The neural processing loop of language is complex but highly associated with Broca's and Wernicke's areas. The left dominance of these two areas was the earliest observation of brain asymmetry. It was demonstrated that the language network and its functional asymmetry during resting state were reproducible across institutions. However, the temporal reliability of resting-state language network and its functional asymmetry are still short of knowledge. In this study, we established a seed-based resting-state functional connectivity analysis of language network with seed regions located at Broca's and Wernicke's areas, and investigated temporal reliability of language network and its functional asymmetry. The language network was found to be temporally reliable in both short- and long-term. In the aspect of functional asymmetry, the Broca's area was found to be left lateralized, while the Wernicke's area is mainly right lateralized. Functional asymmetry of these two areas revealed high short- and long-term reliability as well. In addition, the impact of global signal regression (GSR) on reliability of the resting-state language network was investigated, and our results demonstrated that GSR had negligible effect on the temporal reliability of the resting-state language network. Our study provided methodology basis for future cross-culture and clinical researches of resting-state language network and suggested priority of adopting seed-based functional connectivity for its high reliability. Broca's and Wernicke's areas are two major regions associated with language processing  of resting-state fMRI (RS-fMRI) data is rapidly emerging as a highly efficient and powerful tool for in vivo mapping of neural circuitry in the human brain. Resting-state functional connectivity (RSFC) approaches evaluate the correlation patterns of low-frequency fluctuations in the resting-state BOLD signal and generate highly detailed FC maps of complex functional systems A fundamental feature of the human brain is the presence of both structural and functional asymmetries between two hemispheres at both macroscopic and microscopic dimensions Additionally, in RS-fMRI studies, global signal regression (GSR), a widely used preprocessing step, has been shown to introduce negative correlations in standard fMRI analysis The present work provides a temporal reliability examination of resting-state language network. Firstly, we computed RSFC maps with the seeds located at the Broca's and Wernicke's areas separately. The Intra-class correlation (ICC) was then used to evaluate the test-retest (TRT) temporal reliability of the resting-state language network. Furthermore, we investigated the functional asymmetry of resting-state language network and its TRT reliability. In addition, the impact of GSR on the temporal reliability of the resting-state language network was explored.http://www.nitrc.org/projects/nyu_trt/) was used in the current study. All participants had no history of psychiatric or neurological illness, as confirmed by clinical assessment. Informed consent was obtained prior to participation. Data were collected according to protocols approved by the institutional review boards of New York University (NYU) and the NYU School of Medicine. This data set has been used to examine the TRT reliability of cross-correlations A RS-fMRI data set including 25 right-handed participants  that is publicly available at NITRC (3). Scans 2 and 3 were conducted in a single scan session, 45 min apart, and were 5–16 months (mean 11±4 months) after Scan 1. During the scan, participants were instructed to rest with their eyes open.A Siemens Allegra 3-T scanner was used to obtain three resting-state scans for each participant. Each scan consisted of 197 contiguous EPI functional volumes  and RS-fMRI Data Analysis Toolkit  th slice as reference slice; 2) realignment; 3) normalization by using EPI template and resampling to 3 mm isotropic voxels; 4) spatial smoothing via a Gaussian kernel with FWHM  = 6 mm; 5) removal of linear trend and band-pass temporal filtering (0.01–0.1 Hz); and 6) regressing out nine nuisance covariates ; and 7) temporal “scrubbing” of volumes within each subject's fMRI time series that were associated with sudden head motion The preprocessing was carried out by using Data Processing Assistant for RS-fMRI (DPARSF) z-maps through Fisher's transformation  were considered significant, and masks of significant correlated regions with each ROI were generated.As we focused on the cortical regions which functionally correlated with Broca's and Wernicke's areas, we estimated the RSFC maps for each subject and each functional scan using the standard seed-based correlation analysis. In line with a previous study z-maps of the rBro and rWer were left-right flipped. The asymmetry index (AI) was defined as z-transformed RSFC of left ROI minus left-right flipped RSFC of the right ROI, which can be shown as Eq. (1).The asymmetry of RSFC in the present study was defined in line with previous studies t-test was conducted on individual AI maps to reveal regions which showed significant hemispheric asymmetry and were corrected for multiple comparisons FWEp <0.05 . Before correction, t-maps were masked by an “OR” mask which were generated by merging corrected RSFC maps of left and right seed ROIs.As a result, the left hemisphere of AI map revealed ipsilateral asymmetry, while contralateral asymmetry was demonstrated in right hemisphere. AI maps of both Broca's and Wernicke's areas were computed voxel by voxel across all subjects and averaged among three scans for each subject. One-sample b) and within-subject mean square (MSw), and then ICC values were computed according the following formula:Z scores:To assess the TRT reliability of those seed-based RSFC maps and AI maps, we calculated ICC values, a common coefficient assessing TRT reliability FWEp <0.05 .To evaluate the impact of global signal regression on ICC of resting-state language network, we adapted Eq. (3) to compare the ICC values obtained with GSR or without GSR Z-transformed RSFC maps across subjects were calculated for each scan. r = 0.9022; Scan 1 vs. average of Scan 2 and Scan 3: r = 0.9388).Voxel-wise RSFC analysis demonstrated the whole brain correlation patterns related to the two seed ROIs which represented Broca's and Wernicke's areas. The patterns of the connectivity  with ther = 0.6824, p<10−300; Wernicke's area: r = 0.6960, p<10−300). For Broca's area, regions with high reliability were located at the bilateral frontal cortices, middle temporal gyrus (MTG) and inferior (IPL), left middle occipital gyrus (MOG), precuneus, and posterior cingulate gyrus. And for Wernicke's area, regions with high reliability located at the left supramarginal gyrus (SMG) bilateral MTG/inferior temporal gyrus (ITG), bilateral frontal cortices and bilateral IPL.To evaluate the short- and long-term reliability of the RSFC, we calculated the intra- and inter-session ICC maps for each of the two ROIs  , Table 1The functional hemispheric asymmetry maps of Broca's and Wernicke's areas were shown in t values within each significant cluster were extracted from one-sample t map of left seed and LR-flipped one-sample t map of the right seed. To further evaluate the RSFC strength within each region that revealed significant hemispheric asymmetry, mean Z-maps were shown in To assess TRT reliability of functional hemispheric asymmetry of resting-state language network seeding at both Broca's and Wernicke's areas, we computed both inter- and intra-session voxel-wise ICC of AI of those two regions. Intra- and inter-session ICC maps and the converted FWEp <0.05) or with much looser threshold . In addition, To evaluate the impact of GSR on TRT reliability of the resting-state language network, the ICC patterns identified with and without GSR for Broca's and Wernicke's areas were computed respectively. We calculated the whole brain RSFC and the functional asymmetry for Broca's and Wernicke's areas respectively and evaluated their short- and long-term reliability. In addition, the impact of global signal regression on test-retest reliability of resting-state language network was investigated. We found that there was a highly temporally reliable, both short- and long-term, language network during resting state. Moreover, we found the RSFC of Broca's area was mainly left lateralized, while that of the Wernicke's area was right lateralized. In addition, both Broca's and Wernicke's areas revealed high inter- and intra-session reliability for functional asymmetry in most brain regions. Finally, we showed that TRT reliability of the resting-state language network was little impacted by the GSR.The RSFC patterns based on Broca's and Wernicke's areas were highly similar across three scans. This resting state language network was similar to that of language comprehension and production network which was reported in the reviews Those negative correlation pattern might demonstrate the functionally temporal segregation of auditory and visual perception and that of speech comprehension and production The resting-state language networks were temporally (both short- and long-term) reliable. The highly reliable regions were mainly located at bilateral frontal cortices, IPL, MTG and precuneus. The highly reliable regions were those typically observed to be coactive during task-based studies imilar to other TRT studies of RSFC We examined the functional lateralization of resting-state language network using Broca's and Wernicke's areas as seeds. The functional network of Broca's area was almost leftward lateralized see . This fiHowever, rightward asymmetry was found in most regions within functional network of Wernicke's area. It was not beyond our expectation and evidences of this rightward lateralization can be found in previous studies. Rightward lateralization long-range connectivity density of the anterior Wernicke's region was revealed by Tomasi and Volkow TRT reliability of functional hemispheric asymmetry of language networks was also assessed via ICC. We showed that widely spread cortical regions revealed significant both intra- and inter-session temporal reliability of functional asymmetry. The significant reliable regions included frontal cortices, temporal cortices and precuneus. These cortical areas were all within language network and default mode network. It demonstrated that functional asymmetries of language networks were both short- and long-term temporally stable. Considering that the lateralization of language related networks might be a biomarker of mental disorders, such as schizophrenia, our findings suggest its potentially clinical application in tracing longitudinal effect of treatment and training.The ICC patterns with and without GSR were similar to each other, though GSR would impact the RSFC patterns at both the individual and group level. In previous TRT studies, some preprocessed the resting-state data with The current study is not without limitations. Firstly, a recent study showed that reliability of RSFC can be greatly improved by increasing the scan lengths from 5 min up to 13 min In this study, we established seed-based analysis of language network and its functional asymmetry during resting state, and investigated TRT reliability of the language network. We found there was a highly reproducible language network during resting state. Furthermore, we showed that the RSFC of Broca's area was left lateralized, while that of the Wernicke's area was right lateralized. Moreover, both Broca's and Wernicke's areas revealed high inter- and intra-session reliability of functional asymmetry of RSFC in most brain regions. In addition, the reliability of the resting-state language network was little affected by whether performing GSR during preprocessing.Figure S1Functional connectivity patterns of Broca's and Wernicke's areas. The whole brain RSFC maps of Broca's (A) and Wernicke's areas (B). Results were statistically corrected . L and R represent left hemisphere and right hemisphere, respectively.(TIF)Click here for additional data file.Text S1Derivation of equation for testing significance of ICC.(DOCX)Click here for additional data file.


