Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did.Using a system of These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis. Angioplasty is a procedure designed to treat vascular stenosis, blockage(s), or atherosclerotic lesions, but it may also, simultaneously, cause damage to the integrity of the blood vessel wall. Restenosis is the subsequent narrowing and occlusion of the blood vessel in response to the injury or damage sustained during angioplastic procedures such as balloon dilation . During The characteristic response of VSMC, endothelial cells, platelets, and macrophages at the site of injury is the release of specific soluble growth factors which include platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) -4. VSMC To date, the precise molecular mechanisms that link growth factor-initiated intracellular signaling to ECM-mediated adhesion, migration, and proliferation of VSMC are still unknown. Our previous reports of low-level laminin-5 expression in the intima of normal vasculature and an increased expression of laminin-5 in the neointima of injured vessels suggest that laminin-5, in addition to PDGF and TGF, may mediate VSMC responsiveness following vascular injury -13.in vitro assays to study the role of laminin-5 in modulating these behaviors in rat vascular smooth muscle cells (rVSMC). We report here that PDGF induces differential responses in rVSMC behaviors on laminin-5, but not on fibronectin. In addition, we find that the PDGF-induced responses on laminin-5 are inhibited in a dose-dependent manner by an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD98059, but not on fibronectin.To further elucidate VSMC response to growth factors and the intracellular signaling cascades that may be linked to ECM-mediated adhesion, we used in vitro may be the result of different signaling pathways that are initiated by integrin-mediated adhesion to laminin-5. We suggest that rVSMC binding to laminin-5 may initially activate a MAPK-independent signaling cascade that may make these cells more responsive to MAPK inhibition. In contrast, rVSMC binding to fibronectin may activate a signaling pathway shared by PDGF that ultimately converges in MAPK activation. These results suggest that a complex interaction of ECM and growth factors may closely regulate VSMC behavior following vascular injury and these studies may directly identify define molecular targets that may reduce the incidence of restenosis following angioplasty.These differences in MAPK-sensitive rVSMC responses We have previously reported that laminin-5 expression by rat vascular smooth muscle cells (rVSMC) is upregulated by platelet-derived growth factor (PDGF-BB) and that laminin-5 specifically enhances PDGF-BB-stimulated rVSMC migration . In addiThe level of rVSMC migration was observed over 18 hours in transwell migration filters in the presence or absence of laminin-5, with and without PDGF-BB or serum. Maximal cell migration (chemotaxis) was obtained using cell migration media containing ten (10) percent fetal calf serum (FCS). Similar to our previous reports, laminin-5-coated wells induced a greater than four-fold increase in rVSMC migration over that measured in naked plastic controls in three independent experiments as shown in Figure The mitogen-activated protein kinase (MAPK) pathway is known to be activated by PDGF-BB-stimulation in VSMC . To explTo determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor TGF-β1) for its ability to modulate the PDGF-stimulated increase in rVSMC migration on laminin-5. Our results indicated that the addition of TGF-β1 was able to reduce laminin-5-stimulated rVSMC migration to levels approximating the levels observed in naked plastic controls over all tested ranges (5 – 50 ng/mL), although the most statistically distinct reduction was found over the range of 5 – 25 ng/mL, as shown in Figure  for its in vitro adhesion assays were performed. In the absence of exogenous growth factor stimulation, laminin-5- and fibronectin-coated wells (20 μg/mL) sustained an approximate two and a half-fold increase in rVSMC adhesion compared with negative controls, as shown in Figure To determine whether or not the PDGF-BB-stimulated increase in rVSMC migration correlates with a reduction in rVSMC adhesion to laminin-5, thirty-minute ed wells 0 μg/mL sTo investigate if the relationship between the PDGF-BB-stimulated increase in migration and corresponding decrease in adhesion of rVSMC on laminin-5 may be related to MAPK activation, cells were pre-treated with PD98059 (40 ng/mL) for twenty (20) minutes prior to assay and the adhesion media was supplemented with PD98059 (40 ng/mL). The addition of exogenous PD98059 (40 ng/mL) restored the PDGF-BB-stimulated reduction (25 ng/mL PDGF-BB) in rVSMC adhesion to laminin-5, to approximately eighty-five (85) percent of laminin-5 controls (n = 24, p < 0.05). The addition of PD98059, however, did not restore the thirty (30) percent PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1). Our results indicated that the addition of TGF-β1 (25 ng/mL) was able to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on laminin-5 by roughly the same level as PD98059, eighty two (82) percent versus eighty five (85) percent, respectively. In contrast to PD98059, the addition of TGF-β1 (25 ng/mL) was sufficient to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.in vitro proliferation assays to determine the relative effects of the extracellular matrix (ECM) and exogenous growth factors described above. First, to test the effects of the ECM substrate on rVSMC proliferation in the absence of exogenous growth factors, quiescent cells were plated in cell-culture plates either coated with or without laminin-5 or fibronectin, in serum-free Dulbecco's Modified Eagle's Medium (DMEM) for one (1) to six (6) days.Based upon our observations of rVSMC migration and adhesion, we performed 0(G1) between forty eight (48) and seventy two (72) hours as determined by flow cytometry and determination of [3H] thymidine-labeled nuclei [To induce quiescence, rVSMC were incubated for forty eight (48) hours without mitogen (0% FCS, FBS) at 37°C and quiescence was verified by proliferation controls, cultured for forty eight (48) hours at 37°C with mitogen stimulus as shown in Figure d nuclei .Our results suggest that culture of rVSMC plated on exogenous laminin-5 (coated at a concentration of 20 μg/mL) did not significantly increase cellular proliferation compared with naked plastic controls over a period of four (4) days, as shown in Figure Next, to test the modulating effects of exogenous growth factors on rVSMC proliferation when cultured on these ECM substrates, quiescent cells were plated in cell-culture plates either naked or coated with laminin-5 or fibronectin, in the presence of TGF-β1 or PDGF-BB, both at a concentration of 25 ng/mL, from one (1) to six (6) days. Our results indicated that the addition of exogenous TGF-β1 (25 ng/mL) to the cell culture medium was sufficient to induce a suppressing effect on rVSMC proliferation on naked plastic, as well as laminin-5 and fibronectin, to levels approximating the quiescent growth controls.The addition of PDGF-BB was sufficient to induce an increase in rVSMC proliferation on laminin-5 of nearly one-half (46%) over laminin-5 DMEM (no serum, no mitogen) controls (n = 24, p < 0.01). However, our results indicate that the addition of exogenous PDGF-BB (25 ng/mL) did not have a statistically significant effect on cells plated on fibronectin.To examine MAPK activation, rVSMC were plated onto laminin-5- or fibronectin-coated plates, then lysed after thirty minutes and prepared for immunoblotting. Our results indicated that laminin-5 did not induce a detectable increase in MAPK activation over a thirty (30) minute time interval, as shown in Figure The addition of growth factors, such as PDGF-BB and TGF-β1, have been associated with increases in p44/p42 (ERK1/2) or MAPK activation -18. To din vitro VSMC mitogen and may be responsible for initiating the phenotypic changes in VSMC migration and proliferation during restenosis in vivo [PDGF-BB is an  in vivo ,20. Our  in vivo -13.in vitro [The current study augments the body of evidence that suggests VSMC growth is influenced by an ECM-VSMC interaction  and thatin vitro ,8,10. Moin vitro, driven by PDGF-BB responsiveness, can be blocked by MAPK inhibition (via MEK1 inhibitor: PD98059) on laminin-5, but not on fibronectin.Because PDGF-BB stimulation increases the overall levels of intracellular MAPK, as well as MAPK phosphorylation, we sought to explore this signaling cascade to determine its role in modulating these rVSMC behaviors on laminin-5 . SpecifiAn additional signaling regulator, TGF-β1, has been implicated in the negative regulation and decreased rate of proliferation of VSMC stimulated with serum or PDGF -28. Our 1 phase of mitosis.Several lines of evidence now suggest that the anti-mitogenic effects of TGF-β1 may be dissociated from inhibition of ERK1/2 signaling pathways ,18. ThesAlthough our previous reports linked ERK1/2 to rVSMC adhesion and migration, these studies did not examine the possibility for differential signaling initiated by rVSMC binding to laminin-5 or fibronectin ,13. Expain vitro. Based upon these findings, we postulate that PDGF-BB and laminin-5 binding may initially activate different intracellular signaling cascades, causing rVSMC to be more responsive to the inhibition of MEK1 and MAPK on laminin-5 than those activated on fibronectin, as outlined in Figure Our results indicate that laminin-5 activates different intracellular signaling pathways from those of fibronectin and PDGF-BB in rVSMC and that binding to laminin-5 may modulate rVSMC behaviors that are distinctive from those modulated by fibronectin. Although binding of rVSMC to laminin-5 may not cause an initial increase in MAPK activation levels or proliferation, laminin-5 can augment PDGF-BB-stimulated proliferation and migration of rVSMC In contrast to laminin-5, fibonectin and PDGF-BB may have parallel, reinforcing roles in MAPK activation. Our analysis of the effects of TGF-β1 demonstrated that TGF-β1 does not strongly activate MAPK in rVSMC, but rather strongly inhibits the effects of fibronectin and PDGF-BB-stimulation on laminin-5. Our results support the previous findings that TGF-β1 may inhibit mitogenesis and other VSMC behaviors via mechanisms independent of MAPK activation.These results suggest that a clear understanding of the roles and contributions of each ECM may provide new insights into the mechanisms of regulating rVSMC cell behaviors, including migration, adhesion, and proliferation. Further analysis of the events that trigger and sustain the underlying cellular mechanisms of rVSMC behaviors may help aid in the design of more effective therapies for the treatment of restenosis.2 in humidified chambers. Cells were maintained in DMEM High Glucose, supplemented with 10% fetal bovine serum and 1% L-glutamine (29.2 mg/mL), penicillin G (10,000 U/mL), and streptomycin sulfate (10,000 mcg/mL) (GPS) from Irvine Scientific (Santa Ana, CA). Rat aortic smooth muscle cell explants were a gift from RC Smith and were isolated and passaged as previously described [0(G1) arrest can routinely be induced by incubation of cells for 72 h. in low-mitogen (0.5% FBS) medium [Cells were maintained in 100 mm × 20 mm Corning tissue-culture dishes (Plainfield, NJ) at 37°C and 5% COlutamine .2 mg/mL,) medium ,30, thes5 in each of 96-transwell chamber filters (100 μL of 1.2 × 106 cells/mL solution) with and without ECM in the presence or absence of PDGF-BB at the indicated concentrations (5–25 ng/mL) and allowed to migrate for 18 hours at 37°C. Where applicable, the medium was supplemented with PD98059 (MEK1-inhibitor) at the indicated concentration. Cells were counted at the end of an 18-hour interval as indicated, quantified with the following modification. 