophoresis grade or higher and were supplied by Amresco . SR gel stain and 7 cm 3–10 non linear immobilized pH gradient (IPG) strips were purchased from Bio-Rad .Triplicate rat brains were pulverized using automated frozen disruption, and the powdered tissue fractionated as described previously 2O (ddH2O) and incubated on a rocker for 3×20 min at RT. Staining was by overnight incubation on a rocker in the dark with 40 mL of SR per gel.Reduction and alkylation steps were carried out as described previously High sensitivity imaging was accomplished with the LAS-4000  using standard sub-saturation exposure Delta 2D  software was used for quantitative image analysis. Gel images were submitted for automated protein spot detection and individual protein spot totals were averaged across replicates. 4HCO3 pH 9 containing 25% of the standard trypsin content (i.e. ∼3 ng/µL). Gel pieces were incubated for 10 min on ice followed by 12 h at RT. The tubes were then sonicated for 30 min and the supernatant removed to a fresh tube. Ammonium bicarbonate  was added to the hydrated gel pieces and sonication was repeated. Supernatants were pooled and concentrated by speedy vac. Data analysis was carried out as described with the following modifications p-value. The spectra of proteins identified by a single significant peptide were assessed and annotated but are reported only in Supplementary data (A selection of protein spots that were newly detected following deep imaging were identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Peptides were prepared for MS analysis as described previously ary data .p = 0.0213) were initially below the detection limit but could be detected by a single round of deeper imaging (p = 0.004) detected in the pre- and post excision gels, respectively   . An ‘ave p<0.05) . The repThe issue of proteome coverage using available analytical methods is well established. For decades, stain developments have driven routine detection of diminishing quantities of protein Here we tested a deep imaging approach: could resolved proteins of even lower abundance be detected following the excision of saturating protein spots? By thus extending the saturation threshold , Carbonyl reductase (NADPH)1 (P47727), and reticulocalbin-2 (Q62703) were also identified in a study that sought to enrich low abundance proteins Considering their near-saturation abundance, most, if not all of the excised spots likely contained multiple resolved protein species; this has been established previously by quantitative analysis Considering the extent of proteome resolution shown here, the ability to resolve multiple samples in parallel, and with its routine information on isoelectric point, approximate MW, abundance, isoforms and post-translational modifications, the results here move 2DE well away from the antiquated dogma that sometimes surrounds it in the literature Table S1Protein species identified by LC/MS/MS. A subset of protein species specifically detected by deep imaging were prepared for identification by MS and analysed as described previously Rattus norvegicus species.(DOCX)Click here for additional data file.


Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female’s reproductive life.The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence (increase or decrease) primordial follicle assembly in a neonatal rat ovary culture system. One novel factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect similar physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased.A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems biology approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease. Complex and interconnected networks of gene expression, cellular signaling and other processes within cells and organs are what control biological processes. This raises the concern that the common reductionist experimental approach to biomedical research may not be adequate to fully understand the systems that control these processes. Reductionist experiments will commonly impose single treatments onto the biological entity under study and measure a single response parameter compared to controls. A relevant example from the authors’ own laboratory is the study of the effect that treatment of neonatal rat ovaries with anti-Müllerian hormone (AMH) has on the proportion of primordial follicles formed . ResultsPrimordial follicle assembly is the process by which ovarian primordial follicles are formed. A primordial follicle is composed of an oocyte arrested in prophase of the first meiotic division and surrounded by a single layer of pre-granulosa cells . FolliclDuring the follicle assembly process oocyte nests break down and a single layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles . The majin vitro in a manner that allows primordial follicle assembly to occur. The ovaries were treated with one of several different extracellular signaling factors that have been shown to regulate follicle assembly. Messenger RNA was isolated from the ovaries and used for microarray transcriptome analysis to globally survey gene expression under these different treatment conditions. The effects of each signaling factor treatment were analyzed to determine similarities and differences in gene expression between the treatments. A gene bionetwork analysis subjected all the differentially expressed genes across all treatments to a weighted co-expression cluster analysis to identify groups of genes (i.e. modules) whose expression was regulated in a coordinated and interconnected manner [A systems biology experimental approach was employed in the current study to expand upon the results of these previous experiments that examined single gene effects on the follicle assembly process. A similar systems biology approach has been used previously to identify coordinately and interconnected expressed gene modules and gene networks that regulate the primordial to primary follicle transition which is the subsequent stage of ovarian follicle development . This prd manner -35. In td manner ,36,37. IIn the selection of regulatory factors to be used in the current study one novel factor was considered. Previous research ,30 indicTo determine the gene networks and processes involved in follicle assembly ovaries from zero-day old rats were placed into an organ culture system and exposed to different regulatory factors. The ovaries were treated for 24 hours with one of each of the following regulatory extracellular signaling factors: AMH, CTGF, E2, FGF2, activin A, P4, TNFa, or were untreated as Controls. CTGF, activin A, estradiol have been shown the increase assembly, while AMH, progesterone and TNFa decrease assembly. A 24 hour culture period was used to minimize the impact of differences in follicle numbers , due to the required 2 days of culture to observe detectable morphological differences. After culture the ovaries receiving the same treatment from one culture well were pooled and RNA isolated. There were three different experiments involving different ovaries for each of the seven treatment compounds, and seven different experiments with different ovaries for the controls, for a total of 28 samples (see Methods). Gene expression in the RNA samples was evaluated using Affymetrix© Rat Gene 1.0 ST microarrays. Array data pre-processing and evaluation determined that one array (P4-treated) was abnormal and an outlier, so that array was eliminated from further analysis  to identify physiological processes and pathways that may be regulated during follicle assembly. Pathways that were statistically over-represented within the differentially expressed gene lists included focal adhesion, chemokine signaling, cytochrome P450 metabolism, glutathione metabolism, ECM-receptor interaction and ribosomal components  of genes whose expression is regulated in a coordinated and interconnected manner (see Methods). The cluster analysis scores each gene according to how well across different treatments its changes in gene expression are correlated with the changes in expression of every other gene. High connectivity scores indicate that expression of a particular gene changes in concert with that of many other genes. In addition, the cluster expression analysis identifies gene modules in which the member genes have similar changes in expression in response to the various experimental treatments. Such gene modules are often associated with specific biological processes . To idenGenes whose expression altered in response to treatments were correlated with the genes assigned to each module to determine if specific modules were heavily influenced by