On the other hand, it is well known that MRI imaging plays a very important role in distinguishing abscess loculation from malignancy. The authors present the case of a 13-year-old boy with pain in the right heel for few months. Radiograph of the right foot revealed a lucent focus with sclerotic margin in the right calcaneum. MRI T1-weighted images were inconclusive of penumbra sign to characterise abscess cavity due to the small volume lesion. Whole-body 18F-FDG PET/CT scan showed multiple small avid lesions at the margin of the sclerotic rim in the right calcaneum. Final diagnosis of Brodie’s abscess with Klebsiella culture was confirmed via bone debridement.Chronic osteomyelitis (Brodie’s abscess) is essentially a problem of diagnosis, and there may be considerable difficulty in distinguishing it from other benign and malignant bone lesions. Early diagnosis of Brodie’s abscess is deemed important as the disease has a good curative potential following an appropriate antibiotic treatment. Of late, PET/CT using  Brodie’s abscess is a rare lesion manifesting as a chronic form of intraosseous osteomyelitis surrounded by dense fibrous tissue and sclerotic bone . It is p18F-FDG tracer as glucose analog provide a considerably good complementary functional biomarker in defining the chronic osteomyelitis and is a highly sensitive and specific method for the evaluation of chronic infection in the axial and appendicular skeleton in patients with trauma [18F-FDG PET/CT utility as a potential adjunct to MRI in characterising Brodie’s abscess in a 13-year-old boy in whom diagnosis was proven by isolation of Klebsiella organism culture via bone debridement.The central portions are mainly constituted of necrotic fluid and pathologic organisms . Grey eth trauma . Furtherh trauma . The aut18F-FDG avidity at the margin of the sclerotic margin consistence with granulation tissue formation . Bone debridement yielded the culture of Klebsiella organism and the final diagnosis of Brodie’s abscess was made.A 13-year-old boy presented with a history of right heel pain for a few months. His right foot was swollen and tender. He recalled an accidental step on a rusty nail two years back following which he had pain and swelling. The symptoms subsided following courses of antibiotic treatment. The recent radiography of the right ankle revealed a focus of intraosseous lucency within the right calcaneum . MRI stu18F-FDG PET/CT, foci of tiny rim of avid metabolic activity at the margin of the sclerotic rims points to the formation of granulation tissue [18F-FDG appears to be a highly sensitive method in detecting infective foci in the bone [18F-FDG avidity defines the area of fibroblast proliferation and neovascularisation with mononuclear cell infiltration at the granulation tissue formation whereby these cells utilise most of their energy from the trapped 18F-FDG for cells metabolism [18F-FDG as a biomarker in chronic osteomyelitis is further substantiated by a revelation of an experiment of which findings stated that the maximum uptake of 18F-FDG was observed in chronic inflammation far more pronounced than in acute phase [18F-FDG is exceedingly a sensitive tool in the imaging of chronic infection given its ability in allowing precise anatomical localisation and optimal characterisation of the abscess formation and its extent by its biological 18F-FDG affinity to the chronic inflammatory tissue.This young boy presented with a history of right heel pain for a few months without preceding inciting trauma or symptoms. The past history of trauma to the right heel raises the index of suspicion of an inadequately treated infective bone inoculation which manifested at the present time. Initial radiography of the right ankle was indeed nonspecific as a lucent focus in the right calcaneum may resemble other pathological entity i.e. eosinophilic granuloma or metastatic deposit but these alternate diagnoses can be excluded by the extensive soft tissue inflammation clinically . MRI of n tissue . The sclthe bone . Histolotabolism . The relte phase . In this18F-FDG PET/CT as a form of molecular imaging procedure should be viewed as an important adjunct to an already powerful armamentarium of clinical imaging tools, specifically the MRI imaging. This report underlies the promising method of 18F-FDG PET/CT and its further roles as a potential primary workhorse in chronic osteomyelitis in instances where MRI fails to characterise the differentiating feature of abscess formation from malignancy.The


These energy separations ranging from 10 to 30 meV among the emissive states can be attributed to the coupling of wavefunctions of carriers between neighboring NDs.The picosecond carrier dynamics in a closely packed Si-nanodisk (Si-ND) array with ultrathin potential barrier fabricated by neutral beam etching using bio-nano-templates was investigated by time-resolved photoluminescence (PL). The PL decay curves were analyzed as a function of photon energy by the global fitting method. We show three spectral components with different decay times, where the systematic energy differences of the spectral peaks are clarified: 2.03 eV for the fastest decaying component with a decay time  Semiconductor nanostructures have been paid much attention because of their potential applications to optical devices with improved performances or novel functionalities. Quantum effects in the nanostructures are essential to realize those optical applications. The quantum effects can also affect the carrier dynamics in the nanostructures because carrier transfer or separation can be understood by a combined effect of the spatial distribution of carrier wavefunction with the energy relaxation of carriers. Therefore, the size, density, and interspacing of the nanostructure, which strongly modify the carrier wavefunction, need to be precisely controlled for the device applications. For this purpose, we have recently reported Si-nanodisk (Si-ND) arrays fabricated by neutral beam etching using bio-nano-templates . This meThe Si-ND array was fabricated by damage-free neutral beam etching using a bio-template composed of ferritin supramolecules. Details of the fabrication process are described elsewhere -3. The N−2) are shown in Figure The time-resolved PL spectra of the Si-ND array at 250 K with an excitation power of 50 mW  is meaningfully lower than those of the faster two components . The energy difference between I1 and I2 is 20 meV, and that between I1 and I3 is 30 meV, which are quantitatively obtained from the fittings for the decay-associated component PL spectra with Gaussian functions. These energy differences mean the energy separations among the energy levels of individual emissive electronic states discussed above. The lowest energy state associated to the slowest PL component (I1 with τ1) originates from the electron–hole pairs that are weakly localized in surface states in individual NDs, which corresponds to the ground state in the present high-density ND system, and the wavefunctions of carriers are localized within one ND. Higher-energy levels with faster decay times  are excited states in the ND system, and the coupling of carrier wavefunctions among NDs can form these excited states. Therefore, we consider that the obtained values of 20 to 30 meV of the energy differences among the decay-associated PL components can be explained by the energy differences between the ground state and the excited states in the present coupled Si-ND array structure. We have also confirmed this result from the temperature dependences of the decay-associated PL intensities. The intensities of these three spectral components were measured with varying temperatures, and the results were fitted by calculations taking activation energies between each energy level as well as the barrier level into account. The differences of the activation energy also indicate the energy differences of the lower two states, and the obtained value is 55 meV between the slowest component (τ1) and the middle component (τ2), which agrees with the results obtained from the global fitting method. Further study to test the energetic structure experimentally elucidated here in the Si-ND system is needed using three-dimensional calculations of the carrier wavefunctions based on the actual ND array structure.As can be seen in Figure τ1) and the faster two components . These energy differences for the emissive states can be attributed to the different coupling states of the carrier wavefunctions in the present high-density ND system. The ultrafast carrier dynamics elucidated from the time-resolved PL analysis demonstrates that high-density semiconductor nanostructures precisely designed and fabricated by neutral beam etching using bio-nano-templates are promising for optical applications based on quantum effects such as quantum dot lasing and photovoltaics.The carrier dynamics in the high-density Si-ND array fabricated by neutral beam etching using bio-nano-templates was studied by time-resolved PL, where the PL decay curves were analyzed at various photon energies by the global fitting method. The obtained decay-associated spectral components show clear differences of the peak energy of 20 to 30 meV between the slowest component (ND: Nanodisk; PL: Photoluminescence.The authors declare that they have no competing interests.TK and AM conceived the spectroscopic study, participated in its design and coordination, and drafted the manuscript. TK and YM carried out the time-resolved PL measurement and analyzed the data. MI and SS conceived the fabrication process and participated in its design and coordination. MI fabricated the Si-ND array sample. All authors read and approved the final manuscript.