30 minutes prior to measuring migration, 5 μM calcein AM from Molecular Probes (Eugene, OR) was added to the migration wells at 37°C. To quantitate migration, cells were removed from the top of the filter with cotton-tipped applicators and fluorescence of the incorporated calcein was measured from the bottom of the filter with a fluorescence plate reader. Relative fluorescence values for each experimental condition are expressed relative to controls and untreated samples.Cell migration assays were performed in Costar transwell filter plates either coated with purified matrix (laminin-5 or fibronectin) at a protein concentration of 20 μg/mL for one hour (60 min.) at room temperature, 25°C, and washed twice with phosphate-buffered saline 0.2% Tween-20 and 5% skim milk (PBST) prior to assay as previously described ,32. Cell5 in each of 96-transwell chamber filters (100 μL of 1.2 × 106cells/mL solution) with and without ECM-coating (described above) in the presence or absence of PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or both, and allowed to attach for 30 minutes at 37°C. Where applicable, cells were first incubated for 20 minutes with PD98059 (40 ng/mL), a MEK1 inhibitor from New England Biolabs (Beverly, MA) at 37°C and the adhesion assay culture medium was supplemented with PD98059 at 40 ng/mL. Following adhesion, non-adherent cells were removed by suspending plates upside down in a rotating tank of PBS for 10 minutes at room temperature, 25°C. Adherent cells were then fixed and stained and the relative absorbance was measured using a TECAN-SPECTRAFluor spectrophotometer (TECAN, Durham, NC) at 595 nm.Cell adhesion assays were performed as previously described ,32 using5 in 100 mm2 cell culture plates with and without fibronectin or laminin-5 and allowed to attach overnight (12 h.) at 37°C. Cells were then starved in serum-free DMEM for 48 hours to induce quiescence at 37°C, as outlined in Cell Culture Methods below. The medium was then replaced with fresh medium containing 25 ng/mL of TGF-β1 or PDGF-BB obtained from Calbiochem (La Jolla, CA) and incubated at 37°C. Cells were removed from culture wells with trypsin/EDTA and counted using trypan blue stain from Gibco Life Technologies (Rockville, MD) and a VWR Scientific Counting Chamber (Plainfield, NJ) at 24 hour intervals, from 1 – 6 days.Tissue culture plates were coated with purified fibronectin from Calbiochem (La Jolla, CA) or laminin-5 from Demos (La Jolla, CA) at a 20 μg/mL protein concentration for 1 hour (60 min.) at room temperature, 25°C as previously described . Cells wQuiescent VSMC were pre-treated with culture medium containing 10% FCS, PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or plated on laminin-5- or fibronectin-coated tissue culture plates with DMEM for 30 minutes at 37°C. Subconfluent cell cultures were then lysed using ice-cold, 1X lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, pH 7.5) and boiled in SDS Boiling Buffer prior to analysis as previously described . Proteint distribution. All samples were measured using two-tailed t tests as departure from normality can make more of a difference in a one-tailed than in a two-tailed t test. So long as the sample size is even moderate (>20) for each group, quite severe departures from normality make little practical difference in the conclusions reached from these analyses [The differences between untreated and treated cell populations were measured using a analyses .The author(s) declare that they have no competing interests.KK carried out the migration, adhesion, and proliferation assays, the Western Blot analysis and assisted with experimental design. GEP conceived, monitored, and coordinated the experimental design. Both KK and GEP contributed equally to the writing of this manuscript.


Genomic sequence variation is the hallmark of life and is key to understanding diversity and adaptation among the numerous microorganisms on earth. Analysis of the sequenced microbial genomes suggests that genes are evolving at many different rates. We have attempted to derive a new classification of genes into three broad categories: lineage-specific genes that evolve rapidly and appear unique to individual species or strains; highly conserved genes that frequently perform housekeeping functions; and partially variable genes that contain highly variable regions, at least 70 amino acids long, interspersed among well-conserved regions. The latter we term segmentally variable genes (SVGs), and we suggest that they are especially interesting targets for biochemical studies. Among these genes are ones necessary to deal with the environment, including genes involved in host–pathogen interactions, defense mechanisms, and intracellular responses to internal and environmental changes. For the most part, the detailed function of these variable regions remains unknown. We propose that they are likely to perform important binding functions responsible for protein–protein, protein–nucleic acid, or protein–small molecule interactions. Discerning their function and identifying their binding partners may offer biologists new insights into the basic mechanisms of adaptation, context-dependent evolution, and the interaction between microbes and their environment. Segmentally variable genes show a mosaic pattern of one or more rapidly evolving, variable regions. Discerning their function may provide new insights into the forces that shape genome diversity and adaptation Microbes occupy almost every habitable niche in the biosphere, highlighting their enormous capability for adaptation and survival. This adaptive ability has been refined during millennia of evolution and has resulted in genes that evolve at very different rates. Some, such as housekeeping genes that code for the essential biochemical functions of the organism, are now evolving rather slowly. Others that have to defend against potentially lethal attack by viruses or toxins and adapt to varying environmental conditions, often evolve more rapidly . PathogeAn example of an SVG family is provided by the cytosine-5 DNA methyltransferases . These eTo the best of our knowledge, there has been no systematic attempt to identify, catalog, and classify similar SVGs in the sequenced microbial genomes. Nor has any attempt been made to find potentially common functions among genes displaying this property. Since it is known that many genes involved in defense mechanisms, such as the DNA methyltransferases and the antigens exposed on the surface of bacteria, show such variability , it is thttp://geneva.bu.edu) is also provided with results for several selected genomes, including Escherichia coli, Helicobacter pylori, Neisseria meningitidis, and several others. Each gene is accompanied with schematic diagrams from Pfam . SVGs are defined as genes containing at least one highly variable region, containing more than 70 amino acids, interspersed among well-conserved regions. In any single SVG family, the length of the variable region can differ only within a certain range  space, where the x-axis shows the length of the variable region and the y-axis shows the number of possible homologs of each gene. Lineage-specific genes (filled square in H. pylori (In . pylori , only ap. pylori  with onl. pylori , etc. Th. pylori . A few eH. pylori. The variability lies in its DNA recognition domain (approximately 140 amino acids), which in this case recognizes the DNA sequence CCTC (H. pylori, which has variable regions at both its N-terminus (approximately 200 amino acids) and C-terminus (approximately 100 amino acids). VacB has been shown to encode a 3′–5′ exoribonuclease and is necessary for expression of virulence  to 8.6 MB(Streptomyces coelicolor)  and a low percentage of lineage-specific genes (0.2%). However, when a closely related species, M. pneumoniae, is excluded from the database, its proportion of lineage-specific genes rises to 14%, while the proportion of SVGs remains unchanged. In general, the genomic proportion of SVGs is less affected by the database content.The number of SVGs increases as genome sizes vary, from 0.5 MBlicolor) . For mosIn the following sections, we have selected several SVG families to demonstrate the functional implication of segmental variability.Ralstonia solanacearum [Rs] and Ralstonia metallidurans [Rm] in , e.g., genes encoding methyl-accepting chemotactic protein (MCP)  than in other genomes (see the gene list of V. cholerae at http://geneva.bu.edu), which must correspond to its expanded ability to detect different chemical signals and find favorable environments. Although a few conserved motifs have been detected in the sensing region  and a response regulator. Most HKs are membrane-bound, homodimeric proteins with an N-terminal periplasmic sensing domain and a C-terminal cytoplasmic kinase domain. HKs usually possess a highly variable sensing domain , while the cytoplasmic kinase domain is quite conserved. By diversifying the sensing domain, microorganisms can develop different two-component modules to respond to different signals and interact with small molecules from the exterior. g region , the exaThe biggest family of SVGs is cell envelope-related, including the ATP-binding cassette transporters (ABC transporters), outer-membrane proteins, and virulence-related gene products. For membrane proteins, since part of their sequences are exposed to the outside of the cell and interact directly with the environment, one might hypothesize that the variable portions have evolved rapidly to deal with the changing environmental conditions.hetA and spaB shown in E. coli has been solved (E. coli BtuB overlaps with the 22-strand β-barrel (position 150–360), while the N-terminal hatch domain (position 6–132) and the extreme C-terminal TonB-box domain (position 550–614) are conserved among many homologs  and a large (over 300 amino acids) variable integral membrane domain. Two examples from this group are the multidrug-resistance genes n solved . The varhomologs . The exthomologs  and possgyrB) and DNA topoisomerase A (topA), whose competing actions control the degree of DNA supercoiling  is variable both in sequence content and in length A. The corculosis A. Interelymerase  and in Dlymerase . Since Rlymerase . Deletioresponse . All togVariable regions are sometimes found in DNA processing enzymes with essential and conserved functions. One example is DNA polymerase I, which has a variable region between the conserved C-terminal 5′–3′ polymerase domain and the N-terminal 5′–3′ exonuclease domain. In some polymerases, this region encodes a 3′–5′ exonuclease activity for proofreading replication errors, and conserved motifs can be observed . HoweverE. coli ProRS are known to be involved in the posttransfer editing for mischarged Ala-tRNAPro  . This fa-tRNAPro . ProRS i-tRNAPro . Partialcylation , suggestbilities . As a reN-acetyl muramyl pentapeptide; SpsA: unknown). Owing to the lack of homology in the acceptor binding domains, the substrate specificities encoded by these regions