particular regulatory factors Figure . In mostIl1b , Fn1  and Igf1  , (see Methods). A total of 326 genes were found to form a gene network that linked neighboring genes together with regulatory or binding relationships. While this network was too large and complex for easy visual interpretation  and Vcam (vascular cell adhesion molecule 1) as having many connections, suggesting they may play a role in controlling the physiological processes mediated by the turquoise module. For the blue module , Anxa2 (annexin A2), F3  and Ccnd1 (cyclin D1) have the most neighbor connections. F3 and Ccnd1 are seen to be primarily the recipients of the regulatory relationships suggesting their regulation may be an important output of the blue module network. Although relatively small, the black module also formed a network of connected genes  and Tgfb2 (transforming growth factor beta 2) have many neighbor connections, while Ccnd1 and F3 appear to be outputs of the network  were selected. These genes were then screened for whether they were present in the large network of 326 genes with known regulatory connections derived from the entire list of 1081 differentially expressed genes , Mdm2 and Prkcb (protein kinase C beta). These genes formed a regulatory network among themselves, and were associated with specific pathways and cellular processes. As can be seen in Figure In order to determine those genes whose actions are likely to be the most important in regulating the control of primordial follicle assembly, a combined approach was taken that determined the gene network associated with the most highly interconnected genes. For each module, the top 10% of genes with the highest connectivity index (k.in.)  [Fgf2 expression [In addition to these known regulatory agents, a novel factor was examined and included in the analysis. Previous research suggested FGF2 may be a regulator of follicle assembly. A receptor for FGF2, FGFR2B, has previously been shown to be differentially expressed in 0-day old rat ovaries with oocyte nests compared to 4-day old ovaries that had completed assembly . Treatmenudix 6) , which apression . In ordeOne thousand and eight-one genes were found to be differentially expressed compared to controls when results were combined across the different treatment groups. These genes were predominantly from the functional categories of metabolism and transport, signaling proteins and receptors. The genes differentially expressed in response to each growth factor or hormone treatment were compared across the different treatments. Interestingly, only a small proportion of differentially expressed genes (<10%) were found to be common between any two treatments Figure . This isA cluster analysis of coordinated gene expression grouped the differentially expressed genes into gene modules containing genes whose expression responded in concert to the different growth factor and hormone treatments. This approach of generating a weighted gene network and then clustering genes making use of a topological overlap matrix has been used extensively for uncovering biologically meaningful gene modules ,32,44-46When genes whose expression changed in response to treatments were cross-matched with the genes assigned to each module, it was found that in most cases differentially expressed genes from each treatment group could be found in all the gene modules Figure . This isIl1b (interleukin 1 beta), Fn1 (fibronectin 1) and Igf1 (insulin-like growth factor 1) had the most regulatory connections to neighbors. These genes are considered important in controlling the follicle assembly process as either regulators of the assembly network or as downstream effectors of the network. However, it should be kept in mind that genes that have been extensively studied are more likely to have relationships with other genes in the published literature, and that un-studied genes may in fact be important. Nonetheless, gene networks of regulatory connections provide a good starting point toward understanding the control of processes such as follicle assembly.In order to identify functional relations among differentially expressed genes, an analysis of published literature was used to detect connections among listed genes to form gene networks of putative regulatory relationships. Examination of these networks can yield inferences about how genes interact to regulate primordial follicle assembly, and can identify potentially important control points within these regulatory networks. When the entire list of genes found to be differentially expressed during follicle assembly was analyzed in this way it was found that the genes Fn1, Stat1 , Vcam (vascular cell adhesion molecule 1), Cav1 (caveolin), Anxa2 (annexin A2), F3 (coagulation factor 3), Ccnd1 (cyclin D1), Igf1 and Il1b as being important within their respective modules. Interestingly, genes of the brown, yellow, green, red, pink and magenta modules did not contain gene networks of known regulatory relationships, even though some of these modules contained many more genes than the black module, which did have such a network. This suggests that the genes within each of these modules may have as yet un-characterized regulatory relationships with each other. Furthermore, the genes of the red module were not found to be over-represented in association with any particular known cellular pathways or processes, and many of the red module genes are poorly characterized expressed sequence tags (ESTs). However, genes of the red module had relatively high connectivity scores (k. in.), indicating that this group of genes was quite tightly co-regulated  genes from each module were compared to databases of genes present in specific cellular pathways and processes. This group of highly interconnected genes was found to be over-represented in the processes of apoptosis, cell migration, cell differentiation and cell proliferation Figure . This isAnalysis of a specific gene sub-network of differentially expressed genes in the growth factor, hormone and receptor functional gene categories identified a large number of such regulatory signaling factors that appear to regulate ovarian primordial follicle assembly Figure . Many ofvia inhibition of DUSP6 [Analyses of the differentially expressed genes of the current study implicate specific physiological pathways and gene networks as being important to the follicle assembly process. In order to test the validity of some of these predictions, organ culture experiments were preformed in which neonatal rat ovaries were treated with BCI. BCI has the effect of increasing ERK1/2 (MAPK1) activity of DUSP6 ,39. ERK1http://ovary.stanford.edu/). Seven of these genes were found to be in common with the 1081 differentially expressed genes found in the current study to be associated with follicle assembly (significant at p<0.05 by Fisher’s Exact test). These genes were Tgfbr3 (transforming growth factor beta receptor type 3), Amhr2 (anti-Müllerian hormone receptor type 2), Pgrmc1 (progesterone receptor membrane component 1), Nupr1 , Pof1b (premature ovarian failure 1b), IGF1 and AFF2 . AMH, progesterone and Pgrmc1 are known to play roles in follicle assembly [Pof1b, the gene named for its association with premature ovarian failure (i.e. POI), is linked to follicle assembly in the current study. These observations suggest that some cases of POI may have abnormal follicle assembly as an underlying cause. In addition to specific gene links with POI, a number of links were also made to polycystic ovarian syndrome (PCOS) . Women with POI deplete their pool of primordial follicles prior to age 40 and undergo early menopause ,15. Fortassembly ,18,22,23) Figure . PCOS is) Figure . ObservaIn summary, a systems biology experimental approach can provide an unbiased global view of the relationships important to a particular developmental process. For the primordial follicle assembly process the systems approach evaluated genes that were differentially expressed in response to growth factor and hormone treatments. It was found that different treatments all affected similar cellular pathways and processes, but that each treatment affected expression of different genes within those pathways. Cluster analyses identified modules of coordinately regulated genes and the different modules appear to accomplish distinct cellular functions during follicle assembly. The regulatory gene networks identified provide predictions about important regulatory genes, signaling pathways and cellular processes involved in ovarian primordial follicle assembly. An organ culture experiment in which ovaries were treated to increase ERK1/2 activity confirmed some of the predicted physiological pathways were in fact important in follicle assembly regulation. The regulatory genes and gene networks identified as controlling primordial follicle assembly, when disrupted or altered, were suggested to be linked to the etiology of ovarian diseases such as primary ovarian insufficiency POI and polycystic ovarian syndrome PCOS. Future investigations can now utilize the observations from this systems approach to further elucidate the molecular control of ovarian primordial follicle development and associated diseases.