However, the information content inherent to some relationships between the interrogated gene sets, such as pathway crosstalk, is often underutilized. A gene set network, where nodes representing individual gene sets such as KEGG pathways are connected to indicate a functional dependency, is well suited to visualize and analyze global gene set relationships. Here we introduce a novel gene set network construction algorithm that integrates gene lists derived from high-throughput experiments with curated gene sets to construct co-enrichment gene set networks. Along with previously described co-membership and linkage algorithms, we apply the co-enrichment algorithm to eight gene set collections to construct integrated multi-evidence gene set networks with multiple edge types connecting gene sets. We demonstrate the utility of approach through examples of novel gene set networks such as the chromosome map co-differential expression gene set network. A total of twenty-four gene set networks are exposed via a web tool called MetaNet, where context-specific multi-edge gene set networks are constructed from enriched gene sets within user-defined gene lists. MetaNet is freely available at  Networks that connect biomolecules such as genes and proteins with each other have been increasingly used to understand global cellular systems. Edges in biological networks are either determined from high-throughput experiments such as those for identifying physical interactions between protein pairs and potential transcription regulatory interactions between transcription factors and genes or are constructed using informatics approaches as in the case of gene co-expression networks. Informatics approaches that integrate various edge types as independent sources of evidence have been used to construct functional linkage networks for the prediction of gene function and identification of functionally coherent modules et al. describe a gene set network algorithm for the construction of biological process linkage networks where two GO biological process terms are linked in a gene set network if there are a significant number of edges between the unique gene members of the two terms in the original single-biomolecule network Networks can also be used to describe functional relationships between biological themes et al. Li Here, we describe a novel algorithm for constructing co-enrichment gene set networks that integrate experimentally derived gene lists with literature-curated gene sets and demonstrate the utility of the approach through examples of novel insights gained as compared to co-membership and linkage gene set networks when applied to KEGG Pathway and Chromosome Map gene sets through integration with gene expression microarray data. We take advantage of disease related gene sets in the KEGG Pathway database to compare neurodegenerative diseases based on their transcriptional dependencies with pathways. The relationship between differential expression, pathways, and linear and three-dimensional genome organization is demonstrated via the Chromosome Map co-enrichment gene set network. Next, we describe Phosphorylation Substrates gene set networks where gene sets consisting of substrates of kinases and phosphatases are connected to identify functional relationships between the enzymes, which to our knowledge are novel. We find that co-enrichment of Phosphorylation Substrates gene sets based on integration with gene expression does not provide additional information as compared to co-membership and linkage gene set networks, which is expected considering the post-translational nature of the gene sets.http://blaispathways.dfci.harvard.edu/metanet/.Several tools such as the ConceptGen Here we describe the intuition behind gene set network construction. For details please see Co-membership gene set networks connect a pair of gene sets if there is significant overlap between the corresponding members . These gWe apply the co-membership, linkage (PPI), and co-enrichment  to popular collections of human gene sets, such as Gene Ontology (GO), KEGG Pathway, and WikiPathways, that are often used in gene set enrichment analyses . For claWe also constructed gene set networks where gene sets were determined based on phosphorylation and physical interaction data . A node KEGG Pathway (1556 edges: 201 nodes), WikiPathways (981 edges: 147 nodes), and GO biological process (1852 edges: 396 nodes) co-DE gene set networks have the highest proportion of edges to nodes amongst the considered co-DE gene set networks suggesting that pairs of entire biological processes are more often transcriptionally controlled together than pairs of individual complexes or molecular functions . The perIn the following sections, we demonstrate the utility of gene set networks using the KEGG Pathway, Chromosome Map, and Phosphorylation Substrates gene set collections as examples. We first discuss co-DE as a novel approach for construction of global gene set networks.The KEGG Pathway database contains literature-curated maps of metabolic, signaling, and immune response pathways as well as descriptions of cellular complexes such as the spliceosome and replication and repair machinery. The addition of disease related pathway maps provides for a common database for linking the aforementioned cellular processes with diseases. Furthermore co-membership between pathway gene sets directly lends itself to interpretation as pathway crosstalk, a well-studied phenomenon The KEGG Pathway gene set networks consist of 201 nodes each. After evaluating all 20100 possible pairs for pathways, we determined 2097 and 1556 edges in the co-membership and co-DE gene set networks respectively . While sDespite the surprising similarities between the two gene set networks, considering that shared members are explicitly removed when constructing the co-DE gene set network, there are several unique relationships that are only revealed after integration of gene expression data. For example, the most significant co-DE edge is between the Steroid biosynthesis and Terpenoid backbone biosynthesis metabolic pathways , an edgeIn addition to the novel pairwise relationships between pathways, the KEGG Pathway co-DE gene set network has differences in network topology as compared to the co-membership gene set network. While the overall degree centrality is correlated  space. We collected Hi-C data from Lieberman-Aiden Three-dimensional proximity only partially explains the co-differential expression of chromosome loci pairs since the number of Hi-C reads for co-differentially expressed pairs on different chromosomes is still significantly less than the number of Hi-C reads for same chromosome loci pairs that are not co-differentially expressed  coupled with mass spectrometry The MetaNet tool constructs a gene set network of the enriched gene sets with edges from all pre-computed co-membership, linkage, and co-enrichment gene set networks. The current version of MetaNet contains co-membership, PPI and co-differential expression gene set networks. The KEGG Pathways that are enriched amongst the Ku complex genes are connected in a gene set network with multiple edges from the aforementioned pre-computed gene set networks . The conAll MetaNet results can be downloaded as graphics or text files; the gene set network graph visualization can be downloaded in PDF and PNG and the graph contents and structure can be download in SIF or GraphML format that can be subsequently imported in visualization tools. While MetaNet is not designed to visualize global gene set networks, all pre-computed gene set networks in their entirety and all gene set annotations are available for download in tab-delimited text format, which can be imported into network visualization tools such as Cytoscape Gene set networks map functional relationships between biological themes and can be used to identify novel insights into their organization. While co-membership gene set networks intuitively link similar gene sets, based on shared entities, they cannot be used where the expected overlap between any pair is negligible as in the case of chromosome loci. Co-enrichment and co-linkage gene set networks integrate additional evidence with curated gene sets to identify hidden relationships. To our knowledge, this is the first reported study describing the co-enrichment method for gene set network construction. The advantage of this approach is that the experimental data are included as gene lists, allowing independent instrument and experiment specific processing of the data. Thus, differentially expressed genes lists from next-generation sequencing data can be readily integrated for the construction of co-differential expression gene set networks using the co-enrichment method. Given the flexibility in processing, the approach can be extended to other types of data. For example, co-differential expression is not an appropriate measure of functional relationship between the Phosphorylation Substrates collection of gene sets. In this case, phosphoprotein lists derived from phosphoproteomics experiments can replace differentially expressed gene lists. We tested the approach on protein lists derived from proteomics studies from the PLIPS database A feature of the co-enrichment and linkage gene set network construction algorithms is that the removal of shared genes penalizes highly overlapping pairs of gene sets and thusly facilitates identification of novel relationships between gene sets. However, in the case of GO gene sets there are trivial and uninformative subset relationships, resulting from the hierarchical GO DAG. All three gene set network construction methods require N*(N-1)/2 computations corresponding to all pairs of N gene sets in a collection. The co-enrichment method requires additional M computations at each of the N*(N-1)/2 steps where M is the number of experimentally derived gene lists to be tested for enrichment of gene sets. While there may be ways to reduce the number of computations, current computing power along with parallel processing easily allow all pairwise comparisons of gene sets from popular databases.KEGG Pathway annotations  etal. to select a reduced set of informative GO terms GO gene sets per namespace  were selected such that each term had a cardinality that was closest to 50 within its lineage; i.e. for each term none of its predecessor or successor terms in the GO DAG had a cardinality closer to 50 than its own cardinality and were thus removed. A similar method has been previously used by Linghu A co-membership gene set network for a specified collection of gene sets such as KEGG Pathways connects a pair of gene sets if there are a significant number of genes in common. The procedure is as follows:For a given pair of gene sets in a gene set collection count the number of genes in one gene set, the number of genes in the other gene set, and the number of overlapping genes.Calculate a p-value for significantly high number of overlapping genes using Fisher's exact test. The background for the test is the set of all genes present in any gene set in the collection.Repeat for every unique pair of gene sets and adjust calculated p-values for multiple hypotheses using the Benjamini-Hochberg method.Connect a pair of gene sets if the Benjamini-Hochberg corrected p-value ≤0.05.A linkage gene set network for a specified collection of gene sets such as KEGG Pathways connects a pair of gene sets if there are a significant number of edges in a reference single-biomolecule network, such as a protein-protein interaction network, that link the unique genes of one pathway with the unique genes of the other pathway. The procedure is as follows:Remove all nodes from the reference single-biomolecule network corresponding to genes that are not present in any gene set in the gene set collection.For a given pair of gene sets in a gene set collection remove all shared genes from both gene sets.Count the number of edges, in the reference single-biomolecule network, that link genes from one gene set to any gene, the number of edges that link genes from the other gene set to any gene, and the number of edges that link genes from one gene set to those in the other gene set.Calculate a p-value for significantly high number of edges linking genes from one gene set to those in the other gene set using Fisher's exact test. The background for the test is the set of all edges in the reference single-biomolecule network.Repeat for every unique pair of gene sets and adjust calculated p-values for multiple hypotheses using the Benjamini-Hochberg method.Connect a pair of gene sets if the Benjamini-Hochberg corrected p-value ≤0.05.A co-enrichment gene set network for a specified collection of gene sets such as KEGG Pathways connects a pair of gene sets if there are a significant number of experimentally derived gene lists from a reference set of experiments, such as gene expression microarray experiments, where both gene sets are significantly overrepresented. The procedure is as follows:For a given pair of gene sets in a gene set collection remove all shared genes from both gene sets.Calculate a p-value for significant enrichment of all gene sets, including the pair of gene sets whose shared genes have been removed, in every experimentally derived gene list using Fisher's exact test.Count the number of experimentally derived gene lists where either of the two gene sets from step 1 are significantly enriched, having a Benjamini-Hochberg corrected p-value ≤0.05.Count the number of experimentally derived gene lists where both gene sets from step 1 are significantly enriched.Calculate a p-value for significantly high number of experimentally derived gene lists where both gene sets from step 1 are enriched using Fisher's exact test. The background for the test is the set of all experimentally derived gene lists.Repeat for every unique pair of gene sets and adjust calculated p-values for multiple hypotheses using the Benjamini-Hochberg method.Connect a pair of gene sets if the Benjamini-Hochberg corrected p-value ≤0.05.GRACLUS i and gsj, we construct two sets, KPi and KPj containing the KEGG pathways that genes in gsi and gsj participate in respectively. The Jaccard similarity coefficient between KPi and KPj is used as the KEGG Pathway participation similarity metric.For two loci gene sets gsWe generated 10,000 pairs of random networks by randomizing both the co-membership and PPI Phosphorylation Substrates gene set networks using the edge-swap method (1000 swaps per network) MetaNet was developed using Python 2.6, Django 1.3.1, and jQuery 1.8.6. The underlying server software is Apache 2.2. Graphs are visualized using the Cytoscape Web plugin Figure S1KEGG Pathway neurodegenerative diseases co-differential expression sub-network. Metabolic pathways are in green, non-metabolic pathways are in purple, and the three disease pathways are marked as diamonds.(PDF)Click here for additional data file.Figure S2KEGG Pathway co-membership versus co-differential expression gene set network degree centrality. Each dot indicates a particular pathway. The two aggregate pathways, Metabolic Pathways and Pathways in cancer, are highlighted as large circles. Pathways that have the highest difference in degree centrality relative to either gene set network are also highlighted as large circles.(PDF)Click here for additional data file.Figure S3Phosphorylation Substrates co-membership gene set network. Each node is a kinase (green) or phosphatase (purple) and edges connect enzymes if there is a significant overlap in the substrates they modify.(PDF)Click here for additional data file.Figure S4Phosphorylation Substrates PPI gene set network. Each node is a kinase (green) or phosphatase (purple) and edges connect enzymes if there are a significant number of physical interactions between the unique substrates they modify.(PDF)Click here for additional data file.Figure S5KEGG Pathway proteomics study co-occurrence gene set network. Nodes represent KEGG pathways; metabolic pathways are in green and non-metabolic pathways are in purple. Edges connect pathways if there are a significant number of protein lists from proteomics experiments where the unique components of both pathways are enriched.(PDF)Click here for additional data file.File S1Archived gene set networks. A zip archive of tab-delimited text files containing edges and associate p-values per gene set network.(ZIP)Click here for additional data file.File S2Archived GO co-membership gene set networks using an expected cardinality threshold of 100. A zip archive of tab-delimited text files containing co-membership gene set network nodes, edges, and associate p-values per GO namespace.(ZIP)Click here for additional data file.Table S1Relationship between the number (or percentage) of shared genes and edge presence in a gene set network. All edges per gene set network are divided into two groups: those with lower or higher number (or percentage) of shared genes than the mean number (or percentage) of shared genes for all possible overlapping gene set pairs.(PDF)Click here for additional data file.Table S2PubMed evidence for neurodegenerative disease co-differential expression. PubMed references describing microarray experiments that generated gene lists where KEGG pathways are co-enriched with Alzheimer's, Parkinson's, and Huntington's disease KEGG pathways.(PDF)Click here for additional data file.