for most GTs are still unknown.Variable regions exist in carbohydrate metabolizing enzymes, such as glycosyltransferases (GTs) and glycoside hydrolases (GHs), which respectively catalyze the biosynthesis of diverse glycoconjugates and their selective cleavage . Many paProkaryotic transcriptional regulators form another large group of SVGs. Transcription regulators are usually two-domain proteins with one binding to DNA and one binding to ligand. The DNA-binding domains, which usually interact with DNA via helix–turn–helix, zinc-finger, or other modes, are more conserved than ligand-binding domains. Based on the characteristic conserved DNA-binding domains, transcriptional regulators can be classified into many different families . Even wiH. pylori: 17 genes; N. meningitidis: 32 genes; V. cholerae: 69 genes, etc.; see http://geneva.bu.edu for the full list). In contrast to lineage-specific hypothetical genes, these hypothetical genes contain conserved domains, which suggest their functional importance. Although most of the conserved domains in these hypothetical genes have currently unknown function, there are a few exceptions. Among them are the prokaryotic mechanosensitive channel proteins, which respond to external osmotic pressure . The central regions (approximately 150 amino acids) of these genes are conserved while both the N-terminal region (approximately 100 amino acids) and the C-terminal region (approximately 100 amino acids) are variable . H. pylori is one of the few microbes that can colonize the highly acidic gastric environment , which appear to be segmentally variable. The unique pattern of cell surface glycosylation in S. pneumoniae has been under extensive investigation and plays an important role in pathogenesis . In 2 test. For all genomes examined, there is a strong association between gene duplication and SVGs, suggesting an SVG is more likely to have originated from a duplicated gene.In H. pylori strain 26695, gene products of HP1299 (253 amino acids) and HP1037 (357 amino acids) both have a conserved domain  that is characteristic of the methionyl aminopeptidase (map) family   that is variable among its similar genes  binding  . HP1299 map gene , while tar genes . Additiofunction . A functfunction . In mostA major fraction of bioinformatics research on sequence analysis has focused on the conserved regions in proteins, trying to hypothesize the role of the protein by identifying sequence motifs that have been shown experimentally to correlate with a specific function. Some work has gone into cataloging the groups of lineage-specific proteins that show no similarity to other proteins in GenBank , but theIn this paper we have initiated an effort to identify SVGs, which contain both well-conserved regions and highly variable regions. By looking carefully at a few specific examples where functional information is available from experimental data, we find that the variable region often seems to play a key role in mediating interactions with other molecules, both large and small. Sometimes the variable portions are involved in biological processes with a component of interaction between the cell and agents from the external environment. For instance, the DNA methyltransferases are part of a defense system that recognizes and clears invading foreign DNA; membrane-bound sensory HKs and mechanosensitive ion channels, etc., monitor changes of living conditions. Sometimes the variable portions are involved in intracellular processes that appear to have lineage-specific features. Thus, the variable regions inside DNA GyrB and several types of AARSs probably determine the specificity of substrate recognition. The detailed factors that introduce the molecular variability may go well beyond our explanations here and likely vary from case to case. Some variable regions may have diverged a long time ago and are now kept constant, while others may keep changing. In all of these cases, SVGs are exceptionally worthy targets of further experimental investigation, and such investigations can be greatly aided by the presence of the conserved regions that may suggest a preliminary function to be tested.H. pylori, none of them had hits with a disrupting stop codon in the variable region; 20 of them have hits with genes showing insertions/deletions that cause frame shifts in the variable region. However, the real number is likely to be much fewer, since, based on previous work, many of them may be the results of sequencing errors . We have also examined the amino acid composition, codon usage, and GC content in the variable regions and the conserved regions of the same SVG. While there is no significant deviation of amino acid composition and GC content between the two regions in general, codon usage appears to be biased in the variable regions (data not shown).SVGs usually account for 10%–20% of the total genes in a microbial genome. Currently, we think of the class of lineage-specific genes as being the key factor that distinguishes one strain or species from another. The class of SVGs that we have defined in this paper must now be added to this collection of lineage-specific genes by virtue of the unique segments that constitute their variable regions. They also appear to provide functional elements that help to differentiate among strains and species. This point is well illustrated by considering the restriction-modification systems. Here, the DNA methyltransferases, which have a variable region responsible for DNA recognition, are members of the SVG class. With the help of their companion restriction endonucleases, which typically appear as lineage-specific genes, they serve to keep foreign, unmodified DNA sequences from entering the genome. In this case, the synergy of function provided by members of the two classes highlights the key role that both sets of genes must play in defining the individuality of a strain or species.Our analysis to date is limited to prokaryotes and archaea where SVGs are transcribed and translated as contiguous genomic segments. In eukaryotes, alternative RNA splicing introduces substantial additional complexity into the interpretation of gene structure and protein product, thereby rendering impossible the simple analysis we have applied here. It is tempting to consider alternative splicing as a highly evolved control mechanism to introduce the variability we find in the SVGs and thereby achieve the functional diversity necessary for cell survival under different conditions. In eukaryotes, alternatively spliced exons can be introduced in response to the functional demands of different cell types by merely juggling protein coding regions in the genome, thereby creating an SVG structure. If this view is correct, then it reinforces and highlights the importance of these SVGs to the workings of the cell.In this paper we have provided an initial glimpse of SVGs, which appear to provide an important genetic layer in the adaptation of cells to novel environments and hazardous pathogens. We have focused attention on the biological significance of these genes, especially those that have highly diverged segments. We are currently trying to develop a more refined classification of these genes so as to explore the functional significance of the variability. We would like to know whether extreme variability is required for diverse function or whether more modest variation is sufficient. Such questions require that we can first distinguish positive selection acting on these variable regions from neutral evolution leading to gene decay and eventual loss. Since the variable regions we report are often not amenable to current tools available for alignment, we are exploring new methods that will help us to assess whether positive selection is driving the evolution of these genes.In summary, we have identified an extremely useful way of classifying genes that leads to the identification of those with a high priority for both experimental and computational research.Our method for detecting SVGs includes several steps: (1) identification of similar genes followed by query-anchored multiple alignment using nongapped BLAST ; (2) taxE-value is less than 1E-10. Here we use a strict E-value threshold to avoid possible functional divergence among the homologs. Accordingly, we use the BLOSUM80 scoring matrix in the BLASTP search, although the result does not change dramatically if BLOSUM62 is used. Second, the overall length of the hit sequence does not differ significantly from the query sequence. We define the gap content (GapC) between two sequences:Given a gene, we start by searching for all its similar genes in the nonredundant database (GenBank release 136.0, 15 June 2003) using nongapped BLAST . We use L,l are the lengths of the protein sequences of two genes. It is a measure of the smallest percentage of gaps needed to be introduced into the pairwise alignment. Sequences with a high GapC value indicate significantly different domain structures, possibly owing to domain insertions or losses, and thus are excluded from the set of similar genes. In our current implementation, we require that GapC must be less than 0.2.where Similar genes reported by BLASTP are not evenly distributed among different species. In many cases, highly similar genes from different strains of the same species or highly similar paralogous genes from a particular strain tend to introduce bias into the dataset. We adopted a simple taxonomy clustering by using the NCBI Taxonomy Database  to reducWe collapse all the similar genes from the same species into a single group. Then we choose the gene with the best similarity score to the query sequence as the representative of that species for later calculations. The definition of species follows the hierarchical taxonomy used in the NCBI Taxonomy database (superkingdom → phylum → class → subclass → order → family → genus → species → no rank [strain]). By doing taxonomy clustering, we are able to collect a less biased sample of similar genes from different species.http://geneva.bu.edu). Two unaligned regions in two sequences are considered as the variable regions if they are bounded by similar HSPs at both ends . To avoid the possibility of a large segment containing insertions or deletions, we again require that GapC be less than 0.2 between these two unaligned regions.Query-anchored multiple alignment after taxonomy clustering is performed by aligning the HSPs reported by nongapped BLAST (see (m) it is inside an HSP region and the number of times (n) it is inside a variable region. A high ratio of n over m + n suggests that this position is inside the variable region most of the time. We estimate the statistical significance  of the variability for each position by a binomial distribution:For each amino acid position in the query gene, we can count the number of times q is the probability of an amino acid position being inside a HSP region. We estimate q by averaging the proportion of HSP in each hit sequence among all hits. If the p-value calculated using the above formula is less than the significance level, which we set at 0.05, we then consider this position as a variable position; otherwise, it is a conserved position. A consecutive run of variable positions forms a variable region. The next question is how long the variable region should be to be considered meaningful, as opposed to functionally unimportant regions such as linker regions, which are usually short. From our experience, there is no clear decision boundary between the length of the region and its functional importance. Any choice of cutoffs would have to balance between false positives and false negatives. However, previous studies on the length distribution of protein domains has shown that the most likely length of a protein domain is around 70 amino acids, and regions shorter than this are less likely to form a functional domain (H. pylori.where l domain . Based on over (m + n) for each position and plotting it. We call such plots variability profiles. Sample variability profiles are shown in A direct way of visualizing the variability of a protein sequence is by calculating the ratio of http://geneva.bu.edu. SVGs for several representative genomes are listed there. For SVG lists in other genomes, please request more information from Y. Zheng at E-mail: zhengyu@bu.edu. All the case examples mentioned throughout the paper and Supporting Information have been compiled into one Web page, http://geneva.bu.edu/paper03.html, with hyperlinks. Readers can follow each hyperlink to access additional information from Pfam, BLOCKS, PRINTS, COG, and nongapped BLAST for each gene.We provide a static collection of segmentally variable genes at our Web site, Figure S1Conservation score is plotted under the alignment . The conserved portions are as follows: N-terminal domain, extreme C-terminal domain, and a segment between N-terminal and C-terminal domain. The variable domain  overlaps with the transmembrane 22-strand β-barrel regions.