-6M, Sigma-Aldrich, USA), or P4 (Progesterone). After only one day of culture there are few morphological differences between control and treated ovaries, so measurements of whole-ovary gene expression reflect differences in RNA transcription, rather than differing proportions of cell types due to differing cell proliferation between treatments. After culture the 2–3 ovaries receiving the same treatment from one culture well were pooled and homogenized in one ml Trizol™ reagent , then stored at −70°C. There were three different biological experiments  for each of the seven treatment compounds, and seven replicates of the controls, for a total of 28 RNA samples.Zero-day old female Sprague–Dawley rats  were euthanized within six hours after birth according to Washington State University IACUC approved (#02568) protocols and the ovaries removed and cultured whole as described previously . Zero-daIn order to determine the effect of FGF2 on primordial follicle assembly, ovaries were cultured as above for two days in the absence or presence of FGF2 50 ng/ml). Similarly, in order to determine the effect of increased ERK1/2 signaling on follicle assembly, ovaries were cultured in the presence or absence of BCI . After 2 days culture ovaries were fixed with Bouin’s solution, paraffin embedded, sectioned onto microscope slides and stained with hematoxylin and eosin as described previously [ ng/ml. S. flattened) pregranulosa cells. Early transition primary follicles have initiated development  and contain at least two cuboidal granulosa cells. Primary and preantral follicles exhibit one or more complete layers of cuboidal granulosa cells [The number of oocytes at each developmental stage was counted and averaged in two serial sections from the largest cross-section through the center of the ovary. Oocytes in ovarian cross sections were classified as unassembled, or as assembled into primordial (stage 0), or developing follicles  as previously described . Oocytessa cells ,52. HemaRNA was isolated from whole rat ovaries after homogenization in one ml Trizol™ reagent , according to manufacturer’s instructions. Two or three ovaries from the same culture well (from different rat pups) and receiving the same treatment were pooled and homogenized together. Homogenized samples were stored at −70°C until the time of RNA isolation. After isolation from Trizol, RNA was further purified using RNeasy MinElute Cleanup Kits  and stored in aqueous solution at −70°C.The microarray analysis was performed by the Genomics Core Laboratory, Center for Reproductive Biology, Washington State University, Pullman, WA using standard Affymetrix reagents and protocol. Briefly, mRNA was transcribed into cDNA with random primers, cRNA was transcribed, and single-stranded sense DNA was synthesized which was fragmented and labeled with biotin. Biotin-labeled ssDNA was then hybridized to the Rat Gene 1.0 ST microarrays containing 27,342 transcripts . Hybridized chips were scanned on Affymetrix Scanner 3000. CEL files containing raw data were then pre-processed and analyzed with Partek Genomic Suite 6.3 software  using an RMA GC-content adjusted algorithm. Comparison of all array histogram graphs demonstrated that the data for 27 of 28 chips were similar and appropriate for further analysis. One chip, from a P4-treated sample, was an outlier and so was discarded  (GEO Accession number: Pending), all arrays combined with one accession number, and can be also accessed through http://www.skinner.wsu.edu. For gene annotation, Affymetrix annotation file RaGene-1_0-st-v1.na32.rn4.transcript.csv was used unless otherwise specified.All microarray CEL files (MIAME compliant raw data) from this study have been deposited with the NCBI gene expression and hybridization array data repository  absolute difference of group means ≥10. The less stringent cutoff for fold change avoids loss of important genes at such an early stage of analysis since these candidate genes will go through subsequent systems-level coexpression network and pathway analyses that can further filter noisy signal, as we have shown in the previous study [. Affymetrix probesets) being identified and used for constructing a weighted gene co-expression network [f(x) = xβ. The parameter β of the power function is determined in such a way that the resulting adjacency matrix , is approximately scale-free. To measure how well a network satisfies a scale-free topology, we use the fitting index proposed by Zhang & Horvath [R2 of the linear model that regresses log(p(k)) on log(k) where k is connectivity and p(k) is the frequency distribution of connectivity). The fitting index of a perfect scale-free network is 1. For this dataset, we select the smallest β ( = 7) which leads to an approximately scale-free network with the truncated scale-free fitting index R2 greater than 0.75. The distribution p(k) of the resulting network approximates a power law: p(k)~k−1.29.The cluster coordinated expression analysis was restricted to genes differentially expressed between the control and the treatment groups based on previously established criteria: (1) fold change of group means ≥1.2 or ≤0.83; (2) us study . The uni network ,44. Unli Horvath   were selected. Additional genes were added from each module, above 10%, if those genes had k. in. scores >8. If the list for any module did not include enough named genes (i.e. genes that were not EST’s) to equal 10% of the module size, then additional genes with the highest k.in. scores were added until 10% named genes was achieved.http://vortex.cs.wayne.edu) [http://www.genome.jp/kegg/pathway.html). KEGG pathways were considered ‘impacted’ and were included in analyses for this manuscript if three or more differentially expressed genes were present within a KEGG pathway. Statistical over-representation of differentially expressed genes within a pathway was determined by Fischer’s exact test for two by two contingency tables, and by calculating hypergeometric probability of obtaining exactly the listed number of genes in common with that pathway.Lists of differentially expressed genes for each regulatory factor treatment, as well as for each module generated in the network analysis, were analyzed for KEGG  pathway enrichment using Pathway-Express, a web-based tool freely available as part of the Onto-Tools (yne.edu) . AdditioGlobal literature analysis of various gene lists was performed using Pathway Studio  software, which performs pathway and interaction analysis and identifies genes that have regulatory or binding relationships. Pathway Studio was also used to identify cellular functions and diseases (including polycystic ovarian syndrome) linked in the published literature to the genes in these lists. In addition, Pathway Studio was used to determine over-represented physiological processes for gene lists based on KEGG, PANTHER  and NCBI GO  databases.POI: Primary ovarian insufficiency; PCOS: Polycystic ovarian disease syndrome; AMH: Anti-Müllerian hormone; CTGF: Connective tissue growth factor; E2: Estradiol; P4: Progesterone; TNFa: Tumor necrosis factor alpha; BDNF: Brain derived neurotrophic factor; KITL: Kit ligand; GDF9: Growth differentiation factor-9; FGF2: Fibroblast growth factor-2; k.in.: Connectivity index.The authors declare that no competing interests exist.MKS designed the study. EN performed the ovary cultures and microarray analysis and network design. BZ performed the bionetwork analysis and bioinformatics. EN wrote the manuscript and all authors edited the manuscript. All authors read and approved the final manuscript.Sample histograms and box plots for ovary RNA sample microarray signal values prior to pre-processing and normalization. Note that one of the P4-treated samples was an outlier, and was discarded. X-axis shows hybridization intensity value. Y-axis (Hybridization Frequency) shows the number of genes having a given hybridization intensity.Click here for fileDifferentially expressed genes: A) Genes influenced by treatment with