A large number of predictive indices are used for evaluation of the capability for transition to spontaneous breathing in critically ill, mechanically ventilated patients. The great number of these indices and the difficulties in the interpretation causes significant obstacles and unclear points during the early attempts for transition to spontaneous breathing. In our study we investigated the role of predictive indices that are significant for weaning after long-term mechanical ventilation. The purpose is to determine predictive indices, which have early and significant predictive value concerning successful transition to spontaneous breathing.The study covers 45 critically ill patients who were mechanically ventilated for more than 7 days in our ICU. The weaning efforts were made through a T-circuit for spontaneous breathing according to the local protocol. The patients were allocated into two groups - group A (38 patients with successful 2-hour spontaneous breathing through a T-circuit) and group B (seven patients with unsuccessful 2-hour test of weaning with a T-circuit system). The monitored parameters in this period were: respiratory rate/tidal volume ratio (f/Vt), occlusive pressure (Po.1), inspiratory time/tidal time ratio (Ti/Ttot), pressure time index, pressure time product and work of breathing (WOBp) together with SAPS II score and clinical and paraclinical parameters, concerning successful weaning.Clinical research of f/Vt and WOBp between the two groups gives a reliable index in transition to spontaneous breathing. Changes in Po.1, Ti/Ttot, pressure time index and pressure time product are later and thus less important in the early assessment of withdrawal after long-term mechanical ventilation.Respiratory rate/tidal volume ratio (f/Vt) and work of breathing (WOBp) are the earliest predictive indices for the possible outcome in the process of weaning after long-term mechanical ventilation.


Sc, it was also observed in the initial analysis that L-type BSE displays a distribution pattern of the pathological prion protein (PrPSc), which clearly differs from that observed in classical BSE (C-type). Most importantly, the obex region in the brainstem is not the region with the highest PrPSc concentrations, but PrPSc is spread more evenly throughout the entire brain. A similar distribution pattern has been revealed for H-type BSE by rapid test analysis. Based on these findings, we performed a more detailed Western blot study of the anatomical PrPSc distribution pattern and the biochemical characteristics  in ten different anatomical locations of the brain from cattle experimentally challenged with H- or L-type BSE, as compared to cattle challenged with C-type BSE. Results of this study revealed distinct differences in the PrPSc deposition patterns between all three BSE forms, while the biochemical characteristics remained stable for each BSE type among all analysed brain areas.Besides the classical form of bovine spongiform encephalopathy (BSE) that has been known for almost three decades, two atypical forms designated H-type and L-type BSE have recently been described. While the main diagnostic feature of these forms is the altered biochemical profile of the accumulated PrP Sc) after protease degradation and Western blot analysis. The unglycosylated PrP fraction migrates 1–2 kDa higher in H-type BSE Bovine spongiform encephalopathy (BSE) belongs to the so called “prion diseases”, also named transmissible spongiform encephalopathies (TSEs). These progressive neurodegenerative diseases are always fatal and can affect humans and animals. The most prominent TSE in humans is Creutzfeldt-Jakob disease (CJD). Scrapie in sheep and goats has been known for more than 250 years Sc depositions in the brains of experimentally challenged cattle using one of the approved BSE rapid test systems. The most prominent differences from the C-type BSE associated distribution pattern became obvious for the rhinencephalon, thalamus and basal nuclei, where distinct amounts of PrPSc could be detected in animals infected with both atypical BSE forms Sc in cattle infected with L-type BSE In earlier studies, we performed an initial analysis of the anatomical distribution of PrPSc in natural C-type, L-type and H-type BSE cases have revealed distinct differences in the PK cleavage sites and thus the antibody binding affinities, as well as in the proteinase K (PK) resistance at different pH conditions and PK concentrations Comparative biochemical analyses of the disease-associated PrPSc ‘glycoprofiles’ in the brains of cattle experimentally challenged with atypical and classical BSE.Interestingly, for human TSEs, a variation of the glycoform ratio has been described in different brain regions for the sporadic form of Creutzfeldt-Jakob disease (sCJD) Sc characteristics may change after passage in certain hosts. For example, a conversion of BASE (L-type BSE) to C-type BSE after serial transmission to inbred mice has been described Moreover, transgenic mouse bioassays revealed that the biochemical BSE profile does not always remain stable, meaning that the specific PrPSc depositions in ten different brain regions of four or five animals challenged with C-, H- or L-type BSE, as determined by Western blot analysis.While earlier studies have mainly addressed the description of clinical signs Eleven female Holstein-Frisian calves (six month of age) were inoculated intracranially with 1 ml of a 10% (w/v) brain homogenate derived from the two identified German atypical BSE cases For a comparative analysis of brains from C-type affected cattle, we included the respective brain samples collected from four animals from an earlier oral challenge study with C-type BSE The challenge experiments in cattle described in this manuscript were approved by the competent authority of the Federal State of Mecklenburg-Western Pommerania, Germany on the basis of national and European legislation, namely the EU council directive 86/609/EEC for the protection of animals used for experiments (LALLF M-V/TSD/7221.3-020/06).cutoff value was evaluated separately for every test run according to the manufacturer’s instructions .First of all, the IDEXX HerdChek® enzyme immunoassay (EIA), which is one of the approved BSE rapid test systems, and which had been applied on four selected brain areas of L- and H-type affected cattle in an earlier study Sc precipitation protocol using phosphotungstic acid with a subsequent Western blot (PTA-WB) using the PrP-specific monoclonal detection antibody mab L42 , as previously described Sc.We also applied a highly sensitive PrPThe Western blot signals were analysed and quantified using the VersaDoc™ Imaging System . For the quantification of the signals, the signal intensity should be in the linear range of the VersaDoc™ Imaging System according to the manufacturer’s instructions. Therefore we first ran one initial gel for each sample to determine the signal intensity, followed by four gels with adjusted sample quantities that were used for our analysis. The molecular weight, the relative quantities and the volume  of each of the three PrP bands were determined using the Quantity One® Software. For the analysis of the glycoprofile, the signal intensity of each of the three PrP bands was calculated as the percentage of the overall signal for each brain region and BSE type individually.Sc depositions in the different brain regions, the cranial medulla was set as the reference (100%) and the signal intensities of the other brain regions were determined in relation to this. Arithmetic means were calculated for each sample.For the quantitative analysis of the PrP(t-test) was performed for the achieved WB data of the glycoprofile and the molecular mass.Statistical analysis Sc signal determined after incubation with a final PK concentration of 50 µg/ml, which is used in our standard protocol, was set as the reference point (100%). In the second approach, we incubated reactions with 50 µg/ml PK for 1, 3, 6, 10 and 26 hours. Here, the residual signal obtained after a one hour PK digestion using 50 µg/ml (standard protocol) was again set as the reference point (100%).In a pilot experiment using homogenates from different brain regions, it was revealed that the PK susceptibility did not differ between the analysed brain areas. For the systematic analysis of the PK susceptibility, we therefore pooled different brain areas with the strongest signal intensities of each BSE form. The PK sensitivity was analysed by two different experimental setups: first, we added varying PK concentrations of 0, 25, 50, 250 or 1000 µg/ml to the reaction and incubated these for 60 min at 55°C. The remaining PrPFor each BSE type, testing was done in duplicate, the gel electrophoresis was repeated three times and the arithmetic means of the obtained signal intensities were calculated.This study was aimed at the detailed analysis of the anatomical distribution and the biochemical characteristics of both atypical BSE forms designated L-type and H-type, as compared to C-type BSE.n = 5) the highest OD values were detected in the midbrain, cranial medulla and thalamus. The OD values determined for these examined brain regions showed relatively even overall OD values ranging between 2.47 and 3.05, which is close to the upper limit of the test, and therefore in the non-linear range. In cattle challenged with H-type BSE (n = 5) the highest OD values could also be detected in the midbrain, cranial medulla and thalamus. Like in L-type BSE affected cattle, the OD values were relatively even, but overall lower than determined for L-type BSE, ranging between 1.60 and 2.68. In contrast to the atypical cases, a more distinct difference was seen between the different brain areas of cattle affected with C-type BSE. For the medulla and midbrain region OD values between 2.61 and 3.09 were determined, as opposed to the OD values for the cortex regions between 0.46 and 1.16. The relative signals in relation to the OD value determined for the cranial medulla as determined by the IDEXX HerdChek® EIA are summarized in At first we used the IDEXX HerdChek® EIA rapid test on the ten brain areas analysed in this study. In cattle challenged with L-type BSE  revealed high values for L-type BSE in every brain region, which were distinctly higher compared to the other two BSE forms  determined the mean percentage ratio of the di-, mono- and unglycosylated PrPSc fraction out of the overall signal. Our study revealed glycoprofiles