(2.69 MB EPS).Click here for additional data file.Figure S2H. pylori ProRS. The blue segments are HSPs reported by nongapped BLAST. The yellow segments are the variable region. The gray region is the gap-rich region . See http://geneva.bu.edu/paper03.html for a high-resolution Web figure.The query protein is (4.71 MB EPS).Click here for additional data file.Figure S3(3.12 MB EPS).Click here for additional data file.Figure S4YhdY is currently annotated as a hypothetical protein and contains a conserved domain for mechanosensitive proteins  and two variable domains (N- and C-termini).(2.86 MB EPS).Click here for additional data file.Figure S5map). This is an example of a fully conserved gene.It is the methionine aminopeptidase (type Ia (1.87 MB EPS).Click here for additional data file.Figure S6It is currently annotated as “conserved hypothetical protein.” The N-terminal region is variable. The conserved C-terminal domain is characteristic of methionine aminopeptidase.(2.22 MB EPS).Click here for additional data file.Figure S7Shown as a histogram. Only variable regions inside fully conserved genes and SVGs are included. Pink line shows the domain size distribution in 3D-structure database (data from (643 KB EPS).Click here for additional data file.Table S1(62 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/GenBank/) accession numbers for the genes discussed in atpA (2314285), atpD (2314283), atpG (2314284), dnaX (2313841), flgK (2314271), ftsK (2314237), gyrB (2313611), hetA (2314367), HP1450 (2314626), infB (2314195), M.hpyAVIB , mutS (2313742), NQO3 (2314431), NQO8 (2314432), polA (2314647), rps4 (2314460), spaB (2313717), spoT (2313901), tlpA (2313179), and tlpC (2313162).The GenBank (Agrobacterium tumefaciens (15890351), B. subtilis (16079962), Enterococcus faecalis (8100675), E. coli K12 (16128553), L. innocua (16801788), Mycobacterium leprae (15826988), M. tuberculosis CDC1551 (15840173), Nitrosomonas europaea (22955201), Nostoc sp. PCC 7120 (17228666), P. syringae pv. syringae B728a (23470301), Ralstonia metallidurans (22980570), R. solanacearum (17548875), Synechococcus sp. PCC 7942 (21954778), and Thermotoga maritime (15644402); in case studies, B. subtilis yhdY (2633299), E. coli b1330 (1787591), H. pylori cytosine-specific DNA methyltransferase (2313124), H. pylori HP1299 (2314463), H. pylori HP1037 (2314181), H. pylori prolyl-tRNA synthetase (2313329), and H. pylori VacB (2314413).The GenBank accession numbers for the genes discussed in


It already exists within the US Food and Drug Administration, argues a former clinical reviewer of psychotropic drugs at the FDA It already exists within the US Food and Drug Administration Over the past several years, there has been growing concern about selective publication of clinical trial results ,2. The dThe two most frequently suggested remedies for the selective reporting of clinical trials results have been to register all clinical trials and to make their results publicly available. Registries have been called for at least as far back as 1974; hundreds have in fact already been established . Thus, oIncreasing the pressure on pharmaceutical companies to include more trials in registries, the International Committee of Medical Journal Editors has announced that, as a condition of considering a trial for publication, member journals will require its registration in a public trials registry . FurtherIn this essay, I argue that a highly valuable but underused registry and results database for US trials already exists within the Department of Health and Human Services, specifically within the Food and Drug Administration (FDA).Before a pharmaceutical company can conduct a US trial that it intends to use in support of a new drug application (NDA), it must first register that trial with the FDA. Because the NDA forms the basis for marketing approval, it seems likely that the percentage of industry-sponsored trials that are registered with the FDA is very high. This registration takes the form of an investigational new drug (IND) application . The INDThe FDA then begins the NDA review process, during which a physician, a statistician, and a pharmacologist, among others, generate lengthy review documents . These rA Cochrane systematic review of antidepressants for generalized anxiety disorder  lists onp = 0.039) result on a secondary observed-cases analysis, but a nonsignificant (p = 0.38) result on the primary efficacy analysis defined a priori. The third study was clearly negative, with p-values of 0.33 and 0.57 on the primary and secondary analyses, respectively.Turning to the controlled-release formulation of paroxetine (Paxil CR) for panic disorder, a review article states in its abstract that the drug “demonstrated efficacy in three well designed studies in patients with panic disorder with or without agoraphobia” . In readhttp://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm). These NDA review documents are much more detailed than the resulting package insert and often more detailed than corresponding journal publications.This process occurs entirely outside of the public domain. However, in the interest of making the FDA more “transparent,” and in accordance with the Electronic Freedom of Information Act , the FDAFor example, while the clinical trials section of the package insert is typically a few paragraphs long, the efficacy portion of the clinical review usually runs tens of pages. Because the FDA is made aware of all studies that the sponsor plans to use in support of the NDA before they are conducted, and thus before there can be any selection based on outcome, these reviews cover not only studies that are positive , but also studies whose outcome was negative or indeterminate. The sidebar gives an example of how NDA review documents at the FDA give valuable information about paroxetine for anxiety disorders.In the examples discussed in the sidebar, our having access to the FDA review documents allows us to become aware of, and see beyond, apparent publication bias. It is in the best interest of the public health for the FDA to make as many reviews available as possible. According to the FDA Web site, “As FDA continues to be one of the world's leading agencies in its emphasis on openness and transparency, it is aware that making even more information available to the public will further the Agency's mission to protect and promote public health and improve its credibility. For example, FDA has aggressively implemented the Electronic Freedom of Information Act…” .Unfortunately, the availability of review documents on Drugs@FDA is sporadic. To take additional examples from psychiatry, NDA reviews have been posted on Drugs@FDA for some approved drug–indication combinations, such as fluoxetine for pediatric depression, and aripiprazole and quetiapine for schizophrenia. However, NDA reviews for many other drug–indication combinations have not been posted: the Prozac Weekly formulation of fluoxetine, clozapine for suicidal behavior in patients with schizophrenia or schizoaffective disorder, and quetiapine for mania, among others. A review on paroxetine for pediatric depression, the subject of Elliot Spitzer's suit against GlaxoSmithKline, is not posted. This is probably because this drug–indication combination was not approved; in fact, it is possible that GlaxoSmithKline did not file an NDA to be reviewed. However, I do not understand why, in cases where NDAs were both submitted and approved, such as the ones listed above, some reviews are posted while others are not.I therefore suggest that we increase access to the clinical trials registry and results database that already exist within the FDA. The agency could expand its implementation of the Electronic Freedom of Information Act and make all NDA reviews, at least for approved NDAs, available in the public domain. The act is written into the FDA portion of the Code of Federal Regulations as follows: “The Food and Drug Administration will make the fullest possible disclosure of records to the public, consistent with the rights of individuals to privacy, the property rights of persons in trade secrets and confidential commercial or financial information” .There would surely be obstacles. The pharmaceutical industry would vigorously invoke Exemption 4 of the Freedom of Information Act, the exemption for trade secrets and confidential business information . HoweverIt should be clarified that this resource does not compete with proposals by the AMA and other groups for clinical trial registries—rather, it complements them. The AMA has proposed the creation of a registry that is comprehensive in scope. The FDA's registry and results database are restricted to those trials aimed at supporting US marketing approval or a change in labeling in the US. While data from many studies conducted abroad are submitted to the FDA for this purpose, this is not the case for drugs for which the sponsor has elected not to seek approval for marketing in the US. Nor does the FDA review data from most trials funded by other US government agencies, such as the National Institutes of Health, or by foundations. And drug companies fund investigator-initiated trials that are often not registered with the FDA.To make the FDA review data more accessible and user-friendly, simple formatting changes would be needed. For those (few) reviews that are currently posted on Drugs@FDA, one can determine the indication being evaluated only after opening the document and paging through it.  DespitDespite the limitations of the FDA's database, making it public is a strategy that could be implemented both rapidly and easily by building upon existing infrastructure. While we await the creation of a clinical trials registry and results database that is truly comprehensive, we already have at our disposal one that could serve as a trove of in-depth and unbiased information on many, if not most, drugs currently marketed in the US.