anti-Müllerian hormone (AMH). B) Genes influenced by treatment with CTGF. C) Genes influenced by treatment with FGF2. D) Genes influenced by treatment with ActivinA. E) Genes influenced by treatment with P4. F) Genes influenced by treatment with TNFa. G) Genes influenced by treatment with E2.Click here for fileTreatment and module differentially expressed genes correlated to cellular pathways and processes.Click here for fileversus Control ovaries. Genes with the greatest number of connections (relationships) to other genes have enlarged gene symbols. Network is derived from an un-biased search of literature using Pathway Studio™. Node shapes code: oval – protein; diamond – ligand; irregular polygon – phosphatase; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Grey arrows represent regulation, lilac – expression, green – promoter binding, olive – protein modification, purple - binding.Gene network of known relationships among all 1081 genes found to be differentially expressed in treated Click here for fileGene network of known relationships among differentially expressed genes assigned to specific co-expression modules. A) Blue module. B) Black module. Network is derived from an un-biased search of literature using Pathway Studio™. Node shapes code: oval – protein; diamond – ligand; irregular polygon – phosphatase; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Grey arrows represent regulation, lilac – expression, green – promoter binding, olive – protein modification, purple - binding.Click here for fileGene network of known relationships among genes differentially expressed in ovaries receiving specific treatments, compared to controls. A) E2 (estrogen). B) FGF2. C) Activin A. Network is derived from an un-biased search of literature using Pathway Studio™. Node shapes code: oval – protein; diamond – ligand; irregular polygon – phosphatase; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Grey arrows represent regulation, lilac – expression, green – promoter binding, olive – protein modification, purple - binding.Click here for file


Triatoma infestans, the main Chagas disease vector in the Gran Chaco region, remains elusive. We implemented an intensified control strategy based on full-coverage pyrethroid spraying, followed by frequent vector surveillance and immediate selective insecticide treatment of detected foci in a well-defined rural area in northeastern Argentina with moderate pyrethroid resistance. We assessed long-term impacts, and identified factors and procedures affecting spray effectiveness.The elimination of After initial control interventions, timed-manual searches were performed by skilled personnel in 4,053 sites of 353–411 houses inspected every 4–7 months over a 35-month period. Residual insecticide spraying was less effective than expected throughout the three-year period, mainly because of the occurrence of moderate pyrethroid resistance and the limited effectiveness of selective treatment of infested sites only. After initial interventions, peridomestic infestation prevalence always exceeded domestic infestation, and timed-manual searches consistently outperformed householders' bug detection, except in domiciles. Most of the infestations occurred in houses infested at baseline, and were restricted to four main ecotopes. Houses with an early persistent infestation were spatially aggregated up to a distance of 2.5 km. An Akaike-based multi-model inference approach showed that new site-level infestations increased substantially with the local availability of appropriate refugia for triatomine bugs, and with proximity to the nearest site found infested at one or two preceding surveys.T. infestans in rural areas with moderate pyrethroid resistance requires increased efforts and appropriate management actions. In addition to careful, systematic insecticide applications, housing improvement and development policies that improve material conditions of rural villagers and reduce habitat suitability for bugs will contribute substantially to sustainable vector and disease control in the Gran Chaco.Current vector control procedures have limited effectiveness in the Gran Chaco. Selective insecticide sprays must include all sites within the infested house compound. The suppression of  Triatoma infestans from a well-defined rural area in northern Argentina. After an initial community-wide spraying, we intensively monitored infestation every 4–7 months for 35 months and applied insecticides selectively to the detected foci. In addition to the moderate levels of pyrethroid resistance reported in parallel, we found that selectively spraying only infested sites performed poorly. Prespraying bug abundance and the characteristics of infested sites before the initial interventions were reliable predictors of postspraying site infestation, including the type and use of the site and availability of refuges for the vector. We conclude that professional vector control based on residual insecticide spraying in an area with moderate pyrethroid resistance requires intense monitoring of house infestation, systematic insecticide applications and appropriate management actions. Operational, economic and political constraints to sustainable vector and disease elimination require complementary tools and approaches that favor changes in material conditions which reduce habitat suitability for the vector.Vector-borne transmission of Chagas disease has not been effectively controlled in large parts of Latin America, particularly in the Gran Chaco ecoregion. To better understand the challenges in this region, we assessed the effectiveness of an intensified insecticide-based spraying strategy in suppressing the major vector  Triatoma infestans, one of the major vectors of Trypanosoma cruziT. cruzi mediated by T. infestans, and the extent and intensity of infestations were substantially reduced in some sections of Argentina, Bolivia and Paraguay Field trials carried out in Brazil and Argentina in 1948 demonstrated the effectiveness of hexachlorocyclohexane for suppressing domestic infestations with T. infestans have usually been associated with passive bug transport by people, active dispersal of bugs from residual or untreated foci, and more rarely and with less supporting evidence, from sylvatic foci Triatoma dimidiata, Triatoma brasiliensis and Rhodnius ecuadoriensis, sylvatic foci represent the major source of bugs The major obstacle to attain effective control of the major vectors of Chagas disease using residual insecticide spraying has been the reappearance of triatomine bugs and the difficulties in addressing this recurrent process. Reinfestation of human habitations and peridomestic structures after insecticide application has been documented for most of the main triatomine species T. infestans and Chagas disease persist as a major public health problem in many rural and some periurban communities in the Southern Cone countries T. infestans set by the Southern Cone Initiative in 1991 has not been reached yet in the Gran Chaco region –a 1.1 million km2 semiarid plain covering large parts of northern and central Argentina, southeast Bolivia, and central and western Paraguay T. infestans: suitable environmental conditions, hosts and habitats for bug development; poor living conditions; irregular vector control activities coupled with intrinsic operational difficulties ; relatively few resources assigned to vulnerable populations with low political visibility and high disease burden; diminished effectiveness of pyrethroid insecticides because of environmental conditions Despite progress in vector control status, T. infestans than standard doses and reduced house reinfestation rates in the dry Chaco T. infestans may range from 4 to 6 months depending on temperature and resource availability T. infestans has been detected in northern Argentina and Bolivia T. infestans may be multiple and remain unclear.In the Argentinean dry Chaco, house reinfestation after insecticide spraying was mainly associated with the occurrence of residual foci in peridomestic structures T. infestans in the Gran Chaco, we assessed the long-term impacts on house infestation and bug abundance of an intensified control strategy based on full-coverage pyrethroid spraying followed by frequent vector surveillance and immediate selective treatment of the detected foci in a well-defined rural area in northeastern Argentina. Before initial control interventions, a multi-model inference analysis showed that availability of appropriate refuges for T. infestans, use of cardboard as a building material, and household numbers of domestic