for classical BSE of 60 : 27 : 13%, for L-type BSE 42 : 36 : 22% and for H-type BSE 55 : 29 : 16% for the diglycosylated : monoglycosylated : unglycosylated PrPSc fraction .While we determined distinct differences in the glycoprofiles and the molecular masses of the PrPSc deposition pattern of the inoculum in our analysis; hence no alteration of the specific biochemical characteristics had occurred for the three BSE strains upon subpassage in cattle.Moreover, all cattle of a group challenged with one BSE type revealed the same PrPWith the evaluation of the PK susceptibility of the three BSE forms by applying different PK concentrations  and longIn addition, when we applied a long-term PK digestion , the difSc in ten different brain regions of cattle affected with atypical H- and L-type BSE, as compared to that of C-type BSE. All animals were in the moderate to severe clinical stage of a BSE infection when euthanized. For a comparison of the results obtained after an intracerebral challenge with atypical BSE to those of classical BSE, we selected four representative animals from the oral C-type BSE pathogenesis study performed earlier Sc depositions after intracranial challenged with C-type BSE was assessed in 18 different brain regions by Western blot ). However, it reveals that the cortex areas, especially the occipital cortex, seem to be less affected in orally challenged cattle. On the other hand, the Italian L-type field cases where different brain areas were available for analysis also displayed high PrPSc concentrations in the cortex Sc depositions was higher around the injection canal. It could even be further demonstrated by the use of bovine- versus ovine-PrP-specific antibodies, that the PrPSc depositions around the injection site were indeed newly formed ovine PrPSc formations, rather than the bovine inoculum Sc depositions in the forebrain areas Sc and infectivity are usually washed out from the damaged brain area via the blood stream and dispersed into the periphery In the present study, we assessed the anatomical distribution pattern and the biochemical characteristics of the accumulated PrPSc depositions in ten different brain regions. These analyses verified distinct qualitative and quantitative differences in the anatomical distribution of PrPSc depositions in the brains of cattle clinically affected with C-, L- or H-type BSE. While for C-type BSE the PrPSc depositions are mainly restricted to the brainstem and to a lesser extent to the cerebellum, whereas cattle affected with both atypical BSE forms showed an almost even distribution of the pathological prion protein throughout the whole brain. Although deposition patterns were similar for L- and H-type BSE, L-type BSE displayed stronger overall intensities. Even though the results from these areas may be biased by the inoculation route as explained above, the most pronounced differences were detected in the cortex regions. While cattle affected with C-type BSE showed only faint signals, even in the end-stage of the disease, cattle affected with both atypical BSE forms displayed distinctly higher PrPSc concentrations in the analysed cortex samples. Cattle affected with all three BSE types showed remarkably high PrPSc amounts in the midbrain, medulla oblongata and in the thalamus. However, higher concentrations were determined in the caudal medulla than in cranial medulla for classical BSE. The opposite was true for atypical BSE, where higher PrPSc quantities were found in the cranial medulla as opposed to the caudal medulla. This finding stresses the importance to carefully take the diagnostic sample from the obex, instead of caudal medulla for BSE rapid testing, in particular with regard to L-type BSE, because the thalamus, basal nuclei and midbrain showed stronger involvement than the obex and especially the caudal medulla. This confirms similar findings that have been reported by others We performed biochemical analyses of the PrPSc depositions also in L-type BSE affected cattle. However, in our approach we did not use diluted samples in the BSE rapid test, but we adjusted the protein amounts in the Western blot, so that we were able to quantify the PrPSc amounts in this assay.The IDEXX HerdChek® EIA is one of the EU-approved rapid tests with a comparably high sensitivity for all three BSE forms During the challenge experiment with atypical BSE in cattle, we observed behavioural differences between classical and atypical BSE affected cattle during the end-stage of disease which have already been described in more detail Sc accumulated in the ten different brain regions. As expected non-glycosylated PrPSc of L-type BSE had a slightly lower molecular mass than that of C-type BSE, whereas non-glycosylated PrPSc of H-type was approximately one kDa higher and all three BSE forms were encoded by a distinct glycoform. However, apart from these general differences, no statistically significant sampling effects were revealed for any of the three BSE types. These findings confirm what has been reported for mouse passaged BSE and scrapie strains We then analysed the glycosylation profiles and molecular masses of PK digested PrPSc associated with C-type BSE is more resistant towards PK degradation than that of both atypical BSE forms, irrespective of the experimental setup addressing a PK digestion with higher concentrations of up to 1000 µg/ml, or a long-term PK digestion of up to 26 hours. These findings confirm earlier reports Sc as opposed to C-type BSE. Although it has been postulated that PrPSc is not identical to the infectious agent Sc will also reduce the infectivity titre in a sample.Furthermore, we could show that PrPSc deposition in the brains of cattle affected with the three BSE forms. These differences are sufficiently clear to enable discrimination between classical BSE and atypical BSE, and at least supportive for a differentiation of both atypical BSE forms by Western blot or ELISA rapid test analysis, providing a sufficient number of relevant brain areas are available for analysis. Moreover, these findings are scientifically interesting, since we believe that they mirror the clinical picture observed in cattle affected with either classical or atypical BSE. The differences in protease sensitivity may also have a practical impact on the stability in the environment and possibly on the interspecies transmissibility of the three BSE forms, but these hypothesis need to be elucidated by further analysis.In summary, we could show distinct differences in the anatomical distribution of PrP


Here we address the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. We show that GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, we detected a two-fold increase in the percentage of GSCs in mitotic division compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. We conclude that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance our understanding concerning stem cell dynamics in normal tissues and in a tumor model.Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their  Adult stem cells self-renew and give rise to differentiating daughters that maintain specific tissues throughout the life of an individual. The therapeutic potential of stem cells and the etiological role they may play in cancer biology make studying the behavior of these cells in living animals crucial to our long-term ability to both treat and prevent disease Less is known about how the mitotic activity of stem cells is regulated in vivo. This understudied aspect of stem cell biology is crucial because small changes in the frequency of stem cell divisions can dramatically alter the number of terminally differentiated cells. In mammalian tissues, stem cells are generally thought to be long-lived and to cycle slowly Drosophila gonad are remarkably similar to those found in vertebrates and studies using this model have revealed fundamental insights into stem cell biology. The Drosophila testis is a coiled, tubular structure that contains nine to twelve GSCs at the apical tip which are organized around a group of terminally differentiated somatic cells, termed the hub  that form cytoplasmic extensions around the GSC and into the hub , is cleaved into its active, secreted form by the protease Stet spi or stet do not associate with cyst cells and fail to differentiate. Instead, they accumulate as GSCs, gonialblasts, and early stage spermatogonia, resembling germ cell tumors upd or the TGFβ ligand decapentaplegic (dpp) in germline cells results in their accumulation at early stages. Whereas upd overexpression leads to testes filled with single germline cells that resemble GSCs dpp overexpression leads to clusters of supernumerary spermatogonia Germline tumors in the stet as well as RNAi-mediated knockdown of the EGFR in cyst cells recapitulated the increased mitotic activity of GSCs. These data demonstrate a novel and specific role for EGF signaling: the repression of GSC division frequency.Here, we report on the division dynamics of GSCs in response to attenuated EGF signaling. GSCs in EGF mutant testes contained more cells in M-phase and in S-phase of the cell cycle and it took significantly less time for all GSCs within one testes to complete one round of the cell cycle compared to GSCs in control testes. Confirming the role for EGF signaling in regulating the frequency of GSC divisions, germline-specific expression of EGF ligand rescued the hyperproliferation of GSCs in EGF mutant animals. Mutations in This novel role for EGF signaling is developmentally independent of its previously reported role in promoting germ cell differentiation To address the in vivo division dynamics of stem cells, we quantified the percentage of GSCs in mitosis (M-phase index). Testes were labeled with a hub marker (anti-Fasciclin III), a germ cell marker (anti-Vasa), and a mitosis marker (anti-phosphorylated Histone-H3 (PHH3), 77-20spi. As previously reported 77-20spi mutant animals grown at a restrictive temperature of 26.5°C were small and filled with early stage germline cells (not shown). We discovered that the M-phase index was approximately two-fold higher for GSCs in testes from 77-20spi mutant animals  than the M-phase index for GSCs in 1118w control testes  than the M-phase index observed for GSCs in control testes.To circumvent fluctuations as much as possible and to be able to compare the GSC division frequencies from different sets of experiments, we only used three to ten day old adult males that were raised