The developments in echocardiography or ultrasound cardiography (UCG) have improved our clinical capabilities. However, advanced hardware and software capabilities have resulted in UCG facilities of dubious clinical benefits. Is tissue Doppler echocardiography (TDE) is one such example?TDE has been touted as advancement in the field of echocardiography. The striking play of colors, impressive waveforms and the seemingly accurate velocity values could be deceptive. TDE is a clear case of inappropriate use of technology.To understand this, a comparison between flow Doppler and tissue Doppler is made. To make clinically meaningful velocity measurements with Doppler, we need prior knowledge of the line of motion. This is possible in blood flow but impossible in the complex myocardial motion. The qualitative comparison makes it evident that Doppler is best suited for flow studies.As of now TDE is going backwards using an indirect method when direct methods are better. The work on TDE at present is only debatable 'research and publication' material and do not translate into tangible clinical benefits. There are several advances like curved M-mode, strain rate imaging and tissue tracking in TDE. However these have been disappointing. This is due to the basic flaw in the application of the principles of Doppler. Doppler is best suited for flow studies and applying it to tissue motion is illogical. All data obtained by TDE is scientifically incorrect. This makes all the published papers on the subject flawed. Making diagnostic decisions based on this faulty application of technology would be unacceptable to the scientific cardiologist. Echocardiography or Ultrasound Cardiography (UCG) is a key investigation in cardiology. Its non-invasive nature makes it a widely acceptable and safe form of investigation. In many cases it has surpassed cardiac catheterisation in diagnostic yield and has become the investigation of choice.The rapid development of UCG has made available to the clinician newer insights into the anatomy and physiology of the heart. With the availability of digital technology it is possible to manipulate raw data in different ways. This has also spawned UCG facilities of dubious clinical benefits. Tissue doppler echocardiography (TDE) is one such example.When we consider UCG we are interested in 2 distinct aspects of the heart:1. Structure 2. Blood flow. Both these ultimately indicate cardiac function. B-mode/M-mode imaging or simply Ultrasound Imaging (USI) studies structure. Blood flow is studied by Doppler studies (DS). In each stream every development should incrementally improve our understanding of the heart, its structure and function. Or the development should improve the ease of operation or permit better documentation.In USI we had the following developments and each development added to the diagnostic yield. M-mode – temporal resolution; 2D – spatial resolution; Harmonic imaging – improvement in image quality; B-color – enhanced tissue perception; Ultrasonic tissue characterization and acoustic quantification – quantitative analysis of tissue; Omni-plane M-mode – temporal resolution at different segments and 3D imaging – another dimension of imaging.In DS too we had many developments, each improving our perception. Continuous wave (CW) – detected and measured high velocity flows; Pulse wave (PW) – located the abnormal flow; Color flow mapping (CFM) – made it possible to 'image' blood flows and power amplitude Doppler – to study vascular flow. Contrast, trans-oesophageal and intravascular UCG are the clinical extensions and exploitation of these developmentsWhat we see in TDE is a retrograde development. In the context of UCG, DS is an indirect method to study blood flow since USI in its present state cannot image fast moving blood cells. Spontaneous echo contrasts could mark the beginning of USI of blood cells. By using TDE we are using an indirect method where direct USI methods are better. Besides, TDE is an inappropriate application of the Doppler methodology.TDE is a by-product of color flow mapping (CFM) technology. In CFM, tissue signals are suppressed and flow signals are analyzed. The reverse is true in TDE. Doppler velocities associated with tissue motion are slower than blood flow. Flow signals are eliminated on the basis of signal amplitude. The amplitude of tissue motion is about 40 db greater than the corresponding flow amplitude. Blood flow imaging applies a high pass filter to exclude the strong but low frequency tissue signals and other 'noise' before the signal is input into an autocorrelator that estimates velocity. ErroneouThe first report on the use of TDE appeared in 1989. However all data using this modality is flawed. You cannot even think of designing a study to prove the point because the mensuration technique itself is wrong. So we have to use the scientific methods of comparison and reductio ad absurdum to verify it. Any new modality of diagnosis or treatment should be compared with the existing systems. In clinical trials we usually employ quantitative comparison with statistical methods. In this case, since the data acquisition is flawed we have to use a qualitative comparison. This is accepted practice in clinical medicine – It is like comparing a weak limb with the strong one in neurology. One also needs to understand the Doppler methodology to test the hypothesis.The article explains the flaws in tissue Doppler echocardiography. As the concept itself is flawed, To estimate clinically significant peak Doppler velocity the following steps are required:prior knowledge of the line of motion of the object. Only if you know this line of motion, you can apply the interrogating signal along the path of motion. This is because of the directional bias of Doppler.1. You must have 2. Next you will know the direction of motion – whether the object is moving towards the interrogating signal or away.3. Only after the first two steps you can measure the clinically significant peak velocity.Based on the above discussion it is clear that the following information can be derived from Doppler:1. Is there motion? Does the object move? This is a random application of Doppler. This is one aspect of tissue Doppler. But this information is redundant as we can already see the movement better by ultrasound imaging.2. What is the direction of motion? Is it moving towards or away from the interrogating signal? This is also redundant information in tissue Doppler for the reason given above.significant velocity we must know the clear-cut line of motion. Application of Doppler along this line enables us to measure the meaningful peak velocity.3. To measure the peak anticipated flow. That is also why we do not measure the mitral flow velocity from the parasternal views. Next we come to learn whether the blood is flowing towards or away from the transducer. After all these steps it is possible to measure velocity. In tissue Doppler we can measure several velocities in several directions. But we can never know which is the clinically significant peak velocity.Thus the primary step is to know the line of motion. This is possible in flow Doppler but impossible in tissue Doppler. For example we know for sure that the blood moves from left atrium to left ventricle through the mitral valve. At the mitral valve this is a unique free linear motion towards the apex. This produces a clinically significant velocity. That is why we place the sample volume at the mitral valve from apical views so as to align the beam in the line of TDE is a clear case of misuse of technology. To understand this, a comparison between flow Doppler and tissue Doppler is in order. CFM allows us to 'see' what we cannot see with ultrasonic 'eyes' hence its value is great. In color TDE we see more or less what we already see by USI hence its value is marginal. In CFM the anatomic landmarks are intact as the color is superimposed on the B-mode image. In color TDE the B-mode is eliminated and the entire 'picture' is Doppler information. It is difficult to determine the different anatomic regions on TDE. For example it would be very difficult to delineate the blood-endocardial boundary. In fact, the word 'tissue Doppler' is a misnomer and this is one of the reasons for the prevailing confusion. It gives the impression that only myocardial tissues are studied. The appropriate term would have been low velocity Doppler. Any movement in the low velocity range will be detected by 'tissue Doppler'. Myocardial tissue movement is just one among them. The discriminatory power of this modality is unsatisfactory.Myocardial motion is very complex and not amenable to Doppler studies. In the cardiac motion there are translational, rotational and deformational movements. Besides many tissues near the heart move – due to transmitted cardiac motion, vessel pulsation, respiratory motion and involuntary muscle movements and these interact with cardiac motion further and cause false Doppler shifts. VelocityBlood flow is simple and suitable for Doppler study. In flow Doppler at a particular point the linear projectile motion of free moving blood cells are studied. At the current interrogation points there is a unique unidirectional flow in one part of the cardiac cycle. For example in mitral valve Doppler interrogation, the unique directional signal is obtained only in diastole. If there is a signal in the in systole, it becomes abnormal and this information has great value. In TDE the to- and- fro motion (systole and diastole) of a tethered interconnected syncytium of myocytes is imaged. Such information is useless. This can be even otherwise seen and analyzed by USI.Doppler in its present form, is best suited to study flow.In flow Doppler there are definite 'points of interrogation', which are the normal and abnormal orifices. In TDE there are no such definite points. While in flow Doppler higher velocities are studied, TDE studies lower velocities i.e. the study of hypo functioning myocardial segments. Lower velocities are difficult to appreciate. The higher velocities are easier to appreciate due to aliasing and variance. There are no such indicators for low velocity. Thus hypo function is difficult to analyze. The derivations from flow Doppler are based on accepted hydrodynamic formulae (like Bernoulli's equation) and allows us to get orifice size, amount of flow and pressure gradients, which are clinically of great importance. The derivations from TDE are useless. Thus It is important to note that in all the above papers the data acquired is flawed due to the basic problem in technology application. Hence the correlations obtained in these papers are spurious or non-sense correlations. One paper presents a misuse of statistics when tissue Doppler velocities have been correlated with myocardial interstitial fibrosis and myocyte interstitial beta-adrenergic receptor density.[TDE has shown some promise in the Wolff-Parkinson-White syndrome. But even density. By analoThe prime use of TDE is to study regional wall motion abnormalities. It is claimed that it is possible to quantify wall motion at rest and during stress. However with the above-mentioned limitations it is unlikely to prove more effective than USI. The measurements are ultra sensitive and represent gross movements rather than myocyte contractions. With the advent of omni-plane M-mode it is possible to get temporal resolution in inaccessible planes and study wall motion abnormalities in a better way.The Doppler methods use mathematical formulae to indirectly study motion that cannot be directly measured. Examples are movements of stellar bodies and in the present context movements of blood cells. We use the indirect method based on Doppler principle because we cannot 'image' blood cells by USI. When the imaging of blood cells become possible, Doppler studies would probably be relegated to the background. Color TDE would have been valuable if direct USI was not available.Tissue Doppler has been a disappointing modality in clinical ultrasound cardiology. This is due to the basic flaw in the application of the principles of Doppler. Doppler is best suited for flow studies and applying it to tissue motion is not acceptable. In flow Doppler we suppress the tissue 'noise' and display flow. In TDE ipotential areas for investigation and research.However the author is of the view that TDE could probably have some role in diagnostic UCG. What is required is that Doppler should be used for the purpose it was intended i.e. to study blood flow. Thus we will have 2 types of Doppler studies: High velocity flow Doppler (HVFD) studies and Low velocity flow Doppler (LVFD) studies. Conventional Doppler would be HVFD. TDE would be LVFD and we would be imaging and analyzing very low velocity flows. The possible uses of color LVFD studies would be the study of low flow thrombogenic states, flow in the atria, the blood-endocardial/endothelial interface, low velocity turbulences and blood flow in organs. Pulse wave spectral LVFD could be useful for the detection of low velocity turbulences as a cause for murmurs (not detectable with HVFD). These are It is time to look at TDE on more realistic terms. As a new modality of imaging it appears exciting. But its real clinical utility is doubtful. TDE does not give any additional information over the conventional modalities. In fact due to the above-mentioned deficiencies it could give misleading information. Making diagnostic and therapeutic decisions based on this faulty application of technology would be unacceptable to the scientific cardiologist.CFM Color Flow MappingCW Continuous WaveDS Doppler StudiesHVFD High Velocity Flow DopplerLVFD Low Velocity Flow DopplerPW Pulse WaveTDE Tissue Doppler EchocardiographyUCG Ultrasound CardiographyUSI Ultrasound ImagingNone declared.