hosts were strongly and positively associated with site-specific bug infestation and abundance, whereas reported insecticide use by householders was negatively related to infestation T. infestans were detected As part of a multi-site research program on the eco-epidemiology and control of 2) of the municipality of Pampa del Indio (25°55′S 56°58′W), Province of Chaco, Argentina (see map and photos in T. cruzi in bugs (27.4%), dogs (26%) and cats (29%) was indicative of active domestic and peridomestic transmission .Fieldwork was conducted in a well-defined rural section  were conducted at baseline and every 4–7 months during 35 months. A community-wide spraying with pyrethroid insecticides of all sites within each house compound was conducted immediately after the baseline survey Demographic and entomological surveys were conducted at baseline, during insecticide spraying, and at 4, 8, 12, 17, 22, 28 and 35 MPS. All existing houses were visited and its status recorded . A sketch map of the spatial setting of all sites within each house compound was drawn, and each site was georeferenced and given an individual code in September or November–December 2007 . The sketch map was updated during each visit. A house compound encompassed a domiciliary area with human habitations (sometimes in two separate buildings that counted as two domestic sites) and all sites within the peridomestic area  –usually a storeroom, a kitchen, an oven, one or more sites for chickens and other poultry , one or more corrals, and a latrine. Each of these habitats characterized by some typical physical structure and use was considered an “ecotope”. A site  was any individual structure built and/or given a defined use by householders which might provide refuge for bugs.All sites within each house were searched for triatomine bugs by timed manual collections (TMC) conducted by two skilled bug collectors using 0.2% tetramethrin  as a dislodging agent. Human habitations were inspected by one person for 20 min and each peridomestic site was searched by a second person for 15 min. In practice, each house compound averaged three peridomestic sites inspected and therefore the total search effort averaged one person-hour per house. In addition, most sites were inspected thoroughly before the stipulated time, and therefore search efforts were roughly similar across sites of different size. In several houses, bugs were also collected after the stipulated search time , or by insecticide knock-down during insecticide applications (by the spray team) or a few days later (by householders) 2) applied by vector control personnel using backpack manual compression sprayers  as described elsewhere T. infestans at 4 or 8 MPS (including adjacent sites) were performed with deltamethrin upon completion of the 8 MPS survey. Likewise, sites found infested with T. infestans at 12 MPS (including adjacent sites within the same house compound and other sites that had not been sprayed at 8 MPS) were sprayed with SC β-cypermethrin (the only insecticide available to the vector control program at that time). To assess the impact of double-dose insecticide application on persistent infestations, standard (50 mg/m2) and double-dose (100 mg/m2) treatments with SC β-cypermethrin were assigned at random to infested peridomestic sites while a standard pyrethroid insecticide dose was applied in domiciles.The treatment criteria, insecticides and doses applied at different times are described in T. infestans were treated with insecticide). Double-dose β-cypermethrin was used at 17 MPS. Field and laboratory-based evidence of local pyrethroid resistance 2) –the only effective alternative to pyrethroids available that was authorized by the corresponding federal agency at that time– to the few house compounds still infested with T. infestans at 22 and 28 MPS. At 22 MPS, one house was left unsprayed by mistake, and the owner of another house refused spraying; both houses were sprayed at 28 MPS.In view of the infestation levels recorded, from 17 MPS and thereafter the spray criterion was modified to full-spray coverage of infested house compounds  at time t was defined as the occurrence of infestation in a given site (or house) both at time t−1 and t. Estimates of infestation prevalence were based on the combined results of TMC and knock-down bug collections. Householders' bug collections were only considered when provided with precise information on date and site of capture; these and other data were used to distinguish between occasional invasions and established infestations. Bug abundance was computed as the number of live T. infestans collected in a specific site per 20 (domiciles) or 15 (peridomestic sites) person-minutes of search effort by TMC. As a measure of insecticide spraying effectiveness at site- or house-level, the percentage of sites (or houses) infested and sprayed at time t that were again found infested at time t+1  was calculated for each selective spray round.All data reported correspond only to inhabited houses unless otherwise noted; no public building and only two uninhabited houses were ever found infested with Data on reported insecticide use, ecotope, building materials, refuge availability, household numbers of people and domestic animals, and host resting places were collected in every survey starting on 4 MPS. The corresponding data for 0 MPS were extrapolated from the 4 MPS survey as explained elsewhere t−1 and t−2 surveys was evaluated by means of multiple logistic regression analysis. Apparently new infestations were only considered from 12 MPS onwards; houses found infested at 4 or 8 MPS were excluded from this analysis because they had high chances of being locally persistent foci –at site or house level– after initial interventions. Thus, for this particular analysis, an infestation occurring at time t was considered new  if it was found at 12 MPS (or subsequently) in a house considered uninfested at 4 and 8 MPS (or at t – 1). A multi-model inference approach based on Akaike's Information Criterion (AIC) was used to assess the relative importance (RI) of each variable The association between new site-level infestations detected at 12 MPS or subsequently and refuge availability, reported insecticide use by householders, and distance to the closest infested site at T. infestans ranging from 4 to 6 months). The null model was built maintaining the pattern of infestation at 0 MPS (pattern 1) fixed, and randomizing the status of infestation of houses at 4 MPS (pattern 2) among all existing houses. The O-ring statistic O12(r) r and a given width, centered at each point of the fixed pattern 1, corresponded on average to a random process  = 1); aggregation of 2 relative to 1 (O12(r)>1), or regularity of 2 with respect to 1 (O12(r)<1). This procedure was implemented in Programita th simulations were used as a 95% confidence envelope. A goodness-of-fit test was used to evaluate the overall fit of the observed pattern to the expected distribution The spatial distribution of houses with persistent infestations at 4 MPS was evaluated with respect to house infestation at 0 MPS  houses were vacated between consecutive surveys, whereas newly-built or re-occupied houses represented 4–5% of the total number of inhabited houses at each survey. Very few households refused searches for bugs through the follow-up . The maiT. infestans from 49.5% before interventions to 12.3% and 8.9% at 4 and 8 MPS, respectively , treatment coverage , and insecticide dose . Persistent infestations were detected 4–5 months after selective applications of pyrethroids with standard dose in 2 (4%) of 55 infested sites, and with a double dose in 3 (10%) of 30 infested sites. In sites negative before selective applications, infestations were subsequently detected in 1 of 21 sites sprayed with a standard dose, and in none of 9 sites sprayed with a double dose.We assessed the effectiveness of selective pyrethroid sprays in suppressing site-specific infestations. TMC searches conducted 4–6 months after each of the first four pyrethroid spray rounds revealed that 5–13% of the treated sites were persistently infested between successive surveys . No sign2 = 3.70, df = 3, P>0.30). Infestation persisted to the subsequent survey conducted 4–5 months later in 10% of the 31 infested houses that were fully sprayed with a double dose of pyrethroids at 17 MPS, whereas none of the 292 negative houses not sprayed with pyrethroids at that time had a subsequent infestation.Regarding the effectiveness of selective treatments at house-compound level, TMC searches found persistent infestations (in at