and kept at 26.5°C. Prior to dissection, these males were kept at a population density of 100 males/bottle and fed with fresh yeast paste for three days. All data were reproduced in at least three independent experiments. Under these conditions, the range of GSC M-phase indices was highly reproducible. The GSC M-phase index from control testes always ranged between 6% and 10% (n>300 GSCs/experiment). The M-phase index of 77-20spi testes. We hypothesized that GSC and CySC divisions may be coordinated to ensure that two cyst cells are produced for each gonialblast. CySCs and their daughters associated with early stage spermatogonia express the transcription factor Traffic jam (Tj) in their nuclei 77-20spi testes  was similar to that of CySCs in 1118w testes  was significantly higher than the S-phase index calculated for GSCs from 1118w testes  was already dramatically higher than the S-phase index of GSCs in 1118w testes , whereas only 61% (n = 144 testes) of GSCs from the 1118w testes had detectable BrdU incorporation  using ex vivo labeling with the thymidine analog, BrdU. We found that the S-phase index of GSCs from poration . This stOur S-phase indices are consistently lower than those reported in a previous study Small changes in the frequency of stem cell divisions can have a dramatic effect on cell number and tumor growth. Thus, it is not surprising that mechanisms have evolved to regulate this aspect of stem cell biology. EGF-dependent regulation of GSC division frequency may be important for reducing the frequency of unnecessary cell divisions that increase the chance of mutations being introduced into the germline. Alternatively, it may simply be required for increasing the duration of fitness by mobilizing energy away from sperm production.77-20spi testes 77-20spi testes from animals carrying either the nanos-Gal4-  or the UAS-s-spi-constructs  alone were approximately two-fold higher than those calculated for GSCs from 1118w testes   similar to that observed from GSCs in 1118w testes  . In cont8 testes . These dstet alleles do not have intact testis sheaths stet-allele, 3stet, over an amorphic allele, 1stet. We found that the M-phase index of GSCs in testes from 1/stet3stet males was increased  relative to GSCs in 1118w testes  (JF02384dEGFR) in CySCs and cyst cells increased GSC division frequency. We used the UAS/Gal4-system JF02384dEGFR using the soma-specific eyaA3-Gal4 transactivator displayed all defects characteristic of the loss of EGF signaling (data not shown), including a higher M-phase index  compared to the GSC M-phase indices calculated from control animals carrying either the eyaA3-Gal4-  or the UAS-JF02384dEGFR-construct  alone (JF02384dEGFR in the germline or the somatic hub cells did not result in increased M-phase indices (data not shown). These data strongly suggest that GSC-secreted EGF is received via the EGFR on CySCs, and that this signaling event in turn represses the frequency of GSC divisions.Mutations in the germline specific protease Stet n = 424) . Similar0) alone . Expressrd instar larvae and adults. The testes of Drosophila third instar larvae are round discs that have yet to undergo the morphogenetic events that result in a coiled tube connected to the reproductive tract and genitalia. Although Drosophila males do not reach sexual maturity until after eclosion, spermatogenesis begins during the 1st instar of larval development. By the end of the 3rd larval instar, testes contain germline cells in most stages of spermatogenesis  was similar to that of larval 1118w testes  was similar to the mitotic index of GSCs in larval 1118w testes  or upd  within their germline cells were similar to the M-phase indices of GSCs in testes from control animals harboring either only the nanos-Gal4-, the UAS-dpp-, or the UAS-upd-construct . All UAS-Gal4 expression studies All fly stocks were raised and maintained on standard cornmeal molasses agar medium at 26.5°C. Mutations and transgenic elements are described in Testes were dissected and placed in Testis Isolation Buffer  on ice. Testes were subsequently fixed in 4% formaldehyde in PBT for 30 minutes. Primary antibody incubation took place overnight at 4°C and secondary antibody incubation took place for 2 hours at room temperature. Testes were mounted onto slides using Vectashield mounting medium with DAPI. Tissues were observed using a Zeiss Axiophot microscope. Images were taken with a CCD camera using an Apotome and Axiovision Rel Software. Antibodies and dilutions used were as follows: goat anti-Vasa , rabbit anti-phosphorylated Histone-H3 Ser10 , mouse anti-BrdU , and anti-FasiciclinIII 7G10 . Alexa-488-, Cy3-, and Cy5-conjugated secondary antibodies were used at 1∶1000 (Invitrogen).BrdU ex vivo labeling of GSCs was performed as described by For BrdU in vivo labeling, animals were fed 10 µM BrdU in yeast paste and the plates were replaced every 12 hours. Flies were kept at 26.5°C and dissected after 36 or 48 hours.All experiments were performed on flies raised and kept at 26.5°C. Adult flies were less than ten days old kept in bottles at a density of 100 males per bottle, and fed with fresh yeast paste for three days prior to dissection. The S-phase and M-phase indices were calculated by dividing either the number of BrdU-positive (S-phase) or pHH3-positive (M-phase) GSCs by the total number of GSCs scored. Optical sections were taken, using an apotome in conjunction with Axiovision Software, of the focal plane in which the middle of the hub was detected. We counted an average of three GSCs in the focal plane scored for each testis. All indices represent the cumulative total of three independent experiments. All p-values were calculated using a two-tailed Fisher’s exact test.


In contrast, when a Tie2-cre+ transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre+ recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2- More than sixty years ago Frank Fenner proposed that virus dissemination during acute infection originates from organs replicating virus to high titer (often liver or spleen) early in infection. Although never formally proven, this model has become commonly accepted and was applied to acute virus infections in general. Recently, we challenged this model by showing that - during acute murine cytomegalovirus infection – hepatocyte-derived virus hardly disseminates to other organs. We now applied our well established model of Cre/loxP-mediated green-fluorescence-tagging of MCMV to determine and quantify the role of infected endothelial cells (EC) in transplantation-associated CMV dissemination. We observed an only very poor dissemination of MCMV from the transplant to recipient tissues and vice versa. Interestingly, we observed no evidence for preferential dissemination of EC-derived virus. Significant differences in virus organ titers were found when comparing intravenous infection with transplant-mediated infection. This suggests a preferential dissemination of cell-associated virus in the transplant setting. In summary, our findings argue for a preferential dissemination of cell-associated MCMV but demonstrate that the Fenner model does not apply to MCMV. Human Cytomegalovirus (HCMV), a member of the betaherpesvirus subfamily, represents an important opportunistic viral pathogen in the immune compromised host. Fetuses, AIDS patients, and recipients of both bone marrow and solid organ transplants are at high risk for the development of debilitating and potentially life-threatening CMV disease. Depending on the risk constellation and immunosuppressive regimen, CMV disease can occur in up to 60% of heart or kidney transplant recipients. Therefore, HCMV is the most important viral pathogen especially during the first six months after transplantation ex vivo: polymorphonuclear leukocytes (PMNL), monocytes/macrophages, and detached infected vascular endothelial cells (EC). Although PMNL are thought to be only abortively infected, they might still function as vehicles for infectious virus in vitro infected granulocytes or monocytes in vitroLeukocyte depletion of blood products derived from seropositive donors prior to transfusion efficiently prevents transfer of CMV to seronegative recipients Despite the undisputed and unique potential of EC in CMV infection and pathogenesis, it is still unknown whether infected EC are responsible for systemic virus dissemination during primary infection, contribute to this process, or merely represent an epiphenomenon with no causal involvement in the pathogenesis of organ disease in vivo and then trace the viral progeny of that cell type egfp expression cassette (MCMV-flox). Mice expressing Cre recombinase under control of either the Tie2 or the Tek promoter, which is selectively expressed in vascular EC (Tie2-cre and Tek-cre mice), were infected with MCMV-flox. In this in vivo infection model MCMV-flox is efficiently recombined resulting in MCMV-rec only during virus replication in EC. It is important to note that Cre-mediated recombination of MCMV-flox is equally efficient in Tie2- and Tek-cre mice and only mediated by EC - as shown using bone marrow chimeras - thus providing highly concordant results by both mouse strains To quantify and to address the fate of virus produced by specific cells, we developed a Cre/loxP mediated approach to label virus in defined cell types cre-negative donor mouse into an EC cre-positive host or vice versa. Here, we investigated export of EC-derived virus from heart and kidney transplants to recipients as well as import of EC-derived virus from recipients into heart transplants. This was achieved by counting and comparing the contribution of EGFP-positive EC-derived progeny to the total virus load of organs and tissues.Vascular EC are present in all organs. A way to study the role of EC in virus dissemination from one organ to another is to either introduce organs from an EC EC-derived virus from infected heart or kidney transplants readily disseminated to organs of MCMV-naïve recipients. The bulk of virus produced in and disseminated from heart is EC-derived, whereas in kidneys infected EC only provide a minor contribution. Yet, we found no evidence for any preferential dissemination of EC-derived virus from both types of transplants to other organs. The heart transplant was also tested as a target organ of EC-derived virus produced in recipient tissues. To our surprise, in contrast to the strong dissemination of virus originating from an infected transplant there was only