Molecular detection of herpesviruses DNA is considered as the reference standard assay for diagnosis of central nervous system infections. In this study nested PCR and real time PCR techniques for detection of Herpes simplex virus type 1 (HSV-1), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in cerebrospinal fluid of HIV patients were compared.Forty-six, 85 and 145 samples previously resulted positive for HSV-1, CMV and EBV by nested PCR and 150 randomly chosen negative samples among 1181 collected in the period 1996–2003 were retrospectively reassessed in duplicate by real time PCR and nested PCR.Samples giving positive results for CMV, HSV-1 and EBV with nested PCR were positive also with real time PCR. One of the negative samples resulted positive for HSV and one for EBV. Real time PCR showed comparable sensitivity and specificity vs nested PCR.Real time PCR proved to be a suitable method for diagnosis of herpesvirus infections in CNS, showing comparable sensitivity and being less time consuming than nested PCR. Opportunistic infections as well as tumors and vascular and metabolic disorders are common in HIV-infected patients -3. GenerAlthough the rate of CNS complications is relatively low, if compared with the high prevalence of herpesviruses in the population, these viruses represent the most important pathogens associated with viral encephalitis and meningitis ,8, beingOne-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus specific Ig antibodies in CSF for laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis.Although nested PCR is considered the method of choice in terms of specificity ,13,14, sIn the last years, introduction of real time PCR has markedly increased the ease and the speed in the virology laboratory due to the relevant technology that permits rapid temperature cycling within a close system. Considering the importance of relationship between viral load in CSF and severity and outcome of disease, an additional advantage of Real Time PCR is the capability to perform simultaneous qualitative and quantitative analysis.Here is reported our experience gained in the diagnosis of herpesvirus infections of the CNS in HIV patients by means of nested PCR and Real Time PCR, which has been recently applied in our laboratory.A total of 1181 CSF samples collected in the period 1996 – 2003 from HIV patients attending at Luigi Sacco Teaching Hospital of Milan  affected by acute encephalitis or meningitis or encephalopathy or other neurological syndromes were considered.Particularly, they consisted of 684, 954 and 933 CSF samples previously tested for HSV-1, CMV and EBV, respectively, by means of nested PCR. Of these, all the positive samples and 150 negative samples randomly chosen were retested by means of nested PCR and real time PCR. Each sample was re-extracted and run in duplicate.CSF positive and negative samples were stored at -80°C until analysis.Clinical data of patients with positive samples were available only in the 20% of the total. These patients generally recorded meningoencephalitis signs such as central motor or sensory alterations, consciousness loss, seizures defects.Spin-column based QIAamp Mini Kit (Quiagen, Hilden, Germany) protocol extraction for CSF was used as indicated by the manufacturer.This procedure allowed for the rapid purification of DNA from 200 μL of CSF and comprised four successive steps carried out using QIAamp Spin Columns in a standard microcentrifuge. Purified DNA was concentrated at a final volume of 20 μL.glycoprotein D gene of HSV-1 [gp58 of CMV [Bam HI-W region of EBV [Nested PCR was carried out in a 50 μl mixture containing 40 μl of first amplification mix (outer primers, buffer, dNTP)-, 2U/μl Taq DNA polymerase (Roche Diagnostics-Germany) and 5 μl of purified DNA. Primer pairs selecting for of HSV-1 , for the8 of CMV , and forn of EBV  are showAfter an initial 2 min denaturation at 94°C, 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec were carried out, followed by a 5 min extension at 72°C using a thermal cycler . The reaction mixture for the second amplification round was the same as for the first one, except for the "inner" primers used instead of the "outer" primers. In the second amplification round 44 μl of amplification mix and 1 μl of the first amplification round PCR product were used. The thermal cycling was repeated as for the first amplification round but using 30 cycles after the initial 2 min denaturation. Each amplification run contained a negative control, consisting of water and a positive plasmidial control.Analysis for the PCR products was performed by means of 4 % agarose gel electrophoresis followed by visualization with ethidium bromide (0.4 μg/mL) staining and UV illumination to confirm the expected products.For diagnostic real time PCR Taq polymerase RT PCR Kit  was used. Target regions for HSV and CMV were the same as in nested PCR, while EBNA-1 gene was amplified for EBV  as shownThe RT PCR was performed in 25 μl mixture containing 20 μl of amplification mix (buffer, dNTPs, Taq gold polymerase, Rox passive fluorocrome, primers and MGB Eclipse probe) and 5 μl of purified DNA.The amplification program included an initial decontamination with uracile N'-glycosilase at 50°C for 2 min, followed by denaturation at 95°C for 10 min and 45 two steps of 15 sec at 95°C and 1 min at 60°C.t) values were calculated by determining the point at which the fluorescence exceeded a background limit of 0.04. Each analytical session comprised also a negative control (distilled water).The RT PCR products were detected by measuring fluorescence with passive reference dye in Sequence Detection System ABI Prism 7000 (Applied Biosystem). Control threshold  and its concentration determined spectrophotometrically. Then, plasmidic DNA was serially diluted in a stabilizing buffer to the final desired concentration. Amounts of copies/mL in each sample were determined by means of a quantification software , by considering an extraction recovery of 80%.Quantification was carried out by analysing four positive plasmidial standards at 10Of 954 CSFs previously examined for CMV by means of nested PCR, 85 samples resulted positive. Among the 684 CSFs tested for HSV-1, 46 samples were found positive, while, 145 of 933 CSFs tested resulted positive for EBV.Reassessment of these positives and of 150 negative samples confirmed results previously obtained with the same method, with the exception of one HSV-1 positive sample, which resulted negative when retested, as shown in Table Results from real time PCR are reported in Table By considering nested PCR as gold standard ,13, sensIntroduction of PCR into routine diagnostic has rapidly gained a pivotal role for diagnosis of a wide range of diseases, supplanting, in many cases, other methods, such as the classical serodiagnosis. This is particularly true for diagnosis of herpes virus infections in immunocompromised patients, where diminished or suppressed virus-specific antibody responses do not reflect possible reactivated herpes mediated aethiologies .Real time PCR has represented a further step forward, since it allows for quantitative detection of target DNA in a single sample over a large range, remaining possible qualitative detection. Even if contradictory results have been found -21, quanIn the last years several real-time PCR methods have been developed for detection of herpesviruses in different biological specimens -27. RealIn the present work, we compared a real time PCR panel for detection of herpesvirus DNA in CSF with methods employing nested PCR. Our results indicate an overall agreement between the two methods, as reported by other authors ,27,28. DSince extraction panels and primers for HSV and CMV were uniform for the both types of assays, inhibitors present in the DNA preparations could not explain the different results obtained for these samples. Moreover, being nested PCR generally considered as the gold standard for diagnosis of herpes virus in CSF, we tried to use similar primers, chemistry and amplification conditions in order to limit differences for a better comparison of the performance of the two methods. Thus, discrepancies could be likely attributed to the different detection of amplification products, although real time PCR has been reported to be as sensitive as nested PCR . This suData obtained in this study confirms the validity of real-time PCR method for detection of herpesvirus DNA in CSF specimens of HIV patients, being sensitive, rapid and quantitative. Since specific and rapid diagnosis is the main target in the case of CNS infections, real time PCR could be considered the method of choice, due to its high specificity, sensitivity and rapidity, once proper quality controls will be available.The company Amplimedical SpA-Bioline Division-Italy will pay the article processing fees for the manuscript.LD conceived of the study and participated in its design and coordination, AL carried out real time PCR, EDV participated in data analysis and drafted the manuscript, GG performed statistical analysis, RB carried out nested PCR, MRG participated in design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


As shown in previous investigations, in many primates and rodents this class of elements has remained transpositionally active, as reflected by increased copy number and high sequence diversity within and among taxa.Endogenous retrovirus-like elements (ERV-Ls, primed with tRNA leucine) are a diverse group of reiterated sequences related to Here we examine whether proviral-like sequences may be suitable molecular probes for investigating the phylogeny of groups known to have high element diversity. As a test we characterized ERV-Ls occurring in a sample of extant members of superorder Uranotheria (Asian and African elephants, manatees, and hyraxes). The ERV-L complement in this group is even more diverse than previously suspected, and there is sequence evidence for active expansion, particularly in elephantids. Many of the elements characterized have protein coding potential suggestive of activity.In general, the evidence supports the hypothesis that the complement had a single origin within basal Uranotheria. From the perspective of evolutionary biology it is of great interest that some classes of ERVs are known to retain original functions, including the capacity to produce infectious viral particles [env gene [ERV-Ls are retroviral elements (retroelements) lacking the envelope gene (ERV-L by . Similararticles . Others articles . Formatienv gene . Human ienv gene . Thus, Eenv gene .pol) gene sequences from 22 mammalian species revealed ERV-Ls that have expanded in copy number and remained active over long periods of time [Comparison of ERV-L polymerase  fragment using degenerate primers tested in other mammalian orders. Extending our previous work [To investigate whether ERV-L and other retroelements may be useful in resolving phylogenetic questions involving