least one site per house) in 13–37% of the treated houses within 4–6 months after each of the four selective spray rounds . No signThe impacts of the initial community-wide pyrethroid spray on house infestation were not homogeneous across the study area . Houses T. infestans at 22 MPS that were immediately sprayed with malathion, two were infested at 28 MPS (see Of the eight sites (each in a different house) infested with Peridomestic infestation prevalence exceeded that in domestic sites during the entire follow-up after initial interventions, even though prespraying infestation was slightly higher in domiciles . The mos2 = 63.14, df = 4, P<0.0001). These variations were mainly caused by an increase in the number of households using domestic insecticide aerosols at least every two months. Householders reported applying insecticides in domiciles, kitchens and storerooms.The reported use of insecticides by householders varied significantly from 41.4% to 69.0% during the follow-up   of the occasions; >1 adult in 50 (46%) occasions, and nymphs in 40 (37%) of their bug collections. Householders captured bugs most frequently in domiciles (72 of 108), followed by kitchens, storerooms and ‘nideros’.TMC significantly outperformed householders' bug detection before and after interventions except in domiciles after initial insecticide spraying . HoweverFor each of the first four selective spray rounds (8–22 MPS) we assessed the hypothetical effects of spraying all sites within a house compound that at least had an infested site, and spraying all sites within a given radius around the detected focus. The number of hypothetically infested sites declined gradually up to a radius of 2–3 km while the total number of hypothetically sprayed sites increased considerably . Such deOverall figures of the impacts of insecticide spraying on infestation provide a broad picture while hiding some relevant observations. Below we report on several specific cases (sites or houses), and relate their infestation status to defined events or processes:Recurrent infestations at house-compound level detected two surveys after selectively spraying infested sites only: Of 19 house compounds with ≥1 site selectively sprayed and with no site found infested in the subsequent survey, 11 house compounds were infested again two surveys later .Persistent infestations at house-compound level after selectively spraying infested sites were suppressed only after spraying the entire house compound: After selective treatment of infested sites only, 10 house compounds had one or more sites infested in the subsequent survey . Six of Persistent infestations after spraying the entire house compound: Three house compounds infested and re-sprayed with pyrethroids at 17 MPS were still infested at 22 MPS: two had a persistent infestation at site level, one of them caused by pyrethroid resistance  despite the occurrence of lower baseline infestation levels than in other similar rural areas with no recent vector control actions T. infestans populations scattered through most of its current distribution range has gradually increased We recorded a large degree of spatial and temporal heterogeneity in infestation, the apparent effectiveness of insecticide spraying, and people's practices related to infestation . The spaT. infestans foci occurring at various scales T. infestans inferred from the spatio-temporal dynamics of reinfestation patterns T. infestans during spring-summer Selective pyrethroid sprays of sites infested only performed poorly. Frequently the treated bug colonies were apparently suppressed, yet in a few months other infestations became detectable in adjacent sites within the same house compound , either The simulation results further suggest that enhanced bug control would be achieved if all suitable sites within 500–1,000 m of the detected foci were sprayed. Similarly, a longitudinal study conducted in the Argentinean dry Chaco reported significant spatial aggregation of reinfested sites at 25–500 m around residual foci, and recommended extending selective insecticide sprays up to a distance of 450–500 m around the detected foci Refuge availability, the main bug habitats, and prespraying bug abundance were closely related to postspraying site-level infestation after initial interventions, in agreement with existing evidence House infestation prevalence fell below 1% only after multiple inspection and selective spray rounds with pyrethroids and finally, selective treatment of highly persistent foci with malathion. During the follow-up, several other local events potentially affecting infestation may have confounded the specific effects of insecticide spraying. Some of the cases analyzed  illustraTimed manual searches conducted by skilled personnel using a dislodging agent is the standard method used to assess infestations in intervention trials despite its limited sensitivity and precision, especially at low bug densities T. cruzi to humans and dogs may occur at very low infected-bug densities in high-risk areas T. infestans “is not supported by rigorous evidences but rather derived from data on Triatoma infestans in Brazil without scientific justification” One limitation of our study is that we have not assessed the impact of interventions on parasite transmission, as vector-borne transmission of T. infestans in the Gran Chaco ecoregion T. infestans in an area with moderate pyrethroid resistance. The chronic limitations in personnel and resources in the Latin American health sector  pose serious obstacles to vector and disease suppression efforts. Research on the real functioning of disease control programs and their capacity to operate and modify what they do and how they do it is needed to push knowledge closer to its effective application T. infestans, and the eventual emergence of insecticide resistance. In addition to careful, systematic residual insecticide applications, our findings confirm that housing modifications and development policies that improve material conditions of rural villagers and reduce habitat suitability for T. infestansEvidence of the obstacles to suppress Figure S1Examples to illustrate persistent infestations with T. infestans at house-compound level. Each row corresponds to a georeferenced (identifiable) site. Only sites ever found infested at least once in the selected houses are shown. Colors indicate infestation status at each house and survey according to collection method.(DOC)Click here for additional data file.Table S1Number of houses inhabited, inspected by timed manual collections (TMC) and sprayed with insecticides in Pampa del Indio.(DOC)Click here for additional data file.Table S2Insecticide use between successive surveys as reported by householders.(DOC)Click here for additional data file.Text S1Details on relevant cases regarding insecticide spraying effectiveness and householders' practices.(DOC)Click here for additional data file.


Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen , sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers  and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter  were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter.Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech ( These approaches have been carried out on a variety of animal digestive systems Fagus sylvatica, a broadleaf deciduous angiosperm species) and spruce  forest soils, converted into cDNA then cloned and 10,000 of them per library sequenced using the Sanger technology. The sequence datasets were specifically analysed with respect to the diversity of enzymes implicated in the breakdown of lignocellulosic material and downstream monosaccharide membrane transporters, as well as enzymes and transporters involved in the mobilization of the organic forms of other elements . In addition, since many microbial enzymes produced for soil nutrient acquisition, and/or toxic soil compounds inactivation (e.g. P450 monooxygenases) are also of biotechnological interest, we performed a global evaluation of the sequence dataset with respect to its biotechnological potential.Since the primary degradation of lignocellulosic materials in soil is believed to be carried out essentially by fungi, we present a first metatranscriptomic analysis of forest soils focussing specifically on eukaryotic organisms whose polyadenylated mRNA can be separated from prokaryotic and non coding ones, thanks to their poly-A tails 5 and 5 105 plasmid clones were obtained using poly-A mRNA extracted from beech and spruce soils, respectively. A significant proportion of cDNA inserts were between 400 and 700 bp-long, as