minimal seeding of host EC-derived virus progeny to the transplant. Interestingly, this was independent of whether transplantation was performed prior to or after systemic host infection. In summary, our data argue against a privileged role of EC in virus dissemination.cre mice were transplanted heterotopically into non-infected syngeneic C57BL/6 mice (5 PFU/g organ) of predominantly recombined virus (∼85%) were observed, confirming that the transplantation procedure itself did not affect MCMV replication in general and demonstrating a very high recombination efficiency (cre mice 6 PFU Transplantation of organs from HCMV seropositive donors to seronegative recipients (D+/R-) is a known situation in transplantation medicine and represents the “high risk constellation” because up to 60% of the recipients can develop CMV disease L/6 mice . Four daficiency . This iscre mice into non-infected C57BL/6 mice, recipient organs were analyzed for the presence of disseminated virus . Collectively, the findings after transplantation of two different organs did not support the hypothesis of a predominant role of EC in virus dissemination during the first four days of infection. Only in blood a significantly higher proportion of MCMV-rec was found on day four post kidney transplantation in 3 out of 4 mice. However, as the absolute virus titers were close to the detection limit, any interpretation has to be seen with caution.Next, we studied dissemination of MCMV following kidney transplantation. Kidneys represent the majority of transplanted organs in medicine. Similar to heart transplantation, the transplantation of kidneys from seropositive donors to seronegative recipients is associated with a high risk to develop CMV-related complications ed virus . In conted virus . This locre+ heart to virus dissemination during the situation of systemic infection of C57BL/6 recipients. Under these conditions, all organs, including the transplanted heart, become infected simultaneously. Thus, MCMV-rec, wherever found, must have originated from ECs of the transplant. Note that under such conditions the infection of the transplant does not have a head start. Four days after transplantation mice were systemically (i.v.) infected with MCMV-flox and four days later they were sacrificed and virus titers determined  infection was the extent of virus production in different organs. In contrast to i.v. infection, which resulted in peak titers in the lung and high titers in heart, kidney, liver, spleen and adipose tissue , virus dcre-positive heart to a cre-negative mouse no significant contribution of virus dissemination from the heart transplant to other organs was observed. To study not only cardiac EC but EC in general as a source of virus dissemination, Tie2-cre or Tek-cre recipient mice received a non-transgenic heart. Recipients were then infected i.v. with MCMV-flox  infection, local virus production might simply outnumber secondary import of EC-derived virus. To address this issue and to initiate the activation of immune functions, systemic infection was performed prior to transplantation. Specifically, Tie2-nfection . Strikinnfection  to ∼60% nfection . This avnfection . It is infection  were on nfection . SimilarTotal titers in the transplant decreased when transplantation was delayed from 20 h to 3 days after infection, reflecting the situation at day 5 and 7 p.i., respectively. In two animals transplanted three days after infection, virus titers in the heart transplant even fell below the detection limit of 10 PFU/g organ, probably reflecting enhanced control by the host immune system at day 7. This is supported by the relatively low virus titers in spleen, kidneys and adipose tissues as well as by the lack of detectable virus in blood .In conclusion, we were surprised to see that ongoing virus replication and the accompanying immune response in the transplanted heart did obviously not alter the absolute amount of EC-derived virus originating from recipients' tissues by secondary viremia. These data demonstrate that virus dissemination between organs – originating from both endothelial and non-endothelial cells – has only minor effects on organ viral load following systemic infection.et al. were the first to propose a two-step dissemination model for systemic virus infections. Primary viremia transports the virus from the site of entry to liver and spleen where the virus replicates. Secondary viremia then causes dissemination from liver and spleen throughout the body One hallmark of CMV infection is the ability of the virus to infect many cell types and tissues from which again the virus may spread. Apparently, immune control defines to which extent this potential is realized in a given scenario. Therefore, the various clinical conditions need to be considered to explain CMV pathogenesis. Blood specimens play an important role in CMV diagnostics. Proper usage of the information gained by this analysis should monitor or even predict events that happen in organs. However, it is currently unclear under which conditions CMV is spread via blood. Fenner In the present study the vascular EC were analyzed for their claimed role in contributing to the CMV load in organs, and in disseminating the virus via primary or secondary viremia. Our salient findings are as follows: EC-derived virus significantly, ∼50% of the body virus pool, contributes to total virus load during acute infection. This contribution was quantified for the first time for the major organs. Yet, there was obviously no preference for dissemination of EC-derived virus over virus produced by other cell types. In addition, and similar to hepatocyte-derived virus, EC-derived virus was poorly disseminated via secondary viremia. These data raise doubts on the indiscriminate applicability of the primary and secondary viremia concept to virus infections in general.Properties of EC have enticed scientists to consider them as key production sites for virus dissemination, as they may release free virus particles directly into the blood stream or may detach from the vessel wall and transfer virus to other organs via the blood stream Here, we provide the first quantitative analysis of organ- and cell type-specific virus dissemination. From an infected organ EC-derived virus readily disseminated to the other, uninfected organs. In the specific cases shown here, the infected transplanted organ (heart or kidney) created the condition of a primary viremia initiating from a defined source. EC-derived virus remained a stable fraction in both heart (∼80%) and kidney (∼20%) throughout the first week of infection Infected EC might detach from the vessel wall and circulate. In fact, HCMV-infected EC were considered as a parameter for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infection Do our findings formally exclude any prominent role of EC-derived virus? The answer is both yes and no. Yes, we can exclude this role in the mouse model and for the temporal conditions of our experiments. Unfortunately, the more time passes after initial infection of the animal the definition of virus as being EC-derived virus becomes more and more indirect. EC-derived virus progeny keeps the marker independent of the cell type in which the virus replicates in further replication rounds. Thus, with our experimental setup we cannot study later phases of infection when other conditions of virus productivity and dissemination may prevail. However, according to our previous experience, second and third replication rounds contribute less and less to the viral load in the immune competent host due to the onset of immune control Nevertheless, by comparing the virus titers in different organs following transplant-derived and i.v. infection, we observed striking differences. Systemic (i.v.) infection with tissue culture produced virus preparations resulted in a uniform distribution of virus to many organs, whereas transplant-derived virus appeared to preferentially colonize spleen, lung and liver but not heart, adipose tissues and kidneys. This cannot be explained by known differences in organ specific virus kinetics. Therefore, cell-free virus, which is usually used for experimental infection, is apparently able to efficiently colonize all organs, whereas virus leaving an infected organ via a natural route reveals a different kind of spread. What could be the cause of the difference between i.v. infection with a solution enriched in isolated virions and the spread of infection from an infected organ? The most plausible explanation is that the virus leaving an infected organ during systemic infection is predominantly transported in a cell-associated manner. Yet, this difference in organ and tissue distribution shows no preference for EC-derived virus and is altogether marginal with respect to total virus load in an organ.This study was carried out in strict accordance with the recommendations and guidelines for the care and use of laboratory animals according to Tierschutzgesetz . All animal experiments were approved by the responsible state office (Regierung von Oberbayern) under permit number 55.2-1-54-2531-19-07.crecreCre-transgenic mouse strains were maintained on the C57BL/6J background. Experiments were performed with gender matched pairs of mice at 3 to 12 months of age. C57BL/6J mice were obtained from Janvier. Tek-cre mice were obtained from Jackson Laboratories (nr. 4128).M2-10B4  and BALB/c-derived mouse embryo fibroblasts (MEF) were grown as described previously 5 PFU in a volume of 300 µl.All viruses were derived from the molecular clone pSM3-fr cre or Tek-cre mice that were maintained on the genetic background of C57BL/6 mice.Syngeneic transplantations of hearts or kidneys were performed between C57BL/6 mice and Tie2-Heart transplant model: Abdominal-heterotopic cardiac transplants were performed, as previously described Kidney transplant model: The murine kidney transplantation was performed as described previously Virus load in organs was determined by plaque assay as described previously Mice were anaesthetized and the peritoneal cavity was opened. After injection of 50 µl of heparin into the inferior vena cava, abdominal aorta and vena cava were cut cranially of the transplant. After all organs were perfused with 5 ml PBS via the vena cava the heart transplant was removed and perfused separately with 3 ml PBS.The percentage of MCMV-rec compared to total virus organ load per group, mean values, and standard deviations were determined.