uranotheres at multiple taxonomic levels, we utilized an ERV-L polymerase gene . No identical sequences were shared among taxonomic groups. All nine Asian elephant and all 10 manatee clones were unique. However, one Asian elephant clone, designated Max3 (accession number AY394573), was a recombination product of clone Max2 (accession number AY394572) and clone Max6 (accession number AY394576). Whether this represents a PCR artifact or is a genomic recombination event is not known. However, it is not expected that recombinational PCR would be observed in modern undamaged DNA .Procavia.Among the African elephant runs, four clones differed at 0–1 positions. As PCR errors probably account for these minor differences we assume only 6 unique ERV-Ls were discovered for this individual. Similarly, the hyrax sample yielded 3 groups comprised of 2 identical sequences, while two other sequences differed at 5 positions. Thus, 5 unique ERV-Ls were also obtained for gag and pol genes (MuERV-L, GenBank no. Y12713). Twelve clones were in frame with no stop codons. However, only 6 of the total 12 were unique  is known to be expressed in specific tissue types [The most important finding resulting from this study is that elements that have undergone expansion–i.e. have remained transpositionally active–are the ones that are most likely to group monophyletically and those that have not tend to be consistent in their higher-level taxonomic distribution . Thus, there are different elements within the same family demonstrating different evolutionary trajectories. It is reasonable to suppose that for some groups, such as rodents, primates, and uranotheres, continued expansion of active ERV-L elements was tolerated by the host. By contrast, in other ungulate and carnivore lineages active elements were not inherited or were silenced early during their evolution and ERV-L expansion did not occur. Older elements tend to evolve as typical orthologous sequences. An advantage of the great diversity of elements is that with a single PCR, cloning, and determination of multiple clone sequences one retrieves multiple independent sequences with which to do phylogenetic analysis. In these regards, the uranothere evidence is consistent with results previously reported for primates and rodents in which older elements and elements that have undergone bursts of transposition were found coexisting. In each of these groups there is now good sequence evidence for retroelements that have retained coding capacity, which is of some interest since HERV-L ue types . This inIn terms of our general results, elephants and the manatee were found to contain the most diverse sequences, while hyrax showed comparatively less diversity. This finding is consistent with results from our previous study involving extinct elephantids . ElementAlthough this study shows that the value of ERV-Ls for the narrow purpose of phylogenetic reconstruction is limited at higher taxonomic levels, ERV-L is only one class among many different groups of ERVs in mammalian genomes. Some regions of ERVs have been used successfully to reconstruct phylogenies at lower taxonomic levels . AdditioLoxodonta africana) DNA was supplied by N. Georgiadis of the M'Pala Research Centre, Kenya. Asian elephant (Elephas maximus) blood was provided by J. Hektor of the Tierpark Hellabrunn, Munich. Manatee (Trichechus manatus) blood was provided by D. Murphy of the Lowry State Park, Florida. Hyrax (Procavia capensis) muscle was provided by G. Amato of the Bronx Zoo, New York.African elephant (One ml of blood or approximately one gram of tissue was incubated in 1–2 ml 10 mM Tris-/Cl (pH 7.5), 10 mM EDTA (pH 8.0), 50 mM NaCl, 2% SDS, and 0.6 mg/ml Proteinase K overnight at 37 C, extracted with phenol and chloroform, and subsequently concentrated with 50 ul Millipore Ultrafee MC 30,000 NMWL columns or precipitated in 2.5 volumes ethanol and 1% NH4 Oac.pol gene are described in [PCR primers for the ERV-L ribed in . Three μribed in . Five μlRepresentatives of each clade determined by Bénit et al.  were incMaximum parsimony and neighbor joining analysis was performed using PAUP 4.0b . HeuristADG oversaw the molecular genetic studies, coordinated the study, participated in the sequence alignment and phylogenetic studies and co-drafted the manuscript. CCE participated in the phylogenetic analysis. RDEM participated in the design and coordination of the study and co-drafted the manuscript.


Defining and classifying disease is at the heart of medical practice, but the process is slow and laborious. A new "open source" approach could be faster and more democratic Defining and classifying disease is at the heart of medical practice. But the standard approach to classification is slow and laborious. A new approach promises to revolutionise the way in which we classify disease. It involves the free and public sharing of information via the Internet—the so-called open-source, or, perhaps more appropriately termed, “free community,” approach .http://www.ICDNS.org). The main characteristics of the ICDNS are free collaboration via the Internet, online access to all collaborative tools via the World Wide Web, global participation, and democratic decision making [The International Society for Neuropathology is the first worldwide professional medical organisation to adopt such an approach with its International Classification of Diseases of the Nervous System , are an example of a disease subset now defined by a common molecular pathology. The large field of pathology and the neurosciences are two areas where the translation of morphological phenotypes into molecularly defined entities is already well underway. With the pace of change accelerated by advances in molecular science, we need a much more effective way to develop the debate about medical classifications.One effective way to further develop this debate is to adopt the approach used by the free-software and open-source movements , which hhttp://www.opensource.org) is a nonprofit venture dedicated to managing and promoting the open-source idea (http://www.fsf.org). The creation of the GNU/Linux operating system (http://www.gnu.org and http://www.linux.org) has resulted from the work of both.The development of global computer networks and the World Wide Web, in particular, have fostered the evolution of free and global sharing of intellectual property. The Open Source Initiative (rce idea . A relathttp://www.opensource.org).“The basic idea behind open source is very simple: When programmers can read, redistribute, and modify the source code for a piece of software, the software evolves. People improve it, people adapt it, people fix bugs. And this can happen at a speed that, if one is used to the slow pace of conventional software development, seems astonishing… Open source software is an idea whose time has finally come. For twenty years it has been building momentum in the technical cultures that built the Internet and the World Wide Web. Now it is breaking out into the commercial world, and that's changing all the rules.”  SourceThe ICDNS was inspired by the free software and open-source approach to software development. The Council of the International Society for Neuropathology approved the ICDNS as a community activity at its last meeting in Turin, Italy, in September of 2003 . The imphttp://www.ICDNS.org, where the global community of neuropathologists can judge them  as well as expert interest groups propose core definitions, which are then posted so that the global consultation process begins via the World Wide Web. After an online discussion period, ICDNS members holding a specialist certification in diagnostic neuropathology are invited to vote. Comments made online by contributors become part of the history of a disease definition so that nobody is left out and divergent views are not forgotten. The discussion process is thereby open and democratic, allowing wide participation—including from individuals in developing countries who are often excluded in traditional academic discussions.Different countries around the world show variations in their use of diagnostic criteria and medical classifications, which, in turn, can lead to different treatment approaches. This can certainly create problems when trying to reach a global consensus. However, free access to the information held under the ICDNS open licence may help to minimise these problems by promoting and stimulating collaborative research and the exchange of scientific ideas across the globe.Certain diseases are far more common in particular parts of the world. In India, for example, neurotuberculosis, cerebral malaria, fungal infections of the central nervous system, human rabies, encephalomyelitis, and cerebrovenous thrombosis are more common than in most developed countries. While the conventional pathology of these diseases is well known—and in some cases we also have expert knowledge on their morphological phenotypes—their exact pathogenesis is not understood, and cellular as well as molecular knowledge is missing. The ICDNS is expected to stimulate local researchers to engage in collaborative international projects in which they can receive feedback via the global consultation process.Publication of ICDNS criteria occurs under a general public licence. This means that all text can be freely downloaded and republished, avoiding the need for defining basic facts over and over again. Both core definitions and comments may be used immediately for diagnostic purposes as outlined in the guidance section of the ICDNS Web site.In the future, definitions of histopathological phenotypes may be linked to clinical as well as molecular biological datasets, such as those obtained from microarrays and combined with imaging parameters. It is obvious that expert consensus on histological phenotypes is required before “genome matching” and similar procedures can be applied to complex diseases in a meaningful way. Most diseases are presently still defined on the basis of their histopathology. Online forums to find diagnostic consensus will provide a very effective means of correlating descriptive data with molecular data. Subsequently, statistical clusters representing “signatures of disease” may be extracted from multidimensional data spaces that will be available online. This opens new roads to link with diagnostic and therapeutic approaches.It seems reasonable to propose that the adoption of the ICDNS paradigm by other medical specialties would facilitate the development of a comprehensive, global body of medical knowledge. Free access to this knowledge would allow novel, collaborative approaches to be developed to address the most pressing medical problems through supranational concerted efforts. The rather lowly role traditionally ascribed to the exercise of defining and classifying diseases would give way to an appreciation for the key importance of this process as a potentially powerful driver of change.