estimated by agarose gel electrophoresis (64% and 48% of cDNA for beech and spruce respectively). Following sequencing and cleaning of the sequences , 7905 (beech) and 8606 (spruce) cDNAs were retained for further analyses. Contamination levels by eukaryotic and bacterial rRNA sequences represented at most 8.8% of the sequences (beech). These rRNA sequences could have been cloned because they processed A-rich sequence stretches, and therefore may not reflect the taxonomic diversity of the soil microbial communities. Finally the average read length was 430 bp for beech and 482 bp for spruce cDNAs.cDNA libraries of ∼9 10versus the numbers of sequences had the shapes of straight lines  18S rDNA fragments PCR amplified from reverse-transcribed soil RNA , and ii. The lasWhatever the sequence dataset and the studied soil, the Opisthokont group  dominated, representing 71% (beech) and 77% (spruce) of the unambiguously annotated PCR amplified 18S rDNA and 60–61% of the annotated cDNAs . FurtherSequences from each “protist” group were identified at least once in each of the different beech or spruce sequence datasets and protist sequences contributed to the different datasets between 3% (cDNAs) and 12% (amplified 18S rDNA) of the unambiguously annotated sequences. It should however be stressed that the use of a single primer pair to amplify eukaryotic rDNA genes is known to capture only a fraction of the true diversity Using the default parameters and the BLASTX results against the NCBI-nr protein database as input file, the MEGAN software attributed a taxonomic annotation to 46% (spruce) and 37% (beech) of the cDNAs. Among these annotated cDNAs, only 2.5% were attributed to “protists” and 22–29% could not be unambiguously attributed to a single taxonomic group  and 39.5% (beech) cDNA sequences returned a positive hit in BLASTX searches against the GenBank nr protein database. Among the sequences with no homolog in this preliminary analysis, an additional 227 (spruce) and 238 (beech) contained a registered protein motif identified using InterProScan.Using an e-value threshold of 10Using Blast2GO, a Gene Ontology (GO) term  could be assigned to 31.5% (beech) and 39% (spruce) of the cDNAs. The five most abundant “biological process” and “molecular function” GO categories were the same for the beech and spruce datasets and altogether accounted for respectively 44% and 43% of the sequences annotated with either a “biological process” and “molecular function” GO term . The twoFinally, 12–13% of the sequences in each datasets encoded proteins with enzymatic activities to which an Enzyme Commission (E.C.) number could be associated . Two hunOur annotation effort concentrated on gene categories encoding proteins with biochemical activities directly linked to the breakdown of plant compounds and other complex nutrients  and polypeptides involved in the mobilisation of soil nutrients such as transmembrane transporters. This targeted annotation was essentially performed through sequence homology searches against specialized protein databases, which compile members of each of these different protein categories, or by using reference sequences when such specialized databases did not exist (in the case of phytases and dioxygenases). Results were further filtered manually and/or by using the MetaBioME database This annotation strategy identified a large variety of enzymes involved in the above-mentioned processes including many different enzymes classes active on most plant cell wall polymers  but also on starch and chitin which, as a component of arthropods exoskeleton and fungal cell wall, represents an abundant nitrogen source in forest soils . AltogetGiven that organic matter input in soil also results in the input of toxic secondary metabolites, we searched for enzymes classes potentially involved in their detoxification. While we only identified a single putative fungal dioxygenase (cathechol dioxygenase) among sequences from the spruce soil, we identified a total of 26 P450 monooxygenases belonging to 12 different families . For both spruce and beech soils, more than 3% of the sequences returned a positive hit in this analysis and the two most represented CUE classes were the oxidoreductases and the hydrolases . About h−6) of putative membrane transporters as identified by blasting all cDNA sequences against the Transporter Classification Database  encoding cDNAs, belonging to families CE1 (accession no. FR865947), GH5 (FR865941), GH7 (FR865943-4), GH11 (FR865942), GH45 (FR865945), GH61 (FR865936-40) and PL1 (FR865946), were identified and sequenced entirely. The longest cDNA clone was 1671 bp-long and encoded a putative GH7 cellobiohydrolase. Except for the CE1 family member, which is thought to be an intracellular enzyme with S-formyl glutathione hydrolase activity, all other 11 predicted protein sequences possessed a N-terminal signal peptide in agreement with the potential polysaccharide degrading activities of the corresponding enzymes.The putative taxonomic/phylogenetic origin of these 12 cDNAs was assessed using Bayesian (MrBayes) and/or maximum likelihood (PhyML) phylogenetic analyses of the deduced amino acid sequences. Protein sequences used in the phylogenetic analyses included not only the CAZyme best BLASTX hits from GenBank nr database but also homologous sequences identified among GenBank non fungal ESTs in addition to the gene models predicted from 57 recently released fungal and non fungal  genome sequences .Each of the 12 full-length environmental sequences were homologous to fungal sequences and to sequences from other taxonomic groups either closely related to the Fungi, such as the Metazoa (GH7 and GH45) or the choanoflagellates (CE1), or to more distantly related groups such as the Plantae  or the Bacteria . With the exception of the Carbohydrate Esterase family 1 (CE1) genes, all other studied CAZyme genes were not present in all genomes analysed and, when present, frequently occurred as gene families from which between one and three members per species were used for the phylogenetic analyses.In fine, only the phylogeny of the CE1 family fitted to the overall phylogeny of the Fungi In many cases, sequences from one specific taxonomic group (eg Fungi or Bacteria) did not group together to form a single homogeneous and statistically well-supported clade . This coTwo of the environmental CAZymes potentially originated from the soil fauna. This was the case for one of the two analysed GH7  and targeted annotation identified a large variety of genes of interest in a context of soil ecosystem functioning and more specifically in the turnover of plant biomass and soil nutrient cycling and utilization. These nutrients encompass inorganic  and organic  forms of nitrogen and phosphorus, sulfates and simple sugars resulting essentially from either rhizodeposition or lignocellulose breakdown. We also noticed that genes representative of other key pathways such as the nitrate assimilation one  were however missing in the datasets which could indicate that the corresponding nutrient represents a minor N source in the studied acidic forest soils.versus spruce) on the relative abundance of transcripts related to plant cell wall degradation or nutrient assimilation (eg sugar transporters). It is indeed known that these two species differ with respect to the nature and proportions of polymers present in their cell walls Enzymes participating to plant cell wall deconstruction are of special interest not only from an ecological point of view to quantify and understand soil C turnover, but also because they are among the most widely used enzymes in the industry and are instrumental to the development of second generation biofuels. We identified among the beech and/or spruce soil cDNAs representatives of enzymes active on the main plant cell wall polymers: lignins, cellulose, hemicelluloses and pectins. Genes encoding carbohydrate active enzymes clearly outnumbered those encoding enzymes directly active on lignin although members of the three lignin oxidase families were identified in at least one of the two metatranscriptomes . GlycosiThe use of Sanger sequencing of cloned cDNAs allowed us to easily identify several full-length genes among which several CAZyme ones. Phylogenetic analysis of the coded protein sequences suggests that many of them are distantly related to protein sequences present in databases. This further emphasizes the interest of eukaryotic metatranscriptomes as a source of novel enzymes with potentially interesting catalytic properties. This also indicates that organisms studied thus far at the