The system consists of six transgenic lines of Anopheles stephensi, each with a single piggyBac-Gal4 element in a unique genomic location; six lines with a single piggyBac-UAStdTomato element; and two lines, each with a single Minos element containing the piggyBac-transposase gene under the regulatory control of the hsp70 promoter from Drosophila melanogaster. Enhancer detection depended upon the efficient remobilization of piggyBac-Gal4 transposons, which contain the yeast transcription factor gene Gal4 under the regulatory control of a basal promoter. Gal4 expression was detected through the expression of the fluorescent protein gene tdTomato under the regulatory control of a promoter with Gal4-binding UAS elements. From five genetic screens for larval- and adult-specific enhancers, 314 progeny were recovered from 24,250 total progeny (1.3%) with unique patterns of tdTomato expression arising from the influence of an enhancer. The frequency of piggyBac remobilization and enhancer detection was 2.5- to 3-fold higher in female germ lines compared with male germ lines. A small collection of enhancer-trap lines are described in which Gal4 expression occurred in adult female salivary glands, midgut, and fat body, either singly or in combination. These three tissues play critical roles during the infection of Anopheles stephensi by malaria-causing Plasmodium parasites. This system and the lines generated using it will be valuable resources to ongoing mosquito functional genomics efforts.Transposon-based forward and reverse genetic technologies will contribute greatly to ongoing efforts to study mosquito functional genomics. A  Plasmodium parasites continues to be a major component of malaria control efforts. Understanding the genetic and molecular genetic basis of insecticide resistance, olfaction, reproductive physiology and the immune system of Anopheles mosquitoes figures heavily into contemporary ideas for developing new strategies for controlling mosquito populations and Plasmodium transmission . In the case of malaria, controlling the mosquito vectors of malaria-causing Recent advances in mosquito molecular genetics have depended upon the availability of whole-genome sequence data and a host of technological advances, including transcription-profiling and RNA-based gene-silencing technologies  without having to physically isolate and characterize the regulatory elements , and larvae were provided with pulverized fish food  ad libitum, while adults were provided with 10% sucrose continuously. Adult females were occasionally allowed to feed on adult mice to obtain a blood meal, which was necessary for reproduction. The use of mice was with the approval and oversight of the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland, College Park, operating under the National Institutes of Health’s Office of Laboratory Animal Welfare guidelines. Mosquito blood-feeding protocols involving mice were not terminal, and animal pain and distress were minimized with the use of anesthetics with the approval of the IACUC.An. stephensi originally isolated in Pakistan and selected in the laboratory for susceptibility to Plasmodium falciparum infection (This is a wild-type strain of Minos gene vector from pMi[3xP3-DsRed]-hsp70-piggyBac (piggyBac-transposase open reading frame (ORF) under the regulatory control of the promoter from the hsp70 gene from D. melanogaster  under the regulatory control of the 3xP3 promoter (piggyBac (GenBank J04364). The second module consisted of the Gal4 ORF from pGaTB attached to the 3′ UTR of the hsp70 gene of D. melanogaster  , along with a marker gene consisting of the enhanced yellow fluorescent protein (EYFP) under the regulatory control of the 3xP3 promoter. This vector was also constructed using Gateway recombination cloning technology  involving the simultaneous recombination of four recombination modules into a destination plasmid. The first module contained 690 bp of the 3′ terminal sequences of piggyBac. The second module contained 1.5 kb of the tdTomato ORF from ptdTomato  inserted into pUAST-attB at the EcoRI/NotI sites between the promoter region containing five UAS elements and the 3′ UTR of hsp70 from D. melanogaster. The third module contained EYFP under the regulatory control of the 3xP3 promoter, and the fourth module contained 671 bp of the 5′ terminal sequences of piggyBac  by injecting preblastoderm embryos of SDA 500 An. stephensi with vector-containing plasmids and plasmids expressing piggyBac transposase (phsp-PBac) , and the DNA sequence of their integration sites were deposited in GenBank (accession numbers JX242566–JX242578)Transgenic sp-PBac) . VectorsPB-Gal4–containing individuals  were mated en masse with ∼100 piggyBac transposase-expressing individuals of the opposite sex (DsRedUMITF-PB-M5 and DsRedUMITF-PB-F2). Approximately 100 individuals heterozygous for both PB-Gal4 and DsRedUMITF-PB-F2  or DsRedUMITF-PB-M5 were mated to ∼100 PB-UAStdTomato individuals en masse, and the resulting progeny were screened as third or fourth instar larvae and as adults for tdTomato expression. Although piggyBac transposase was under the regulatory control of the promoter from the hsp70 gene from D. melanogaster, heterozygous individuals containing both PB-Gal4 and piggyBac transposase were not heat-shocked. Earlier work showed that heat shock was unnecessary for transposase expression and piggyBac remobilization using these and similar lines  Tu at Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, and now publically available on VectorBase   49001 ET-CFP , 49002 ET-GFP , 49003 ET-EYFP , 49005 ET-DsRed  as well as a Zeiss Axiom Imager A1 fluorescent compound microscope with Zeiss filter set 20  and filter set 38HE .The patterns of Gal4 enhancer-trap–containing lines were created, each with a single piggyBac element. Similarly, six UAStdTomato-containing lines were created, each of which contained a single UAStdTomato transgene  was observed in the germ line of females compared with the germ line of males . When enhancer-trap events were detected, they were almost always represented by a single individual among the progeny. Of the 317 progeny with tdTomato expression, we estimate that most resulted from independent transposition events.We screened a total 24,250 larvae and adult progeny for the presence of remobilized 5 and F2 . As obseof males   . As we reported in an earlier study  was very small in the genetic screens reported here. Multiple progeny with an identical pattern of tdTomato expression, containing piggyBac in the same genomic position and found among the progeny of a single genetic cross, were rarely recovered. At this point, the temporal patterns of piggyBac transposition within the germ line of An. stephensi are unknown, although our observations suggest that transpositions are not occurring early during germ line development. The promoter from the hsp70 gene from D. melanogaster regulates the piggyBac transposase transgenes in lines F2 and M5; however, its expression did not require heat induction in An. stephensi. Future studies will explore the relationship between the timing and frequency of heat shock and the amount and timing of piggyBac remobilization and enhancer detection. Despite the fact that there are aspects of this enhancer-trap system that remain to be determined, it promises to be quite useful for creating Gal4-expressing An. stephensi lines with widespread utility.The frequency of enhancer detection in th males . AlthougAn. stephensi by vector biologists remains somewhat challenging because there are relatively few promoters that have been isolated and characterized, and the creation of transgenic An. stephensi remains technically demanding. Most transgenic lines of An. stephensi created to date have been single-purpose lines with limited utility beyond their intended function, which further increased the costs and inefficiencies associated with using transgenic technologies in this species. The development and use of a Gal4-based enhancer-trap system increases the utility of transgenic technologies in An. stephensi by providing researchers with many more options for expressing transgenes of interest in temporal and spatial patterns. For example, we described lines in which enhancers were detected that regulated Gal4 expression in the adult female midgut, salivary gland, and fat body, three tissues that play critical roles in Plasmodium infection and transmission. Lines UMITF-C2F8 and UMITF-2MCL14 had Gal4 expression in the midgut but not in the salivary glands or fat body of adult females. Lines UMITF-C2F41 and UMITF-2MCL6 had Gal4 expression in the salivary glands but not in the midgut or fat body of adult females. Line UMITF-AEA1 had Gal4 expression in the fat body but no expression in the midgut and only weak expression in the salivary glands of adult females. Line UMITF-MBL24 was particularly interesting from the perspective of Plasmodium infection of An. stephensi because Gal4 expression occurred specifically in the adult female salivary glands, posterior midgut, and fat body. This line will permit transgenes to be expressed in three of the most important tissue compartments of An. stephensi with respect to Plasmodium infection within a single adult female. Line UMITF-MDL8 is expected to be useful because it has Gal4 expression ubiquitously throughout most tissues of both larvae and adults. Although most of the lines reported here were chosen to illustrate the utility of this technology to the study of mosquito-parasite/pathogen interactions, lines UMITF-C2M8 and UMITF-DEA9A had Gal4 expression exclusively or almost exclusively in larval tissue, whereas line UMITF-2MBL3 had Gal4 expression in a specific subset of cells associated with scales and sensillae. The binary nature of this system permits the effort spent on creating transgenic lines to be minimized while enabling investigators repeated opportunities to express their transgene in a variety of patterns simply by mating their UAS-regulated transgene–containing line to any Gal4-expressing line. This modularity is perhaps the most important feature of this system.The genetic manipulation of piggyBac transposase is currently not limited to the germ lines of lines M5 and F2. Because the transposition activity of the enhancer-trap element is sufficiently high in somatic cells, clones of Gal4-expressing cells in various tissues are frequently seen (Gal4 expression patterns involved relatively small numbers of cells resulting in a subtle but significant pattern of reporter gene expression, are of interest, then the somatic clones frequently observed with our current system could be a liability by making such patterns difficult to recognize. Limiting transposase expression to the germ line of An. stephensi could be accomplished by using regulatory sequences that result in germ line–specific transcription (UAStdTomato transgene in a piggyBac vector can be disadvantageous. For example, when piggyBac transposase is present, the UAS-containing piggyBac element can become unstable and be remobilized to new genomic locations. Also, performing splinkerette-PCR or using any other method to identify integration sites of piggyBac elements containing the enhancer-reporter can be confounded by the presence of piggyBac elements containing UAS-regulated reporter genes. Although this current system could be improved by incorporating all UAS-regulated reporter genes into vectors other than piggyBac, careful genetics and accounting for chromosomes containing the piggyBac transposase transgene will avoid any undesirable remobilization of other system components. Although having other transposon platforms upon which to build system components is convenient, the highly effective enhancer-trap systems created for D. melanogaster were based on a single transposon platform (The system described here, although effective at detecting enhancers, could be made more effective with two modifications. First, tly seen . If enhaplatform .The abundance of genome information and the ease with which it can now be obtained makes the need eminent for technologies that enable progress to be made in pursuing questions relating to functional genomics. For Anopheles mosquitoes, there are relatively few tools available for empirically assessing gene function within the context of the whole organism. The enhancer-trap system described here is a valuable first step in increasing our capacity to explore the biology of Anopheles mosquitoes using forward and reverse genetic approaches.