The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs).A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery.Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation.These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans. The need for a safe and effective prophylaxis or treatment for dengue virus (DEN) infection, a category A mosquito-borne human pathogen, is a critical global priority. DEN causes dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is associated with heterologous secondary DEN infection and affects thousands of people worldwide. The incidence of DHF/DSS is increasing in the western hemisphere and, although many different approaches are being tried to develop prophylactic DEN vaccines, none have been licensed for public health and there are no specific antiviral treatments available.DEN belongs to the family of Flaviviruses and is an enveloped single plus-stranded RNA virus with four distinct serotypes. The DEN genome of approximately 11,000 nucleotides encodes a polyprotein (C-prM-E-NS1-NS2a-NS3-NS4a-NS4b-NS5) consisting of three structural proteins (C, prM and E) and seven nonstructural proteins. The open reading frame is flanked by a 100 nucleotide-long noncoding region (NCR) at the 5' end and a 400 to 600 nucleotide-long NCR at the 3'end [in vivo gene-silencing approach using short interfering RNA (siRNA) to decrease DEN replication. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21–25 nucleotide fragments with characteristic 5' and 3' termini [argonaute2, that cleaves the target mRNA [We reasoned that an effective antiviral approach aimed at attenuating the DEN virus burden might protect infected subjects from DHF/DSS and, therefore, we examined the potential of an get mRNA ,5. This get mRNA ,7.in vivo leading to specific gene silencing. However, plasmids transfect mammalian cells poorly, and adenoviruses produce an acute inflammatory response and an immune response to viral vector-encoded antigens [Plasmid DNAs or adenoviruses encoding appropriate DNA sequences allow transient siRNA expression in cells and antigens ,8. We thNot I and the larger fragment was ligated to the synthetic adapter containing in order, Not I-Kpn I-Apa I-Xho I-Hind III-EcoR I-Bam HI-Sac II-Sac I-Cla I-Sal I-Bgl II-Not I. The U6 promoter was obtained by PCR amplification, using specific primers with the desired restriction sites from the template pSilencer 1.0-U6 (Ambion), and inserted into the adaptor at the Kpn I and Apa I sites to get a novel plasmid pCMV-U6.The pCMV-MCS plasmid (Stratagene) was digested with Apa I/Xho I and Xho I/EcoR I respectively. The modified pCMV-U6 plasmid was then redigested with Not I and the smaller fragment was ligated to the 2.9 kb fragment of pAAV-MCS (Stratagene) obtained following its Not I digestion to generate the corresponding si-vector for EGFP, RSV and DEN. HEK-293 cells were cotransfected with the helper plasmid and the si- plasmid to generate recombinant AAV.Pairs of oligos were synthesized to develop siRNA constructs. The nucleotide sequence for each siRNA is as follows: siEGFP: 5'-GGC GAT GCC ACC TAC GGC AAG CTT CTC GAT TCG AAG CTT GCC GTA GGT GGC ATC GCC CTT TTT G-3' ; siRSVNSHEK293 cells were cultured with DMEM (Cellgro) plus 10% FBS (Cellgro) and cotransfected with pSMWZ-siDEN, pHelper and pAAV-RC (Stratagene) by standard calcium phosphate transfection. Cells were harvested 48 hr post-transfection and the cell pellets were stored at -80°C.Cells were lysed by 5 cycles of freezing and thawing to release the virus. Crude viral lysate were collected by centrifugation at 27,000 × g for 30 min, and the supernatants were harvested and put onto a CsCl gradient (density 1.20/1.50) in fresh tubes and centrifuged for 16 h at 100,000 × g. Opalescent bands were collected after ultracentrifugation. Titers of purified AAVs were measured using an AAV titration ELISA kit (Progen Biotechnik, Germany).2 the nonadherent cells were removed and the adherent cells were cultured with fresh DMEM supplemented with 10% FBS (Cellgro), 200 ng/ml IL-4 (BD-Pharmingen) and 50 ng/ml GM-CSF (BD-Pharmingen) for 7 days prior to infection with DEN.Conditions were similar to those described previously . Buffy c5 Vero cells or DCs were seeded into six-well tissue culture plates and infected with different numbers of recombinant AAV carrying the DEN-siRNA silencing cassette. After 2 days the cells were infected with DEN-2 virus (strain16803) at an MOI of 0.1.1 × 10Cells were harvested and centrifuged for 10 min at 150 × g. The cell pellets were washed with PBS and resuspended with antibody to DEN-2 virus envelope protein, (Microbix Biosystems Inc, Clone No 3H5) on ice for 40 min. The cells were centrifuged and pellets were washed with PBS and then resuspended with secondary antibody conjugated with FITC (Sigma) for an additional 30 min. The number of infected cells was measured by flow cytometry 5 days post-infection. DCs were also stained with CD11c antibody conjugated with PE (BD-Pharmingen).2 for 5 days. Afterwards, 1% neutral red (Sigma) was added to each well and the plaques were counted 48 h later.The supernatants from DEN-2-infected DCs were collected at day 5 post-infection and 10-fold serial dilutions were allowed to adsorb to monolayers of Vero cells in six-well culture plates for 2 h. The medium was then removed and replaced by an agarose-containing overlay , 1% low melting-point agarose (Gibco BRL) and the plates were incubated at 37°C/5% COInfected-DCs were harvested on day 5 of infection with DEN-2 virus. Aliquots of DCs were put onto slides using a Cytospin and fixed with 4% paraformaldehyde. Apoptotic DCs were determined using the terminal dUTP nick end-labeling assay (TUNEL, Promega, Madison WI) and the annexin V apoptosis detection kit (BD Biosciences, CA).Supernatants from infected-DCs were harvested at 24 h, 48 h, 72 h and 96 h post-infection. Cytokine concentrations were measured by cytometric bead array (CBA) and ELISA (BD-Pharmingen) following instructions in the manuals.t test. Values of p < 0.05 were considered statistically significant.Data were expressed as arithmetic mean ± SEM. Levels of significance of the differences between groups were determined by the student In order to develop an AAV-siRNA system, a plasmid pSMWZ-1 was engineered that comprised a mouse U6 promoter linked to a siRNA cassette Fig. . To testTo test whether AAV-siDEN-2 expression decreases DEN-2 virus infection in cultured Vero cells, cells were infected with recombinant AAV carrying siDEN-2 (MOI 10) or siEGFP (MOI ~1000) silencing cassettes. After 2 days the cells were infected with DEN-2 virus at an MOI of 0.1. Five days later, the numbers of DEN-2 virus infected cells were quantified by flow cytometry using anti-DEN-envelope protein. Cells pre-infected with AAV-siDEN3UT, but not AAV-siEGFP, showed a significant reduction in DEN infection, and the reduction was dose dependent  silencing cassette. After 2 days the cells were infected with DEN-2 at an MOI of 0.1. Five days later, the numbers of DEN-2 infected cells were quantified by flow cytometry using DEN-2 antibody. Cells preinfected with AAVsiDEN3UT showed a 50% reduction in the number of infected cells  and ELISA assays. As showed in Figure The significant findings of this report include the development of an adeno-associated virus-based siRNA approach for downregulating gene expression. A recombinant AAV-siDEN3UT was utilized to induce significant decreases in DEN infection compared to control in both Vero cells and human DCs. The results indicate that siRNAs may be used to attenuate DEN infection in human DCs and may have therapeutic value.in vitro. In this AAV system, we incorporated the mouse U6 promoter, which is important for transcription and folding of the suppressor RNA, into a plasmid pCMV-U6.Interference of gene expression by siRNAs is a novel strategy to knock down specific genes in cells or tissues, and the specific silencing of pathogen genes using siRNA is a very attractive approach for the clinical treatment of infectious diseases. Long dsRNAs (of >30 nt in length) activate a dsRNA-dependent protein kinase and 2', 5'-oligoadenylate synthetase in mammalian cells, which leads to a non-specific reduction in levels of mRNAs . The endin vivo study [The choice of appropriate target genes is necessary for the success of the siRNA strategy, and two siRNAs derived from either the pre-M or the 3' NCR region of DEN-2 were used in our study. An internal deletion of 3' NCR nucleotide sequences was found to be lethal for DEN virus replication in an vo study . The 3' vo study ,19. ThusAedes aegypti mosquito bite, and resident skin DCs are an early target of DEN infection [DEN is transmitted through nfection . Immaturnfection . Replicanfection , and we nfection . Increasin vivo.DCs are one of the most powerful of APCs. After infection with virus in the periphery, iDCs process viral antigens, then differentiate into mature DCs and migrate from peripheral tissues to lymph nodes where they prime naïve CD4 and CD8 T lymphocytes to maintain protective antiviral cytotoxic T cell memory ,24. ThusOne of the important features of AAV vectors is the lack of inflammation following infection. We failed to detect significant IFNγ or IL-12 production in the supernatants of AAV-siDEN-infected DCs. This is in accordance with previous data -28, whicIn conclusion, we developed a novel AAV-mediated siRNA delivery system. Our results demonstrate significant downregulation of DEN protein expression in Vero cells and human DCs, which strongly suggest that our AAV vector can be useful for siRNA delivery and that this AAV system may be applied in clinical settings to attenuate DEN infection.AAV, adeno-associated virus; DCs, dendritic cells; DEN, dengue virus; DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome; MOI, multiplicity of infection; pEGFP, enhanced green fluorescent protein; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.None declared.WDZ constructed the si-plasmids, performed cell culture, isolated and administered the virus and isolated and cultured dendritic cells. RS assisted with virus handling and cell culture, and performed flow cytometry. GH did TUNEL assays. XK did cytometric bead array assays and ELISAs. HSJ measured virus titer by plaque assay. RFL, SW, KP and SSM designed and implemented the experiments, performed troubleshooting, and did the analysis and interpretation of the data.