molecular level in a context of plant cell wall deconstruction may not be representative of the organisms at work in the soil ecosystem. It is particularly interesting to consider that at least two out of the 12 full-length CAZymes (putative cellobiohydrolase and endocellulase) could originate not from fungi but from animals. A number of recent publications have indeed reported the existence of plant cell wall degrading enzyme genes in the genome of different groups of invertebrates which are represented among the soil fauna i.e. vegetation type, soil characteristics and climate and evaluate the relative contribution of these different variables on different soil processes. In this respect, high-throughput sequencing technologies should be implemented.In conclusion, this first report of a eukaryote-specific analysis of soil metatranscriptomes highlights the potential of this experimental approach in two research fields. In the field of environmental biotechnology, eukaryotic metatranscriptomes represent diversified multigenome resources for many different gene categories used in the bioindustry. In this context, construction of environmental cDNA libraries whose potentially large inserts can be sequenced (this study) or directly expressed in heterologous hosts Fagus sylvatica, Quercus sessiliflora, Betula verrucosa, Corylus avelana) was clear-cut and replanted in 1976 by separate, single forest tree species stands, among which spruce (Picea abies) and beech (Fagus sylvatica). On July the 10th 2007, 14 (spruce stand) and 16 (beech stand) soil samples  were collected along a systematic sampling grid. For each stand, the uppermost 3–7 cm thick organic matter-rich horizon of each core was sieved (2 mm mesh size) and mixed together (100 ml per core) to obtain a composite sample which was immediately frozen at −70 °C. Pedoclimatic parameters, soil characteristics and sampling strategy are detailed in Soil samples were taken from an environmental research observatory forest site located in central France . This site, initially covered by a mixed broadleaved tree forest (Sfi1 and ligated into the Sfi1-digested pDNR-LIB plasmid vector (Clontech).Total RNA (∼100 µg) was extracted from ∼90 g of each of the two composite forest soil samples as described in E. coli cells  and 10,000 randomly selected cDNA clones were sequenced from their 5′ end using the M13-20 primer and Sanger chemistry .Each cDNA library was introduced in electro-competent ca 560 bp-long fragment located at the 5′-end of the eukaryotic 18S rDNA gene was amplified by PCR using primers Euk1A (CTGGTTGATCCTGCCAG) and Euk516R (ACCAGACTTGCCCTCC) described by 2, 0.25 mg.ml−1 of bovine serum albumin, 0.625 U of Pfu DNA polymerase, the appropriate buffer (Fermentas) and one tenth of the reverse-transcription mix. Amplification reaction started with an initial denaturation step of 3 min at 96°C, followed by 25 cycles comprising 45 sec at 96°C, 45 sec at 56°C and 2 min at 72°C, and finished with an elongation step of 5 min at 72°C. Amplification products of the expected size from 15 PCR tubes were pooled, isolated from an agarose gel  and ligated in the plasmid pCR-Blunt II-TOPO  that was used to transform electro-competent DH10B E. coli cells (Invitrogen).350 (beech) and 730 (spruce) ng of soil non-polyadenylated RNA were reverse transcribed using 0.2 µg of random hexamers and 200 U of M-MuLV Reverse Transcriptase according to the manufacturer instructions (MBI Fermentas). A ca 560 bp rDNA inserts of 96 clones were entirely sequenced  using universal primer M13-20. Sequences were manually corrected and edited. BLASTn searches were performed against GenBank nr nucleotide database at NCBI (http://www.ncbi.nlm.nih.gov/) using default parameters except for word size which was set to 7. Sequences were analyzed with the rRNA Database Project CHECK_CHIMERA program (http://rdp8.cme.msu.edu/). Potential chimeras were further analysed by blasting separately the two dissimilar segments of the sequences against GenBank. Confirmed chimeras and artefacts were eliminated from the sequence datasets. All remaining sequences were submitted to EMBL and are available under accession numbers FN393180–FN393221 (beech) and FN393323–FN393380 (spruce).For each rDNA library, the http://www.tigr.org/tdb/tgi/software) with default parameters to eliminate poly-A sequences at the 3′end of cDNA, vector, adaptor and primer sequences and undetermined nucleotides. Sequences shorter than 100 nucleotides were eliminated. Contaminant ribosomal RNA sequences were identified by BLASTN analysis  of all sequences against LSUrdb and SSUrdb_SSURef100 libraries described in http://services.cbu.uib.no/supplementary/community-profiling/), as well as against NCBI-nr nucleotide database . The resulting “cleaned” sequence dataset was submitted to EMBL and is available under accession numbers FR706059–714330 (beech) and FR697056–706058 (spruce).All cDNA sequences were trimmed using TIGR SEQCLEAN tool .Cleaned sequences were clustered using Cd-hit (http://www.ncbi.nlm.nih.gov/), using 2 different strategies, (i) Q-BLAST via BLAST2GO −6 and overlapping length >33% to the corresponding best hit and, (ii) NetBlast 2.2.22 . As both analyses gave similar percentages of annotated cDNA sequences, we only considered the Q-BLAST results. In addition to raw BLASTX analyses, we also used BLAST2GO to perform Gene Ontology  against NCBI nr protein database  against various specialized databases. Searches were made against Carbohydrate Active Enzymes  against a curated database of commercially useful enzymes  of the corresponding cDNAs against GenBank nr and/or Swissprot protein databases. We only retained cDNA sequences similar to the fungal targeted enzyme using a E-value threshold of 0.001. For cytochrome P450, lipases, proteases, phytases and dioxygenases, identified cDNAs were further filtered by BLASTX . 18S sequences were then attributed to major eukaryotic phyla  were performed against the GenBank nr nucleotide database at NCBI  and non human EST one (TBLASTN). Additional homology searches (BLASTX) were performed against the proteomes of fully sequenced organisms available through different websites . Dependiwww.phylogeny.fr; Alignments were performed on the phylogeny.fr platform  Rarefaction curves plotting the no. of cDNA sequences against the no. of clusters showing that most sequences are unique; (B) and (C), size distribution of the clusters showing that few of them contains more than 4 sequences.(PDF)Click here for additional data file.Figure S2The five most represented Gene Ontology (GO) categories are the same for the spruce and beech datasets.(PDF)Click here for additional data file.Figure S3Global biotechnological potential of the cDNA datasets. Distribution of cDNA sequences homologous to “Commercially Useful Enzymes” (CUEs) in the MetaBioME database according to enzyme activity (E.C. no.). Analysis was performed separately for all cDNAs and for those affiliated to the fungi (see ungi see .(PDF)Click here for additional data file.Figure S4Putative phylogenetic origins of nine full-length environmental CAZymes. Environmental sequences (in orange) belong to families PL1 (A), GH11 (B), GH5 (C), CE1 (D) and GH61 (E). Maximun likelihood (PhyML) phylogenetic trees include protein sequences from different taxonomic groups, each identified by a specific colour; red, Fungi Basidiomycota; pink, Fungi Ascomycota; orange, other Fungi; Black, Choanoflagellida; brown, Bacteria; green, Plantae. Correspondence between numbers and species names is given in (PDF)Click here for additional data file.Table S1Characteristics of the PCR-amplified 18S rRNA sequence datasets.(PDF)Click here for additional data file.Table S2Taxonomic affiliation of the 18S rRNA and cDNA sequence datasets.(PDF)Click here for additional data file.Table S3An illustration of some of the key enzymes identified in major KEGG metabolic pathways relevant to either C, N or S metabolism.(PDF)Click here for additional data file.Table S4An illustration of the diversity of potential organic matter degrading enzymes, other than plant cell wall and polysaccharide active ones.(PDF)Click here for additional data file.Table S5Origin of the CAZyme sequences used in the phylogenetic analyses.(PDF)Click here for additional data file.Table S6Percentages of conserved, identical and similar, amino acid positions between the 12 full length environmental CAZyme proteins and one or two of their closest phylogenetically related neighbours.(PDF)Click here for additional data file.Table S7Stand and sampling characteristics.(PDF)Click here for additional data file.